United States
Environmental Protection
Agency

Research and Development
Environmental fitonitoring
and Support Laboratory
P.O. Box 15027
Las Vegas NV 89114
EPA-600/4-78-043
August 1978
Environmental
Monitoring Series

Quality Assurance
Guidelines for
Biological Testing

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                    RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development,  US. Environmental
Protection Agency, have been grouped into nine series.  These nine broad categories
were established to  facilitate further development and application of environmental
technology.   Elimination of traditional grouping was consciously  planned  to foster
technology transfer and a maximum interface in related fields.  The nine series are:


      1    Environmental Health Effects Research
      2.   Environmental Protection Technology
      3    Ecological Research
      4    Environmental Monitoring
      5.   Socioeconomic  Environmental Studies
      6    Scientific and Technical Assessment Reports (STAR)
      7,   Interagency Energy-Environment Research and Development
      8.   "Special" Reports
      9.   Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL MONITORING series This series
describes research conducted to develop new or improved methods and instrumentation
for  the  identification  and quantification of environmental pollutants at the  lowest
conceivably significant concentrations. It also includes studies to determine the ambient
concentrations of pollutants  in the environment and/or the variance of pollutants as a
function of time or meteorological factors.
This document is available to the public through the National Technical Information
Service, Springfield, Virginia 22161

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                                           EPA-600/4-78-043
                                           August  1978
         QUALITY ASSURANCE GUIDELINES
            FOR BIOLOGICAL TESTING
                      by

              Tracer Jitco, Inc.
          Rockville, Maryland  20852
           Contract No.  68-03-2462
                Project Officer

              Richard E. Stanley
Environmental Monitoring and Support Laboratory
           Las Vegas, Nevada  89114
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
      OFFICE OF RESEARCH AND DEVELOPMENT
     U.S. ENVIRONMENTAL PROTECTION AGENCY
           LAS VEGAS, NEVADA  89114

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                                   DISCLAIMER
      This report has been reviewed by the Environmental Monitoring and
Support Laboratory - Las Vegas, U.S. Environmental Protection Agency, and
approved for publication.  Approval does not signify that the contents
necessarily reflect 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.
                                      ii

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                              FOREWORD
     Protection of the environment requires effective regulatory
actions which are based on sound technical and scientific
information.  This information must include the quantitative
description and linking of pollutant sources, transport mechanisms,
interactions, and resulting effects on man and his environment.
Because of the complexities involved, assessment of specific
pollutants in the environment requires a total systems approach
which transcends the media of air, water, and land.  The Environ-
mental Monitoring and Support Laboratory-Las Vegas, contributes to
the formation and enhancement of a sound integrated monitoring data
base through multidisciplinary, multimedia programs designed to:

         •  develop and optimize systems and strategies for
            monitoring pollutants and their impact on the
            environment

         •  demonstrate new monitoring systems and technologies
            by applying them to fulfill special monitoring needs
            of the Agency's operating programs

     In preparing these quality assurance guidelines a definite
effort was made to incorporate into this one document the various
aspects of quality assurance necessary to produce biological
data of known quality.  This required, in addition to the usual
quality assurance considerations, appropriate consideration of the
many peculiarities existing among those more commonly used test
organisms.  Considering the broad scope of this endeavor and the
varied backgrounds of the expected readers, it was difficult to
avoid some repetition among sections, and to determine the details
necessary to meet the needs of the less experienced scientists
without offending the more experienced.  We believe we have mini-
mized the repetition to the point necessary to permit each section
                                 lii

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to stand alone, and the details given are those we feel are
necessary to clearly address the subject.  The comprehensive
references at the end of each section will permit a more in-
depth coverage of any of the material presented.
                         George B. Morgan
                              Director
          Environmental Monitoring and Support Laboratory
                             Las Vegas
                                 iv

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                                  PREFACE
      Quality assurance is widely practiced in Biological Research and in
Environmental Monitoring as in other areas of scientific and technical en-
deavor.  However, the activity of controlling the quality of results in the
subject areas is accepted as an indispensable part of laboratory management
and is not usually described explicitly as a sub-discipline.  In some related
areas such as analytical chemistry and clinical chemistry, good quality con-
trol-manuals are available.  These Guidelines are intended to contribute to
filling the need for a compendium of quality control practices for use in
biological research.

      These Guidelines draw from the good practices published by analytical
and clinical laboratories and incorporate observations made in a number of
EPA laboratories, contractor laboratories, and biological research laborato-
ries in general.  It was realized early and confirmed by discussions with
experts in various biological fields that the quality assurance aspects of
biological testing depend on the particular test systems being used.
Accordingly, the Guidelines cover the general aspects of quality assurance
(Sections 2 and 3.1), and then devote whole, separate sub-sections to Field
Research (3.2), Aquatic Bioassay (3.3), Microbiologic Assay (3.A), and
Mammalian Bioassay (3.5).

      This format has led to repetition of some concepts many times.  However,
the user with a particular interest in one field of bioassay needs to refer
only to the general sections and to that part of the rest of the Guidelines
appropriate to his field.

      Recognition is given to the assistance given by many laboratories of
the Office of Research and Development and by some of their contractors.
This is a first endeavor at bringing together in one place the good practices
observed in many laboratories, confirmed by experience, and gleaned from the
literature.  With time and  use, the Guidelines should help in maintaining
the validity and integrity of data derived from biological testing.

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                            CONTENT?,

Section                                                     Page

FOREWORD                                                     ill
PREFACE                                                        v
FIGURES                                                        x
TABLES                                                    xi - xiii

1   INTRODUCTION
    1.1   PURPOSE OF THE QUALITY ASSURANCE GUIDELINES
          1.1.1  Valid Data                                    1
          1.1.2  Integrity                                     2

    1.2   DEFINITIONS
          1.2.1  Quality Assurance                             4
          1.2.2  Biological Research                           4

2   QUALITY ASSURANCE ELEMENTS
    2.1   QUALITY ASSURANCE POLICY AND OBJECTIVES
          2.1.1  Laboratory Evaluation                         6
          2.1.2  Organization for Quality                      8
          2.1.3  Training for Quality                          9
          2.1.4  Other Objectives of a Quality Assurance Plan  9

    2.2   DESIGN AND ANALYSIS OF EXPERIMENTS
          2.2.1  Description of Design of Experiments         11
          2.2.2  Steps in the Design of Experiments           13
          2.2.3  Essential Statistical Concepts               13
          2.3.4  Experimental Models                          18

    2.3   SAMPLING
          2.3.1  Background of Sampling                       25
          2.3.2  Randomization Procedure                      25
          2.3.3  Sampling Models                              26
          2.3.4  Selection of Size of Sample                  26
          2.3.5  Management of Sampling                       28
    2.4   PRECISION AND ACCURACY OF TESTS
          2.4.1  Measurement of Precision and Accuracy        36
          2.4.2  Control of Precision and Accuracy            37

    2.5   PHYSICAL ENVIRONMENT OF RESEARCH
    2.6   CHEMICALS AND REAGENTS
          2.6.1  Purchase Specifications                      48.
          2.6.2  Acceptance Specifications                    70
          2.6.3  Control of Chemicals and Reagents            70

                               vii

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Section                                                     Page

    2.7   CONTROL OF TEST SUBJECTS
          2.7.1  Control of Animal Breeding                   73
          2.7.2  Good Animal Care Laboratory Practices        74
    2'. 8   CONTROL OF PERFORMANCE OF EXPERIMENTS
          2.8.1  Quality Control Charts                       75
          2.8.2  Assessing Laboratory Performance             78

    2.9   INTERLABORATORY TESTING                             80

    2.10  DATA HANDLING AND REPORTS                           81
    2.11  REFERENCES                                          82

3   QUALITY ASSURANCE IN BIOLOGICAL RESEARCH

    3.1   LABORATORY MANAGEMENT
          3.1.1  On-site Evaluation/Accreditation             86
          3.1.2  Laboratory Personnel                         94
          3.1.3  Biological Sampling and Testing              95
          3.1.4  Preparation of Study Protocols               97
          3.1.5  References                                   115

    3.2   FIELD RESEARCH
          3.2.1  Field Sampling                               117
          3.2.2  Field Analysis                               129
          3.2.3  Sampling Method                              133
          3.2.4  Functional Tests                             163
          3.2.5  Field Bioassay                               177
          3.2.6  References                                   196

    3.3   AQUATIC BIOASSAY
          3.3.1  Basic Requirements of Aquatic Bioassay       213
          3.3.2  Experimental  Procedures in Aquatic  Bioassay  266
          3.3.3  References                                   308

    3.4   MICROBIOLOGIC ASSAY
          3.4.1  Microorganisms-Diagnostic Environmental
                 Microbiology                                 320
          3.4.2  Microorganisms-Mutagenicity  Testing         344
          3.4.3  Microorganisms-General Toxicity  Testing      362
          3.4.4  Cell Cultures-Mutagenicity Testing           365
          3.4.5  Cell Cultures-Carcinogenicity Testing       376
          3.4.6  Cell Cultures-General Toxicity Testing       387
          3.4.7  Statistical Analysis                         398
          3.4.8  References                                   403

    3.5   MAMMALIAN BIOASSAY
          3.5.1  Experimental  Design Aspects                  417
          3.5.2  Conditions of Test                           420
          3.5.3  Good Animal  Care Laboratory  Practices       426
           3.5.4  Bioassay Methods                            439
           3.5.5  Gross  Observations                           459
           3.5.6  Reproduction  and Teratology  Studies         460
           3.5.7  Mammalian Mutagenicity  Tests                465
           3.5.8  References                                   471

                               viii

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Section

APPENDICES
     A   Check List for Planning Test Programs
     B   Good Animal Care Laboratory Practices
     C   Quality Control Surveillance Check List
         for Microbiology
                             ix

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                             FIGURES

Number                                                             Pagt

1.1    Precision and Accuracy                                        2
2.1    Normal Distribution                                          15
 .2    Generalized Control Chart for Averages                       77
3.1.1  Guidelines on Laboratory Management                          91
 .2.1  Field Data Sheet                                            127
   .2  Chain of Custody Form                                       128
   .3  Laboratory Bench Sheet for Aquatic Macroinvertebrates       151
 .3.1  Field Data Sheet                                            226
   .2  Chain of Custody Form                                       227
   .3  Bioassay Biota Log                                          260
   .4  Bioassay Water Quality Log                                  261
   .5  Bioassay Record Sheet                                       262
   .6  Estimating Median Lethal Concentration                      265
   .7  Hydrolysis of Acetylcholine by Sheepshead Minnow
       Brain Homogenate                                            306
 .4.1  Diagrammatic View of Virus-Concentrator Apparatus           323
   .2  Equipment Configuration for Virus Sample Collection         325
   .3  Casella Slit Sampler                                        327
   .4  Sectional Elevation of Cascade Impactor                     328
   .5  Sectional Elevation, Andersen Sampler                       328
   .6  Inertial In-Stack Cascade Impactor                          329
   .7  Porton Impinger and Pre-Impinger                            330
   .8  Sectional Elevations, Multi-Stage Liquid Impinger           331
   .9  Diagrammatic Section, Litton LVS/10K Air Sampler            332
   .10 Technique of Host-Mediated Assay                            346
   .11 Data Sheet for Alveolar Macrophage Toxicity Testing         391
   .12 Data Sheet for WI-38 Cellular Toxicity Testing              397

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                                 TABLES

Number                                                               Page


 2.1      Quality Policies and Objectives for Biological Research      7
  • 2      Recommendations for Sampling and Preservation of Water
          Samples According to Measurement                            32
  •3      Instrument Calibration                                      39
  •4      Techniques for Quality Control of Instruments               44
  '5      Effects of Housekeeping Practices on Laboratory
          Performance                                                 46
  «6      Techniques for Quality Control of Laboratory Support
          Services                                                    47
  • 7  .    Guidelines for Quality Control of Chemicals and Reagents    53
  •8      Restandardization Requirements                              72
 3.1.1    Elements of Laboratory Management and Quality Control       87
    .2    Parameters of Biological Communities Most Commonly
          Analyzed                                                    96
    .3    Comparison of Chemical Preservatives for
          Biological Parameters                                       98
    .4    Recommended Preservation and Handling Methods               99
  .2.1    A List of Biological Sampling Equipment                    120
    .2    Techniques Recommended for Preservation of Biological
          Material                                                   122
    .3    Instruments and Equipment for Laboratory and Field
          Analysis in Biological Research                            130
    .4    Major Analyses of Common Organisms in Field Sampling
          With Laboratory Analyses                                   131
    .5    Preservation of Phytoplankton                              135
    .6    Preservation of Zooplankton                                140
    .7    Sampling Equipment for Macrophytes                         145
    .8    Methods Frequently Used by Wildlife Biologists for
          Estimating Number  of Animals in the Field                 158
    .9    Major Sources of Standard, Pure or Type Culture
          Collections for Algae and Protozoa                         167
    .10   Methods for Measuring Productivity                         172
    .11   Physical Criteria for Water Quality                        175
    .12   Aquatic Species or Taxa, Freshwater and Marine, Used
          in Studies                                                 181
  .3.1    Quantities of Reagent-Grade Chemicals Required to Prepare
          Recommended Reconstituted Fresh Waters                     218
    .2    Quantities of Reagent-Grade Chemicals to be Added to
          Aerated Soft Reconstituted Fresh Water                     218
    .3    Recommended Procedure for Preparing Reconstituted
          Sea Water                                                  219
    .4    Guide to Selection of Experimental Concentrations,
          Based on Progressive Bisection of Intervals                224
                                     xi

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Number                                                               Page

3.3.5    Guide to Selection of Experimental Concentrations, Based
         on Decilog Intervals                                        225
   .6    24-, 40-, and 96-Hour LC50 Values for the Species of
         Organisms Most Sensitive to Selected Chemicals              229
   .7    The 48-Hour TL50 of Some Herbicides to Six Species of
         Fresh Water Crustaceans at Two Different Temperatures       232
   .8    Acute Toxicity of Various Metals to Fresh Water
         Zooplankton                                                 233
   .9    Acute Toxicity of Some Heavy Metals to Aquatic Insects      234
   .10   Copper Bioassays, Average Exposure vs. Average
         Accumulation                                                235
   .11   Lead Bioassays, Exposure vs. Accumulation                   235
   .12   Silver Bioassays, Exposure vs. Accumulation                 235
   .13   Zinc Bioassays, Exposure vs. Accumulation                   236
   .14   Bioassay Parameters and Correlation Coefficients            236
   .15   Effectiveness of Concentration Factors in Estimation of
         Average Levels of Exposure                                  237
   .16   Aroclor 1016                                                237
   .17   Toxicity of Some Heavy Metal Ions Toward Benthic Organisms  239
   .18   EC50 of Neburon, Diuron, Atrazine and Ametryne on
         Oxygen Evaluation by Marine Algae                           240
   .19   Growth Sensitivity of Algae to Copper                       241
   .20   Comparative Static, Acute Toxicity of NTA to Bluegills,
         Snails and Diatoms                                          241
   .21   Sensitivity of T. Pyriformis, Strain W, to Insecticides     242
   .22   Comparison of Lethal Concentrations of Pollutants on
         Aquatic Organisms                                           244
   .23   Inhibition of Growth of Estuarine Bacteria in Nutrient
         Sea Water Medium by PCB's                                   244
   .24   Effects of Organochlorine Insectic des on Bacterial Growth  245
   .25   Fish Species Recommended for Use in Aquatic Bioassay Tests  246
   .26   Recommended Species and Test Temperatures                   247
   .27   Diets and Feeding Schedule at the Fish Control Laboratory
         for Various Species                                         250
   .28   Test Photoperiod for Brook Trout, Partial Life Cycle        251
   .29   Recommended Prophylactic and Therapeutic Treatments
         for Fresh Water Fish                                        252
   .30   Time Factor in Toxicity Bioassay Tests                      275
   .31   Estimates of Time Required for Cessation of Acute Lethal
         Action in Various Bioassays                                 275
   .32   In Vitro Organophosphate Pesticide Inhibition of
         Sheephead Minnow Brain                                      306
 .4.1    Other Mutagenicity Tests                                    351
   .2    Gene Mutation Detection Systems                             352
   .3    Dose Levels for Host-Mediated Assays                        353
   .4    Some Microbial Indicator Strains Available for
         Mutagenicity Assays                                         361
   .5    Positive Controls Used in Nonactivation and Activation
         Assays                                                      362
   .6    Tetrahymena and Paramecium Assays                           364


                                  xii

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 Number                                                               Page

3.4.7     Induction of Unscheduled DNA Synthesis  by  Various
          Compounds in Vitro                                          369
   .8     Promising Bioassays for Detection of  Chemical
          Carcinogens                                                  377
   .9     Transformation  of  Cell  Cultures  by Carcinogens  in Vitro      381
   .10    Sensitivity  of  Cell Transformation Assays                    383
   .11    Cell Culture Tests for  Air  Pollution                         392
   .12    Cell Culture Tests for  Pesticides                           393
   .13    Other General Cellular  Toxicity  Tests                       398
  5.1     Characteristics of Common Routes of Toxicant
          Administration                                               423
   .2     Space Recommendations for Laboratory  Animals                 428
   .3     Ways in  Which Repeated  Treatment Prior  to  the Peak
          Susceptible  Period of Embryo May Produce Misleading
          Results                                                      462
   .4     New Concept  in  Teratogenicity Testing Based on
          Multilevel Tests                                            463
                                  xiii

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                                   SECTION  1

                                  INTRODUCTION


 1.1  PURPOSE  OF THE QUALITY ASSURANCE GUIDELINES

       The purpose of  the Quality  Assurance Guidelines  is  to provide  concepts
 and methodologies which can be used  to maintain and  improve the  quality  of
 data in  laboratory and field investigations.   It  is  intended  to  provide  in-
 formation needed for  the development of quality control plans adapted  to the
 data needs of a wide  variety of programs in biological research.

       The essential characteristics  of data of quality are validity  and
 integrity.

 1.1.1  Valid  Data

       By valid data we mean data  supported by  objective truth.   That means
 data from a well-planned experiment, obtained  using  standard  methods of  test
 and employing instruments or observational techniques  which have acceptable
 performance.   Acceptable performance implies measurement  systems (method plus
 instrument) which have specificity, have sufficient  sensitivity, precision,
 and accuracy  for their intended use, and are practical.

       Specificity requires that the  test actually measure the property of
 interest.  It also means that the test data reflect  as little as possible the
 effects  of interferences.  Thus it applies to  qualitative properties of  the
 substance being measured.  Specificity is  an inherent  property of the method,
 and it should be investigated before the method is adopted for regular use.
 We mention it here because of its implications for quality data but  will not
 discuss  it further because methods development is outside of  the scope of
 these  Guidelines.

       Sensitivity refers to the ability to detect differences in the quantity
 of a substance in a specimen or to make a  yes  or no  judgment  regarding the
occurrence of an effect.  The smaller the amount to be  detected, the  more
 sensitive the test must be.  However, it is not prudent to have a system more
 sensitive than required.  Sensitivity is a judgmental  requirement that must be
 assessed, usually at  the time the method is being developed or the instrument
 is being acquired, so it will not be discussed further in these Guidelines.

      Precision is the degree of  agreement among repeated measurements made
 using  a  constant measurement system.  The  term may also be used to mean  the
 degree of agreement among repetitions of an experiment.   Precision is  usually

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expressed in terms of a multiple  (usually  2,  corresponding  to  95% probability)
of the standard deviation of the  measurements -  the  smaller the standard devi-
ation, the better the precision.  It  is  stated,  in the  units of measurement,
as a plus and minus spread around the reported value.   The  reported value may
be an individual measurement or an average.   See Figure 1.1 (a).
  O"
  l
g
0)
3

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data, differences in observational results (as between two pathologists) and
changes in interpretation of the data.

      These desirable characteristics of data are central to the purpose of
the Quality Assurance Guidelines.  It is for the purpose of achieving quality
data that we shall cover standardization of methods, calibration of instru-
ments, statistical quality control, sampling, design of experiments, data
handling, training, supervision and all the other elements of a quality assur-
ance program.

      Underlying the requirement for Quality Assurance is the necessity that
data be scientifically verifiable and that they stand up in court if the re-
sults of research are questioned.

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1.2 DEFINITIONS

1.2.1 Quality Assurance

     Quality assurance is defined as all those activities which contribute
to producing correct and reliable data.  Personnel assignment, facilities
design, methods development and equipment selection are all important.  How-
ever, in these Guidelines, emphasis will be on methodology in standardization,
control, and audit of performance of work.  This is consistent with the
basic concept that quality control means making the best use of resources
available.  Efforts are measured and if results are not within acceptable
limits personnel must be retrained, facilities and equipment must be
upgraded, or methods improved.

     The quality assurance program is developed to minimize the variations
that are inherent in all research and testing.  Standard operating  procedures
and statistical techniques are used to identify and control assignable
causes of variation.  Random errors are measured and used to express the
degree of confidence to be placed in results.  The total quality assurance
program is rounded out by regular assessment by program managers of the
degree of success in standardization and control.

     Standardization may appear to be too harsh a concept to be applied to
research.  However, it is needed to assure that the  work will meet the first
requirement of good science, namely that it can be repeated and the results
verified by other scientists.

1.2.2 Biological Research

     Biological research is defined as all types of experimentation in which
the test  subject is a form of life.  In  general, however, biological research
concerned with the environment is performed in non-clinical laboratories.
Clinical  laboratories are understood to be medical laboratories engaged in
the direct examination of the condition of human patients.  Non-clinical
laboratories are confined almost entirely to the use of non-human subjects.
There are exceptions, such as experimentation with human cell cultures,
certain host-mediated assays, and in the health effects area.  There has been
a  great deal of progress in quality control in clinical chemistry, and what
is applicable to biological research, as we define it, has been adapted.

     Biological research is supported by analytical chemistry.  Analytical
chemistry is another area in which there has been much progress in quality
control.  The Quality Assurance Elements in the following Section 2 are
based very largely on experience gained  in the analytical quality control
field.  We begin to build on the analytical base, and in later sections of
the Guidelines* devoted to particular  areas of biological research, we
attempt to bring the user of  the Guidelines up with the state of the  art in
quality assurance of biological research.

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1.2.2.1 Laboratory Research—

     It is convenient, because of the different degree of attention
required to sampling, testing, and control of the experiment between work
done in the laboratory and in the field, to make a distinction between
laboratory and field research.

     Laboratory research is that research done in a fixed laboratory
location equipped with all supporting services and usually environmentally
controlled.

1.2.2.2 Field Research—

     Field research is research done under field conditions, usually with test
subjects in a feral state.  Testing equipment may be deployed in the field
or may be located in mobile laboratories more or less equipped and
environmentally controlled.

     Controlled research is defined as a field research, in which constraints
are placed on test subjects, test environment, and/or application  of
treatments.  Examples are chemical treatment of algae in some areas of a pond,
or treatment of fish in a confined area of a stream with measured doses of a
chemical.

     Effluent observations are defined as research in which existing
contaminant levels and condition of test subjects are measured as they are
found in the field.

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                               SECTION 2

                        QUALITY ASSURANCE ELEMENTS

2.1  QUALITY ASSURANCE POLICY AND OBJECTIVES

     Every laboratory and field organization should have a clearly enun-
ciated policy regarding quality of data.  This should include a statement
by management of its concern for quality.  The purpose of a statement of
quality policy by management is to ascertain that quality control is a
pervasive concern; one that merits attention not only at critical points,
but daily in the routine performance of research.  The statement by top
management to the laboratory must be followed up with continuing visible
evidence of its sincerity to all levels of the organization.  Periodic
meetings should be held in .the laboratory to discuss quality objectives
and:progress toward their achievement.

     Points for a quality policy and corresponding quality assurance objec-
tives are given in Table 2.1.  The objectives are spelled out in more
detail in the following paragraphs.  Appropriate methodologies for the
attainment of the objectives are given in the referenced Sections.

2.1.1  Laboratory Evaluation

     Laboratory evaluation is widely practiced as a basis for certification
or accreditation of laboratories.  For example, in compliance with the Safe
Drinking Water Act, EPA has a State Laboratory Certification Program
(Geldreich, 1975).  Evaluation, whether carried out by outsiders or by
the laboratory itself, can be a useful management tool for improvement.
Such an evaluation technique is available for environmental monitoring
laboratories (U.S. EPA, 1978).  This procedure covers personnel, laboratory
space and facilities, analytical methodology, analytical instruments and
apparatus, and quality control, including interlaboratory testing.

     Adaptions of the latter procedure for use in biological research
laboratories would include a number of features unique to that kind of
laboratory.  In the facilities area, there are requirements for separation
of clean and dirty corridors and separate rooms for isolation of test
subjects by species and of test materials, at least by class.  Acceptable
animal care standards are implied and also appropriate experimental
apparatus and techniques.  The design of bioassay experiments would be
covered.  Proficiency testing, i.e., the submission of blind samples to
the laboratory and taking scores on the tests into consideration in the
evaluation,is accepted as an integral part of a sufficient evaluation
procedure.  In biological research such testing is relatively infrequent
and needs further attention.  Proficiency testing can be continued between

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periodic evaluations as a means of interlaboratory comparison.

     TABLE 2.1  QUALITY POLICIES AND OBJECTIVES FOR BIOLOGICAL RESEARCH
            Policy
       Q.A. Objectives
Section
   To provide adequate
   personnel, facilities
   and equipment
   To develop and use
   rugged methods of
   experimentation,
   sampling and testing
   To provide adequate
   support for a Quality
   Assurance Program
Use laboratory evaluation as a
  management tool                  2.1.1
Organize for quality               2.1.2
Train for quality                  2.1.3

Use statistical consultation in
  design of experiments            2.2
Apply formal sampling plans        2.3
Measure and control precision
  and accuracy of tests            2.4

Maintain good housekeeping and
  laboratory services              2.5
Control test materials, chemicals
  and reagents                     2.6
Control test subjects              2.7
4  To demonstrate good      Use good supervisory practices
   control of research        to assure that protocols are
   and monitoring             followed                         2.8
                            Use care in preparation of
                              materials for measurement
                            Control measurements and take
                              action to correct deficiencies
                            Preserve integrity of data and
                              provide adequate computer support

5  To improve laboratory    Participate in interlaboratory
   capabilities continuously  testing program                  2.9
   To produce reliable
   data and reports
Use statistical expertise in
  analysis of results
Establish regular audits of
  performance
Adopt a system for review and
  publication of reports
 2.10

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2.1.2  OrRanization for Quality

      Quality Is a concern of the whole  laboratory and  responsibility for the
control of quality la shared by all  levels of organization  In accordance
with their capabilities to contribute to It effectively.  For example,
management must set the tone by clear enunciation of quality policy and goals
and support of their attainment by giving adequate attention to  facilities,
equipment, personnel  competence,  standards, operating  p tract Ices, quality
control programs, and performance reviews.  Study directors must plan,
assemble appropriate equipment, select methods,  Instruct  researchers, monitor
and adjust performance and check results.

      Scientists and technicians must follow study protocols, use approved
methods, apply appropriate quality control procedures,  maintain  chain of
custody of test materials and test subjects, preserve the integrity of data,
and use good scientific judgment.  Supporting staff, such as analytical
laboratories, consulting statisticians,  or quality control  specialists, muBt
assist the whole organization to the extent that such expertise  is not avail-
able within the study group Itself.

      Many of the activities identified  as elements of  quality control are
widely recognized as good laboratory practices.  Some of  these practices are
honored by time, and experienced researchers may be expected to  follow them
conscientiously.  Some of them, of which statistical design and  analysis of
experiments may be an example, have  been a part  of the  academic  training of
some scientific disciplines only in  recent years.

      The direct control which the study director may have  over  dally routines
may be diluted by the size of the programs or by commendable delegation of
responsibility to junior scientists  or technicians.  He also depends on
analysts and other experts for support.   Quality control  procedures are as
much directed toward coordination of a multiplicity of  activities as they are
toward providing safeguards against  human fallibility.

      We look upon quality control as a  self-discipline, by the  individual
and the groups of Individuals who conduct a study or contribute  to a labora-
tory program.  Quality control emphasizes the validity  and  Integrity of data
not for the purpose of constraining  research but to enhance verlfiablllty by
the scientific community and credibility should  the data be contested in the
courts.   It supplies the whole study or  the whole laboratory with a disci-
pline which is complementary to the  scientific discipline of a good study
director or researcher.

      Responsibility for quality Is  shared by the entire organization.  How-
ever, to make any plan go, It Is necessary to have a leader.  In large organi-
zations, leadership may be assigned  to a Quality Control Department with well-
defined authority.  In smaller laboratories, quality may be a clearly defined
part  of the job of all Section Chiefs, or of a Quality  Control Committee.
Better  than a committee may be a part-time Quality Control Coordinator who
must  have  sufficient authority to see to It that the laboratory's quality ob-
 jectives  are being met.
                                       8

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2.1.3  Training for Quality

      The people who have an impact on quality (bench reaearchers, super-
visors, etc.) should all be trained in the reasons for the benefits of
standards of quality and the methods by which high quality can be achieved.
This may be on-the-job training for most laboratory personnel but those with
assigned responsibility for leadership In the quality control program should
receive formal training In modern methods of statistical quality control.

      Training courses are offered regularly by the Education and Training
Institute of the American Society for Quality Control, 161 W. Wisconsin
Avenue, Milwaukee, Wisconsin 53203, and by local sections of the Society.
Also full-term courses are offered by many universities.

2.1.A  Other Objectivies of a Quality Assurance Plan

      In biological research, approval of all study protocols prior to their
initiation should be required.  The approval procedure nuty consist of a peer
review and a review by the various supervisory levels.  It should also re-
quire comment by a statistician on the design of the experiment and statisti-
cal analysis to be used.  The statistician must be consulted early in the
planning stages to assure that the design meets requirements of statistical
adequacy and that methods of statistical analysis are specified.

      Because of the high level of variability of biological materials,
special attention should be given to sampling.  A sampling procedure may have
to be designed, much In the way that an experiment has to be designed.
Formal sampling requires attention of a statistician who can assist In deter-
mination of location and frequency of sampling as well as the size and
number of Increments needed in the sample.  A chain of custody should be
established to control the flow of samples from field, through the laboratory,
to storage and eventual disposition.

      The need for close supervision, particularly of long-term chronic ex-
periments and wide-flung monitoring activities, has recently emerged as an
important problem.  The quality assurance plan should be an arm of the super-
visor in accomplishing the aim of producing quality data.  Brief, timely
quality reports are one means of keeping the supervisor advised.  There should
be a good bookkeeping system particularly for collecting observations made
during the conduct of the experiment.  These observations should be made on
a suitable schedule which is frequently oftener than daily.  The observation*
should be assembled frequently, analyzed (by computer if necessary) and
reported promptly to highlight problem areas.  In addition, the supervisor
should be close enough to the operation of the laboratory to be sure that
procedures are being followed as Intended and that the observations are being
correctly assembled.

      The laboratory should have a written plan including the quality assur-
ance procedures detailed In the remaining parts of Section 2.  As part of the
plan, all the documentation should be assembled in a Quality Control Program
Manual.  This manual should be available for use In the laboratory and as a
basis for evaluation of the laboratory's performance in accordance with the
plan.
                                       9

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      Section 2 covers the general aspects of quality assurance methodology -
those parts applying to any type of biological research.  Following Section
2 are specialized Sections which describe methodologies required to meet the
requirements of particular areas of research.
                                     10

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2.2 DESIGN AND ANALYSIS OF EXPERIMENTS

2.2.1 Description of Design of Experiments

     The design of experiments is that part of research planning that has to
do with precise scoping of the work to be done.  This involves the layout
of the number of levels of treatments, number of test subjects per treatment
group, use of controls, replications (repetitions of treatments), duration
of treatment, identification of test materials and test subjects, route of
administration, the response to be measured, and description of special
circumstances surrounding the experiment.

     The design work can be done most effectively if experience is available
from earlier experiments and if done with full attention given to the impli-
cations of the design for later statistical analysis.  Therefore, the design
of the experiment should involve joint efforts on the part of the experiment-
er and a statistical consultant.

     The statistician should be involved, along with the experimenter, in
selection of number and levels of treatment, number of test subjects per
treatment, and the use of controls and replication of treatments.  These
activities influence the selection of the proper mathematical model of the
experiment, the measurement of experimental error, and the significance (in
terms of probability) that can be attached to results .  The other activities
including duration of treatment, identification of the response to be
measured and the test method to be used, selection of test materials and test
subjects, and route of administration are the prerogative of the experimenter.

     The subject of design of experiments owes much to the work of two men,
Ronald A. Fisher and Frank Yates, through work at Rothamsted Experimental
Station since its founding in 1920.  Thus, much of the subject has grown
through its use in an experimental science.  This happens to have been in
biological science, largely agricultural at first, but rapidly expanded
into genetics and all kinds of bioassay.

     Fisher's Design of Experiments (1947) and Yates1 Design and Analysis
of Factorial Experiments (1937) are classics in  the statistical literature.
They have been followed by Cochran and Cox's Experimental Designs (1950),
Finney's Statistical Method in Bioassay (1964). Kempthorne's Design and
Analysis of Experiments (1952) and others.  Most of the complex experimental
designs now available for survey work or in scientific/technical experi-
mentation have grown from the pioneering work done in the biological area.

     Great care should be exercised in designing biological experiments
because the wide scope of biological experimentation, the special methods
of test and observation, the responses measured, the variability of test
subjects, the complexity of biological theory, and the difficulty in stan-
dardizing designs all lead  to problems that require special attention.

     The instances in which the needs of the experimenter are satisfied
entirely by an experimental plan, the analysis of variance (a widely used


                                     11

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statistical method) and standard errors, are comparatively few.  What is
needed is a broad understanding of experiment design and relation of this
subject to the general theory of statistics and to the problem of experi-
mental inference.

     The distinguishing feature of science is its method.  The central thrust
of scientific method is examination of what is known, and the formulation there-
from of hypotheses which can be put to experimental test.  The word
"experimental" is the most important one, and therefore the design of
experiment appears as the crux of scientific method.

     The determination of the relevant aspects of the problem for which a
solution is required and the actual formulation of the hypotheses to be
tested require attention of the keenest sort.  The experimenter must be
very knowledgeable in his biological field.   After formulation of the hypoth-
eses   comes consideration of consequences that are verifiable and, finally,
objective verification.  Here is where the intuition or genius of the
experimenter can be enhanced by help of the statistician.

     Verification of a hypothesis cannot be absolute.   It can only be shown
that observations made are compatible with the theory within the limits of
error to which the observations are subjected.  In other words, it is pos-
sible only to prove that a hypothesis is false, thus, the use of the null
hypothesis in statistics.

     The scientific method is circular, proceeding from observation through
abstracting of essential information as a basis for a logical theory, develop-
ment of the theory, and prediction of new events, back to observation and
through the cycle again.  Statistics enter  at the taking of observations
and at the comparison of the observations with predictions from theory.

     It is essential that the hypotheses and their possible outcomes be
formulated before verification is attempted.  Hypotheses formulated from
or modified by the observations are suspect.  It is one of the basic notions
of statistics that probability statements cannot be made about statistical
tests suggested by the data to which they are applied.  Therefore, selection
of the statistical methods to be applied must be made before the experimental
work is carried out.

     The design of an experiment is the pattern of the observations to be
collected.  There are two types of experiments: absolute and comparative.
In an absolute experiment, repeated observations, which do not agree exactly
with each other, are made on a test subject to obtain the best estimate of
some property of the subject and a measure of the reliability of the
estimate.  A sample survey is an example of an absolute experiment for
determining particular characteristics of a population.

     In a comparative experiment, the outcomes of two or more treatments are
compared in their effects on characteristics of a population.  This requires
taking of controlled observations, where control is effected on all treatments
to the same degree by either fixing all the variables in the experiment or
controlling them statistically by randomization.

                                     12

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     The economic aspect of experimentation must be emphasized.  The experi-
menter is usually in the position of being able to spend only a certain
amount of time, effort and money on his investigations.  There are more
efficient and less efficient ways in which he can go about the work, leading
to greater or lesser degrees of certainty in his results.  He must consider
the cost of obtaining a given level of certainty, whether it is worth the
cost, and at what stage the cost of increased certainty is too great.

2.2.2  Steps in the Design of Experiments

     A statistically designed experiment consists of the following steps
(Kempthorne, 1952):

     • Statement of the problem
     • Formulation of hypotheses
     • Devising of experimental technique and design
     • Examination of possible outcomes and reference back to the reasons
       for the inquiry to be sure the experiment provides the required in-
       formation and does so to an adequate extent
     • Consideration of the possible results from the point of view of
       the statistical procedures which will be applied to them, to
       ensure that the conditions necessary for these procedures to be
       valid are satisfied
     • Performance of experiment
     • Application of statistical techniques to the experimental results
     • Drawing conclusions with measures of the certainty of estimates of
       any quantities that are evaluated, careful consideration being
       given to the validity of the conclusion for the population of
       subjects to which they are to apply
     • Evaluation of the whole investigation, particularly by comparing
       it with other investigations of the same or similar problems

     A check list of the detailed activities required in carrying out these
steps is given in Appendix A.

2.2.3  Essential Statistical Concepts

     It is not intended here, or in Section3.4.7 on Statistical Analysis,
to give a complete description of statistical theory.  Some familiarity
with statistics on the part of the experimenter is assumed.  The requirements
for statistical theory which go beyond what can be expected of the average
experimenter are the reason for recommending that statistical advice be
sought at the very beginning of planning an experiment.

     It is worthwhile to consider, briefly, the elementary statistical con-
cepts that are essential to the design and analysis of experiments.

     The first concept is that of a population.  A population is an
assemblage of the objects of possible observation or measurement, or some
attribute of those objects.  The individual objects in the population may be
arranged according to the size of a measurable characteristic, and the function
giving the relative frequency of the individual measurement is called the

                                     13

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distribution of the individual objects.  From this distribution, we may obtain
the proportion of measurements less than a chosen value, or the proportion
lying in any chosen interval of values.  A distribution may be continuous or
discrete:  for example, the ppm bioconcentrations of a toxic substance in test
objects under treatment will be continuous or variable measurements, whereas
counts of the number of neoplastic lesions under a treatment will be integers,
with a discrete distribution.

     Other kinds of discrete measurements include ranks (observations ordered
according to magnitude), or attributes  (yes or no responses to a treatment).

2.2.3.1  Normal Distribution Statistics—

     Distribution curves should be familiar to users of the Guidelines, for
example, the symmetrical bell shaped curve for the normal distribution
illustrated in Figure 2.1.

     The most useful measure of central tendency of a distribution is its
average  (mean), v.  The measure of spread of a distribution most generally
used is  the variance, a2, which is the mean square distance of the population
individuals from the average.

     The median, M, is a measure of location useful in biological research.
It is the measurement in an ordered array that has an equal number of measure-
ments on either side of it (it divides the distribution in half).  The median
would be preferred over the average, for example, if an animal behaviorist is
studying the time from the beginning of an experiment until each individual
responds.  He can obtain the median time of performance without waiting for
all the animals to respond and then calculating an average.  Thus, if the
experimenter knows what the total sample size is, he can get an estimate of
the central tendency without waiting for slowest responders.  Moreover, some
may never respond, so calculation of a true average may be impossible.

     A distribution is characterized by a mathematical form containing quanti-
ties called parameters which, when known, describe the distribution completely.
The estimation of the parameters from sample data is one of the most important
functions of statistical theory.

     The most used distribution is the normal distribution which has the
advantage that the average, and standard deviation (square root of the
variance, or root mean square deviation), a, describe it completely.  The
quantity that best estimates the average of a population from a random sample
size n, is the average of the sample, x, and this estimate has a variance
equal to the variance of the individual measurements divided by n.

     The estimate of the variance, 82, is [l/(«-l)]I(a:-zr)2. where n is the sample
size, x is an individual measurement, and x~ is the sample average.

     A test of significance of a sample average (of its difference from the
population average, or "true" value) is based on Student's distribution where:
       t  =  (ar- v) / (s/^n)                                      Eq. 2.2.1
                                      14

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               u
               OJ
               O"
               
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      Of perhaps even wider  application  than  tests of  significance is the
usage of confidence  limits.  The  limits  on  either side of  the sample average
are, from the above  formula,

      *L = tsh'n                                                    Eq. 2.2.3

where the value of t comes from the  table depending  on the level of signifi-
cance selected by the experimenter and the  degrees of  freedom in the sample.

      The least significant  difference between  two averages  is  given by
      LSD = te  Snl + n^  I  Sn^ «2                                   Eq. 2.2.4

      Another arrangement  of  this  formula, when  one  is concerned with the
size of sample  necessary to achieve a  desired  level  of significance in an
average, is to  solve for n:

      n - (*8/D)2                                                   Eq. 2.2.5

where e is an estimate of  the standard deviation from early data and D is the
allowable difference between  the average of  the  sample and its "true" (popu-
lation) value.

      The economic aspect  of  experimentation has been mentioned earlier.  In
a statistical sense, the value of  a better experiment is determined by the
ability to predict a result of one or  several  treatments with greater pre-
cision.  Another measure is that of the quantity of  information, for which
Fisher (1925) suggested  nl =  n/s2, from which  it derives that the information
per observation is I/a .   The economic experimenter, therefore, increases n
within limits of resources and reduces 8 by  use  of sound experimental design,
precise instrumentation  or careful observation,  and meticulous supervision
of the conduct  of the work.

2.2.3.2  Statistics of Other  Distributions—

      The test  for the significance of differences between two sample
variances or the differences  of means  of several samples is the F test:

      F - 8j2/  822                                                  **• 2-2'6

      The degrees of freedom  are «i -  1 and  n2 - 1.  This test is used in the
analysis of variance of  designed experiments.

      Standard values of F may be  looked up  in standard statistical tables.
Several variances may be compared  by Bartlett's  test.

      The x2 (Chi-square)  test is  applied to problems in which we wish to de-
termine whether the frequency with which an  event has occurred is significant-
ly different from that which  was expected.

      Chi-square » £ (0  -  E)2 / E                                   Eq. 2.2.7
                                      16

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where 0 is the observed frequency in a group and E is the expected frequency.
The degrees of freedom are the number of groups minus 1.  The standard values
of Chi-square are found in the standard statistical tables.

     Discrete experimental data frequently conform to the binomial, Poisson,
or negative binomial distribution.  The binomial is the distribution of the
number of observations of either a yes or no character (say morbid or healthy
animals following a treatment) in n trials.   The chance of a favorable obser-
vation (success) is p.  Then the estimate of the average of the distribution
is p and the variance is pq (where q is equal to 1 - p).  When n is very
large the binomial approaches the normal distribution.  The t test for signi-
ficance of an average portion is:
     t  =  (p - fc) /
where k is some desired proportion.  The formula for sample size is:

     n  -  t2pq / D2                                                Eq. 2.2.9

     The Poisson distribution provides probabilities of the number of
observations per unit of time, area, volume, etc., for example, the number
of "bacterial colonies per unit area or volume of a culture.  The average
count and the variance are the same, a.

     The t test for significance of a count per unit is:

     t  -  (o - m) / Jojn                                          Eq. 2.2.10

where m is some desired count.  The formula for sample size is:

     n  =  t2c/V2                                                  Eq. 2.2.11

     The negative binomial distribution is applicable because of clustering
(or contagion) of "successes" of an otherwise binomial distribution, for
example, deaths of insects.  An example of its application to biological
research is given in Bliss and Fisher (1953).

     It is not correct to treat data from these discrete distributions as
though they were normal.  Many of the commonly used analytical methods such
as the analysis of variance, are based on a number of assumptions.

     Among the assumptions underlying the use of the analysis of variance
are:

          • The sampling of individual items must be at random
          • The experimental error must be a normal random variable
            (the individual measurements must be independent)
          • The variances in groups of samples must be equal
          • Effects of treatments must be additive (if interactions
            are present they must be taken into account) .
                                      17

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      If these assumptions cannot be maintained, it may be possible to use a
distribution-free test.  Tests based on ranking of the measurements include
the Kruskal - Wallis test, the Mann - Whitney U-test, and the Wilcoxon two-
sample test.  See Sokal and Rohlf (1969).  Other tests include nonparametric
multiple comparisons by STP, Friedman's method for randomized blocks, and
Wilcoxon's signed-rank test for two groups (Ibid.)-

2.2.3.3  Data Transformation—

      The measurements to be analyzed may frequently be transformed to meet
the assumption of the analysis.  The entire analysis can then be carried out
on the transformed measurements.  A fortunate fact about transformation is
that very often several departures from basic assumptions are cured
simultaneously by the same transformation to a new scale.  When a transforma-
tion is applied, tests of significance are performed on the transformed data
but estimates of the averages are usually reported in the original scale.

      The most common transformation is conversion of the measurements into
common logarithms.  This transformation is useful in studying the growth
of organisms.

      When the data are counts, such as of blood cells in a hemocytometer,
the square root transformation is frequently useful.  Such data follow the
Poisson distribution where the variance equals the mean.  Transformation
makes the variances independent of the means.

      The arcsine transformation is especially appropriate to percentages
and proportions where, for example, the measurement may be the percent
fertile in a vial of eggs of Drosophila.

2.2.4  Experimental Models—

      The simplest possible experiment is application of a single treatment
to a group of two or more objects, for which the framework is

                        Treatment with a Toxic Material

                                    Animal 1
                                    Animal 2
                                    Animal n

      A simple linear expression provides an analytical model for this
experiment:  y = y + T + e.

      The meaning of this model is that a single measurement, y, can be
decomposed into the average, a fixed deviation of the measurement from the
average (T) and a random deviation of the measurement from its expectation
(e) which is y -f T.
                                      18

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     Analyses possible with this model include:

     Before the experiment
     •  Calculation of the number of animals required to estimate the
        average within desired limits (only if a prior estimate of the
        variance is available).  See Section 2.3, Equations 2.3.1 and 2.3.5.

     After the experiment
     •  Significance of the average.  Eq. 2.2.1
     •  Confidence limits for the average.  Eq. 2.2.3
     •  Sample size for further experimentation.  Eq. 2.2.5; 2.2.9; 2.2.11

     There are two models of the analysis of variance, as first defined by
Eisenhart (1947).  In Model I, it is assumed that the differences among
group averages are due to fixed treatment levels.  The purpose of the
experiment is to estimate the true differences among the group averages.

     The basic form of Model I is given by:  y^j  =  v + T^ + 6j({,)
where i, takes values from 1 to m, the number of treatments, and j takes
values from 1 to n, the number of individual objects per treatment group.
The parentheses about i read "j's random within the -i's."

     Examples of Model I in biological research include treatment of groups
of animals with different concentrations of a toxic substance.  The model
also fits exposure of plants to different levels of stimulant or culture
of bottles of insects at different temperatures.  Another example is
comparison of the body weights of several age groups of animals.

     The design framework is:

                                  Treatment

               Level 1              Level 2 	Level m

               Animal 1.1           Animal 2.1       Animal m.l
               Animal 1.2           Animal 2.2       Animal m.2
                   •                   •               •
                   •                   •               •
               Animal l.n           Animal 2.n       Animal m.n

     Model II assumes that in place of fixed treatments there are randomly
selected treatments different for each group.  The basic form for Model II is:
Via  =  M + T^i  + e(ij), where,  again,  the parentheses indicate randomness.
An example is the determination of DNA content of rat liver cells from three
preparations from the liver of each of five rats:  m = 5 and n - 3.  The rats
were selected at random and the preparations were made from aliquot portions
of the livers.

                              Design Framework

          Rat Liver 1        .1          .3          4          5
Prepara-     1.1            2.1        3.1        4?1        5?1
tion         1.2            2.2        3.2        4.2        5.2
             1-3            2.3        3.3        4.3        5.3

                                      19

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     Both of the models presented above are single factors with replication.
In experiments involving two or more factors, the models may be mixed, having
both fixed and random factors.  Obviously, no measure of experimental error is
provided without replication.  In two-factor or larger experiments, it is
possible to use higher order interactions in place of error to test the sig-
nificance.  Also, in mixed models, the replication error may not be the pro-
per denominator in the F-test.  However, it is recommended that plans for
replication be included in all biological experiment designs.

     The variance tables for the two single-factor models look the same al-
though there is a formal difference in the estimation of expected mean squares
for treatments and the hypotheses tested are stated differently.  The variance
table for a single factor with replication is:

                               Variance Table
           Sum of       Degrees of
Source     Squares      Freedom        Mean Squares   Expected MS   F Test

  T        SSi          m - 1          SSi/(m - 1)    a2e + no2T    MST / MSe

  e
Totals     SST          mn -  1

     -  Zm <£« x)2 / n -  (EwSwx)2 / rm

     »  883 - SSj

SST  =  Z7" l?hs.2 -  (£m£wx)2 / im

     The computational methods required may be found in almost any statistical
textbook or a computer program may  be used.

     In Model I the hypothesis tested is:  H0  : T^  =  0

     In Model II:  H0 : 02T   -  0

     One-factor models with replication are sometimes described as between-and
within-group models, and the mean squares are designated as between and within
variances, 8-^ and «w2, respectively.

     Experiments may involve  two or more factors and may involve mixed, fixed,
or random factors.  Also  these factorial designs may be supplemented by many
available random blocks,  splitplots, square designs, nested designs, response
surface designs, and others.  These experiments are adaptable to both qualita-
tive and quantitative factors and the analytical methods used when the experi-
mental results are in must depend on this and on the nature of the responses
measured or observed.  Here again,  the need for a good statistician to assist
with the data analysis is obvious.
                                       20

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      Most biological research  may involve  single- factor experiments where
 all factors  but  the treatments of interest are  controlled  by balancing or
 randomization.   However,  in  the interest of obtaining most information from
 experiments, it  may be  possible in many experiments,  with  slight  additional
 attention to the structure of  the experiments,  to  use more complex models
 effectively.
      The  general models  for  two  factors are:
     Fixed Factor:   y. .k  -  y + A.
                    ''IT,           1
interaction of the  two  factors.
           AB..+e
             13
     Random Factor:  y . .,
+ A. + B. .  + e . .
   ^    ^«7
                                                      .. where AB  is  the
                     2 Factors, Fixed, with Replication

                                  Factor A

                      Level 1              Level 2

             Level 1  Animal 1.1.1        Animal 2.1.1
                             Level m
                           Animal m.1.1
                      Animal 1.1. n

Factor B     Level 2  Animal 1.2.1
          Animal  2.2.n

          Animal  2.2.1
                                                           Animal m.l.n

                                                           Animal m.2.1
                      Animal 1.2.n

             Level r  Animal l.r.l
          Animal 2.2.n

          Animal 2.r.l
                                                           Animal m.2.n

                                                           Animal m.r.l
                      Animal l.r.n
          Animal 2.r.n
                                                           Animal
                                     21

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Source S.S.*
A SS.L
B SS2
AB SS_
e SS.
4
Totals SS

Level 1
Factor B Level 1
Animal
Animal
Source S . S .
A SS-j^
B SS2
e ss?
Totals SS •
Bennett and
the EMS, leading
complexity :
Variance Table
D.F. M.S. E.M.S.
m - 1 SS/DF a2 + no2
e AB
T - 1 SS/DF a2 + no2
e AB
(m-D(r-l) SS/DF a2 + no2
e AB
ra» (n-1) SS/DF a2
e
mm — • 1
2 Factors, Random
Factor A

Level 2 ... Level r Level 1
1.1.1 1.2.1 I.P.I TO. 1.1
• • • •
• • • •
• • • •
• • • •
1.1. n 1.2.n l.r.n m.l.n
Variance Table
D.F. M.S. E.M.S.
OT - 1 SS/DF a2 + no2 + n
& a
m(r - V SS/DF o2 + no2
e B
mr(n - V SS/DF a2
'""• 	 ™" «3
mm - 1
Franklin (195A) give the following s
to the proper test of significance,

F
+ nro2 MSA/MSAB
A
+ nmo2 MSB/MSAB
Ji
MSAB/MSE



Level 2 ... Lever r
m.2.1 m.r.l
• •
• •
• •
• •
m.2.n m.r.n
F
ra2. MSA/MSB
A
MSB/MSE
teps for arriving at
for experiments of any
* Refer to computational framework in any standard statistical text  for
value SS. through SST.
                                       22

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     Factors are designated by capital letters (A, B, etc.).  Levels are
designated by small letters (jn»f»n9 etc.).  Effects are designated by lower
case letters relating to the respective Factors (a, b, etc.).  A replication
is indicated by parentheses, interactions as products (ab, etc.).

     A table is prepared with Factors as column headings and Effects as row
designations.  Under each column heading is space for indicating the number
of levels and the model, fixed or random.

     The following rules are followed in filling out the table.

        • In each column write opposite any row not containing the same
          letter as the heading, the number of levels.
        • In each row containing an effect in parentheses, write 1 where
          letters are common to row and column.
        • In remaining spaces, write 1, if the type is random; write 0, if
          the type is fixed.
        • The EMS is obtained by multiplying in rows, all figures except
          those in columns having letters in common with the row, as
          illustrated.

               Two-Factor Mixed Model, with Replication

Factor         A        B            E

Level          m        T            n

Type           R        F.            R       EMS

  a            1        r            n       a2  + w a2 A
                                               e        A
                        bA                     2  -i *   2   _i_ —*^ - 2.
               Tfl        U            7Z       (J"T"7t0-j^ mWQ _


  ab           1        0            n       a2  + n a2
                                               e       AB

  e(ab)        11            la2
                                               e

     Effects a and ab are tested by the error term.  Effect b  is tested by
the interaction term.  The analysis of data from biological experiments is
often complex because of non-linear variables, non-linear responses, high
levels of variability, small sample sizes and other  things  that make care-
ful application of statistics a necessity.  The tests mentioned in  this
section are among the basic, most widely used ones.  Each biological testing
program has special requirements.  For example, in bioassay of rats and mice
for carcinogenicity of chemical substances, the following statistical methods
are applied.

     Survival probabilities are estimated by the product limit procedure of
Kaplan and Meier (1958) and presented in the report  in  the  form of  graphs.
Deaths due to accident or scheduled sacrifice are treated as  censored


                                      23

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observations and all other deaths are uncensored.  Statistical tests of
differences in survival between groups are made using the method of Cox
(1972) for 2 groups and an extension of this method by Tarone (1975) for
more than 2 groups.

     The number of animals with tumors is analyzed as percentage of the
number of animals pathologically examined.  For some sites, such as liver
or lung, the animal IB entered in the denominator of the proportion of
tumors at the site only if that site had a hlstologlc examination.  For
tumors that may appear at several sites, any animal that had at least one
such site hlstologlcally examined is entered in the denominator of the
proportions given for that tumor.

     Statistical analysis of tumor incidence is made using the Fisher exact
test (Cox, 1970) to compare the controls to each dose level.  In addition,
the Armitage and Cochran test for linear trend in proportions with continuity
correction (Armitage, 1971) is used.  This test, assuming a linear trend,
determines if the slope of the dose-response curve is different from zero
(P < 0.05).  The method also calculates the probability level of a departure
from linear trend.

     A conservative adjustment for simultaneous comparisons of several
treatments with a control is the Bonferronl inequality (Miller, 1966).  For
the comparison of k doses with a control, this correction requires a
significance level less than or equal to 0.05/k for the overall comparison
to be significant at the 0.05 level.  This adjustment is not made in the
tables where the  fisher exact test results are shown but is discussed in the
analysis when appropriate.

     Other statistical methods are discussed in connection with specific
biological experiments in later Sections.

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2.3 SAMPLING

2.3.1 Background of Sampling

     In U.S. EPA (1973a), Weber presents the concepts of sampling In bio-
logical research very concisely:

     •  An experimental unit is an object on which a measurement or
        observation may be made
     •  The set of all experimental units of Interest In a study is the
        universe, or population
     •  A sample is a sub-set of experimental units* or of the measurements
        made on those units, usually only a small fraction of the population
     •  The sample must consist of a sufficient number of units (sample size)
        to represent the population, with the required precision and accuracy
     •  Sampling units or sampling points must be selected with known
        probability
     •  Random selection is necessary to satisfy the requirement for known
        probability
     •  A random sample, selected using a device such as a table of random
        numbers consciously has no bias

     Experimental units may be discrete objects, such as test animals or, if
Interest is in spatial distribution or density of a population, or rate of
change, may be units of space (volume, area, etc.)-  If the population is a
bulk material, such as water, air, or feed, the sampling unit cannot be known
until a sampling device is applied.  Furthermore, it Is necessary to take
into account the dynamic nature of living populations.  There are evident
benefits to be gained from taking sampling considerations into account early
in the planning stages of a study.  The experimenter may often benefit from
the advice of a statistician at this point.

     For random sampling, it is necessary that each unit In the population
have an equal probability of being selected.  This means that the population
must be identifiable.

2.3.2. Randomization Procedure

     A simple randomization procedure is as follows (alternatively, random
numbers might be generated by a computer program):

     •   Identify and number all the measurement units in the population.
        The total number of such units is M
     •   Determine the sample size, n
     •   From a random number table select numbers equal to the number of
        measurement units required for the sample.  (See any mathematical
        or statistical textbook for the table.)
     •   Start at any random point in the table and read numbers
        consecutively in any direction
     •   Once a number has been selected Ignore the recurrence in the table
        and read on until « numbers have been picked
     *   The correspondingly numbered units in the population constitute the

                                     25

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2.3.3 Sampling Models

     Two models will probably satisfy most sampling requirements in biological
research: simple (or unrestricted) random sampling (Model I), and stratified
random sampling (Model II)  (Bicking, 1976).  Simple random sampling is used
when the population is not  subdivided.  Stratified random sampling is used
when the population is divided into strata or when a material is in divided
or packaged form.  Knowledge of the nature, content, and variability within
strata is necessary in selecting the sampling scheme to be used.  A pilot
study may have to be made to obtain information about stratification.  As a
general rule, strata should be bounded in such a way that measurements are
most alike within strata and most different between strata.  In aquatic
field situations, for example, stratification may be based on depth, bottom
type, isotherms, or other variables  (U.S. EPA, 1973a).

     In field studies, a modified form of simple random sampling (systematic
random sampling) may be desirable.  A transect is laid out to be assured of
including an adequate cross-section yet retaining ease of sampling.  Place-
ment of the transect should be at random.  Also, a random starting point
should be selected.

     Randomness is used to  reduce the possibility that large constant or
systematic errors contribute to inaccuracy of the sample.  Since accuracy
also includes a component due to the variability of the measurement units
within the sample, precision is also important.

2.3.4 Selection of Size of  Sample

     All the information necessary for the selection of a sample with the
desired precision may not be available prior to sampling.  As experience is
acquired, even though there may have been very little information at first
on the distribution of the  property being measured in the population,
sampling can be adjusted to meet precision requirements more exactly and more
economically as information is acquired in early stages of the study.  A
valid estimate of precision can be made from the sample itself if it has
been drawn according to an  appropriate statistical probability model.

2.3.4.1 Sampling from a Normal Distribution Population—

     If the population is homogeneous, a single sample unit may represent it
adequately.  However, even  for water and other simple liquids (single phase
liquids) it is possible that under certain conditions temporary stratification
(caused by poor mixing or temperature gradients) nay exist.  This is the
reason for arranging to get a composite sample by the act of sampling at
several locations or several levels and compositing the subsamples thus
obtained.  This is always a good practice if the purpose is to obtain an
average value for the property of the material.

     If the population is not homogeneous, then a number of sample units
should be drawn and analyzed separately, or composited and analyzed.  If a
prior estimate of the standard deviation is available, the sample size n


                                     26

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is calculated (using Model I) by:
       n = (ta'/D)2                                           Eq. 2.3.1,
where o' is the prior estimate of the standard deviation of the material,
D is the maximum allowable difference between the estimate to be made from
the sample and the actual value, and t is a probability factor to give a
selected level of confidence that the difference is greater than D.  See
Bicking (1968).

       Suppose that repeated sampling of a certain population had resulted
in a standard deviation of 0.187 in measurements of the property of interest.
The number of items required to assure with 95% confidence that the average
quality of the population lies within the limits  0.15 of the average of
the determinations is, from Eq. 2.3.1
        n = (2 x 0.187/0.15)2 = 6.25 or 7 items.

       When sufficient items have been tested to estimate the standard
deviation from the data itself (say 30 as a minimum), sample size may be
recalculated, if desired, using Eq. 2.2.5.

       If the population is divided into distinct units or may be so
divided in some suitable way or if it is stratified, and from these primary
units (strata) secondary units (increments) may be taken by sampling, the
most economic increment number and sample size are given by the following
equation (using Model II):
       k = a' / a' VQ[  /  c,                                 Eq. 2.3.2.
            W    D   A      *                                  ^
       „ =   o

where aw2 is the variance within secondary units averaged over all primary
units;  o^2 is the variance between primary units; Cj is the cost of prepar-
ing a primary unit; 02 is the cost of taking a secondary unit; N is the
number of primary units available for sampling; D is the allowable uncertainty
in the sample result; and t is the probability factor.  Equation 2.3.2 gives
the number of secondary units per primary unit and Equation 2.3.3 the
number of primary units in the sample.

       The total cost of the sample can be represented by:
       a - noi +  nhs2                                        Eq. 2.3.4.
       Accordingly, sample schedules can be set up for any set of conditions for
which variances and cost can be determined, to make possible selection of
samples with predetermined precision at minimum cost.

       Consider a stream section having a series of ten pools.  It is
desired to determine the wet weight in mg of chironomid (midge) larvae in the
bottom sediment of the stream.  An Ekman dredge is to be used.  In a
previous experiment three dredge samples from each of four pools provided
estimates of variance within pools equal to 0.84 mg and between pools equal
to 2.35 mg (U.S. Geological Survey, 1973).  The question is:  how many pools
should be sampled and how many dredge hauls be made per pool to determine the
average chironomid weight per dredge haul within 1.0 mg?  It was also known
                                     27

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from previous experiments that the cost of moving the dredge and setting it
up at a different pool costs 10 times as much as to collect a single sample
where the dredge is already sited.

       The number of dredge hauls per pool is calculated from Eq. 2.3.2 as:
       * = /O. 84/2. 35 x  /I07T  -  1.89, or 2 hauls

       The number of pools to be sampled is  (Eq. 2.3.3):

       n =  10  (0.84 + 2 x 2.35) /  [10 x 2 (1.0/2)2 +  2 x  2.35]
         =  5.7, or 6 pools.

       Therefore, to minimize cost and the known error of sampling plan, two
dredge -hauls from each of six pools are required.  The average weight in mg of
these samples would be reported as the weight of chironomids per substrate
area sampled per dredge haul.

2.3.4.2  Sampling from Non-normal Distributions —


       For the  bionomial distribution  (example:  proportion of occurrence
in the population of an  effect due to a treatment) , sample size may be
calculated, based on a prior estimate of presence  of the effect, by Model I:

       n = t2   PQ/D2                                                Eq. 2.3.5

where P is the  estimate  of the presence of the effect, Q = (1 - P)^ and t
and D are as in Eq. 2.3.1.

       After the study is in progress, n can be  recalculated, using the
data itself, from Eq. 2.2.9.

2.3.5  Management of Sampling

       The importance of sampling cannot be overlooked although there may be
reasons why biological researchers have not always recognized probability
based sampling  as a necessary part of quality of results.  A research lab-
oratory is not  like a service laboratory where the samples usually have
been collected  by someone from outside the laboratory and may even be
blind samples for which  the laboratory's main responsibility is analysis.
Even in such circumstances, however, and much more so in a research labora-
tory, the validity  of results is dependent not only on the precision and
accuracy of tests and observations but also on the precision and accuracy of
the sampling.   Experimentation with improperly collected samples may well be
wasted.

       In the same  sense that experiments should be designed, sampling should
be designed.  Looked at  as a Model II design (i.e., a random factor design),
the dependence  of the final data output on the populations involved, the
samples and the tests, is illustrated as follows:
                                     28

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                        Generalized Sampling Design

Population           1	m

Sample    1«1  	••••  !•**                m-^-  	m'n

Analysis  1.1.1  ...  1.1.T    l.n.l  ... \.n.T    w.1.1  ... m.'L.T m.n.\.  ... m.n.T

The errors propogate throughout the system.  Thus, the variance of the re-
sult is made up of components due to the test method, due  to the sampling
procedure and due to the non-homogeneity of the populations.  It is the ob-
ject of quality control to minimize these components or, where they cannot
be made smaller, to balance  the experiment so that their effects are felt
to the same extent in all parts of the experiment.

      In some biological research, samples are collected in the field either
by the researcher or by a part of a team responsible to him.  The necessity
for good sampling practice begins in the field and extends to all aspects of
the selection of test materials and test subjects, and even, in some
Instances, to selection of data.

      The basic sampling models described in this section will require elabo-
ration, particularly in field sampling.  In the parts of the Guidelines deal-
ing with specific areas of research, more details are given.  The sampling
sections of the biological testing methods given in Standard Methods (Rand
et al., 1975) are very useful.  Also, there are some very  good recent EPA
publications which should be referred to for sampling approaches in
practice (U.S. EPA, 1973a, 1973b, 1974b, 1975).

      Sampling usually presents a statistical problem, often substantial
enough to require advice of a statistically trained person.  The reason for
this can be seen.by reference to the basic formula for calculating sample
size, n = (ts/D) .  There must be Information on the variance in measurements
on similar samples (a ), there must be a determination by  the experimenter of
the difference that is important to him (D), and a selection must be made of
the probability level (determines size of t) at which decisions are to be made.

      In some areas of biological research, particularly in new areas, or when
new methods are being tried, very little information may be available on the
variance of results.   The experimenter must depend on experience and on theory
to get early estimates of variances.  One expedient is to  err on the safe side
and use very large sample sizes.  This may be feasible in  some areas, such
as microbiological research where organisms are found in nature in very
large accumulations or reproduce very rapidly.   This way out becomes more
difficult as the test subject becomes larger, or more expensive and the cost
per test unit becomes larger.  The point is that there are physical and eco-
nomic limits on what  can be done with increasing sample size.  Where statisti-
cal theory is applicable,  the sampling should be based on probability.  Where
background information consists of the scientist's input based on theory and
experience,  that should be used.  Many sampling procedures designed without
statistical help are  very good because the scientist knows what he is dealing


                                     29

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with.  However, sampling should never be haphazard because then there is no
control of errors or of costs and sampling may be overdone and wasteful or
underdone and unreliable.

2.3.5.1  Chain of Custody—

     One of the principal concerns in management of sampling is maintenance
of systematic control of samples as they proceed from the field, through
the laboratory tests, to disposition or storage.  The control system is what
is referred to as the Chain of Custody.  Written records of the chain of
custody are very important if results of sampling ever become evidence in
litigation.

     The chain of custody is very important in field sampling, when different
organizations may be responsible for the sampling and the subsequent test-
ing.  It is also very important when it is necessary to maintain parts of
the original samples as reference samples for future checking or for inde-1
pendent investigators.  It is equally necessary that good procedures be
used in biological research where samples of various kinds are important.

     Test substances,should be carefully controlled because identity,
stability, inventory;control, integrity of the sample and safety are
•important.-             . .    —   ........

     Test subjects may be obtained from supply laboratories or may be
bred or cultured within the laboratory.  Identity of individual subjects,
the record of treatments, observations on individuals and groups, remains
of sacrificed or dead subjects, all need to be controlled by a good system.

     Keeping'in mind that in a biological research laboratory the samples
may be chemicals, organisms in treatment groups, samples of organisms or
parts thereof and organs, tissues, etc. for clinical tests or histo-
pathology, the problem becomes a general one of responsibility, record
keeping, secure storage, and all other activities necessary to maintain
integrity of results.

     Some of the important aspects of a chain of custody system for bio-
logical research are:

     •  Clear assignment of responsibility of keeping track of
        samples of all kinds at all program stages
     •  Designation of secure storage space for all research
        materials when not in actual course of experimentation
     •  Handling of samples by a minimum number of persons
     •  When samples are transferred, receipt or dispatch should
        be handled by one person who keeps a complete record of
        all transactions
     •  All samples should be appropriately identified and the
        identification should be recorded in a permanent log book
     •  While in the course of experimentation all samples should
        be in possession or view of the experimenter or

                                     30

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                appropriately secured
             •  The record should include accounting for unused portions of
                samples and disposition of samples when a program is
                completed
             •  All residual materials and records should be retained until
                an agreed-upon retention period expires

      The Chain of Custody record is an important part of the complete
record system.

2.3.5.2  Sample Preservation and Handling—

      For the water environment, recommendations for preservation and holding
of samples are given in Table 2.2 (EPA, 1974b).  The holding time given in the
table is interpreted as the recommended maximum period between sampling and
anaylsis.  Preservatives, where specified, are required to ensure stability
for the holding time.  If holding times are exceeded, a notation of that
fact should be made on data sheets before they are transmitted.

      For some tests, to exceed the maximum holding time would very
seriously compromise the accuracy of the measurement.  The parameters to
which this applies include the following:

             Biochemical Oxygen Demand
             Cyanide, Total
             Chlorine, Total Residual
             Phenols
             Turbidity
             Streptococci Bacteria
             Coliform Bacteria
             Temperature

      Microbiological sampling requirements are to be found in Section
405 of "Standard Methods" (Rand et al., 1975) and radiological sampling
requirements in Sections 200 and 300A of the same reference.

      For biological organisms, the pertinent information will be found
in Section 3.1.4 and the other sub-sections of Section 3 dealing with
specific biological areas.
                                     31

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  TABLE 2.2  RECOMMENDATIONS FOR SAMPLING AND PRESERVATION OF WATER SAMPLES
               ACCORDING TO MEASUREMENT(1) (U.S. EPA, 1974b)
Measurement
Acidity
Alkalinity
Arsenic
BOD
Bromide
COD
Chloride
Chlorine req.
Color
Cyanides
Dissolved oxygen
Probe
Winkler
Flouride
Hardness
Iodine
MBAS
Volume
Required
(ml)
100
100
100
1000
100
50
50
50
50
500
300
300
300
100
100
250
Type of
Container
P,
P,
P,
P,
P,
P,
P,
P,
P,
P,
G
G
P,
P,
P,
P,
G<2)
G
G
G
G
G
G
G
G
G
only
only
G
G
G
G
Method of
Preservation
Cool, 4°C
Cool, 4°C
HNO, to pH<2
Cool, 4°C
Cool, 4°C
H7SO, to pH<2
None required
Det. on site
Cool, 4°C
Cool, 4°C
NaOH to pH 12
Det. on site
Fix on site
Cool, 4°C
Cool, 4°C
HNO 3 to pH<2
Cool, 4°C
Cool, 4°C
Holding
Time (6)
24 hours
24 hours
6 months
6 hours (3)
24 hours
7 days
7 days
No holding
24 hours
24 hours
No holding
4 to 8 hours
7 days
7 days
24 hours
24 hours
Metals
     Dissolved    200
     Suspended

     Total
100
             P,  G
Filter on site
HN03 to pH<2

Filter on site

HNO- to pH<2
 6 months


 6 months

 6 months

(continued)
                                     32

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TABLE 2.2  (Continued)
Volume
Required
Measurement (ml)
Mercury
Dissolved 100




Total 100




Nitrogen
Ammonia 400

Kjeldahl 500
(total)
Nitrate 100

Nitrate 50
NTA 50
Oil & grease 1000

Organic carbon 25

pH 25

Phenolics 500
Type of Method of
Container Preservation


P, G Filter
HN03



P, G HNO,
J



P, G Cool,
H2S04
P, G Cool,
H2S04
P, G Cool,
H2S04
P, G Cool,
P, G Cool,
G only Cool,
H2S04
P, G Cool,
H2SOA
P, G Cool,
Det.
G only Cool,
to pH<2



to pH<2




4°C
to pH<2
4°C
to pH<2
4°C
to pH<2
4°C
4°C
4°C
to pH<2
4°C
to pH<2
4°C
on site
4°C
Holding
Time (6)

38 days
(Glass)
13 days
(Hard plas-
tic)
38 days
(Glass)
13 days
(Hard plas-
tic)
24 hours (4)

7 days <4)

24 hours (4)

24 hours (4)
24 hours
24 hours

24 hours

6 hours

24 hours
                    H3PC-4 to PH<4
                    1.0 g. CuSOA/l
                                         (continued)
           33

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TABLE 2.2  (Continued)
Measurement
Phosphorus
Orthophosphate
Dissolved
Hy dr oly zab le
Total
Total
Dissolved
Residue
Filterable
Non-filterable
Total
Volatile
Settleable
Matter
Selenium
Silica
Specific
Conductance
Sulfate
Sulfide

Sulfite
Temperature
Volume
Required
(ml)
50
50
50
50
100
100
100
100
1000
50
50
100
50
500

50
1000
Type of
Container
P, G
P, G
P, G
P, G
P, G
P, G
P, G
P, G
P, G
P, G
P only
P, G
P, G
P, G

P, G
P, G
Method of
Preservation
Filter on site
Cool, 4°C
Cool, 4°C
H2SO, to pH<2
Cool, 4°C
Filter on site
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C
None required
HNO- to pH<2
Cool, 4°C
Cool, 4°C
Cool, 4°C
2 ml zinc
acetate
Det. on site
Det. on site
Holding
Time (6)
24 hours (4)
24 hours (4)
7 days <4>
24 hours (4)
7 days
7 days
7 days
7 days
24 hours
6 months
7 days
24 hours (5)
7 days
24 hours

No holding
No holding
                                         (continued)
            34

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                      TABLE 2.2  (Continued)

Measurement
Threshold odor
Turbidity
Volume
Required
(ml)
200
100

Type of
Containter
6 only
P, G

Method of
Preservation
Cool, 4°C
Cool, 4°C

Holding
Time (6)
24 hours
7 days
(1)  More specific instructions for preservation and sampling are found
     with each procedure as  detailed in this manual.  A general discus-
     sion on sampling water and industrial wastewater may be found in
     ASTM, Part 24, p. 72-91 (1973)

(2)  Plastic or glass

(3)  If samples cannot be returned to the laboratory in less that 6
     hours and holding time exceeds this limit, the final reported data
     should indicate the actual holding time

(4)  Mercuric chloride may be used as an alternate preservative at a
     concentration of 40 mg/1,  especially if a longer holding time is
     required.  However, the use of mercuric chloride is discouraged
     whenever possible

(5)  If the sample is stabilized by cooling, it should be warmed to
     25°C for reading, or temperature correction made and results report-
     ed at 25°C.

(6)  It has been shown that samples properly preserved may be held for
     extended periods beyond the recommended holding time
                                35

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2.4  PRECISION AND ACCURACY OF TESTS

2.4.1  Measurement of Precision and Accuracy

     A laboratory must have a well-organized and clearly defined program to
check the validity of the data it produces.  Validity is usually expressed.
in terms of precision and accuracy.  Precision is the reproducibility among
replicate observations and accuracy is the difference between observed and
known, or actual, values.

     An analyst initially may establish the precision of a particular method
by a minimum of 5-10, preferably 30, replicate determinations on a single
sample.  Generally, it will be necessary to repeat this procedure on each type
of sample that will be analyzed by a given method and preferably on several
samples of each type from each source.  Comparison of the precision obtained
with reference standards and that obtained with actual samples will reveal any
interferences from contaminants in the samples.

     The standard deviation of the individual measurements is the basic num-
ber for expressing precision.  The smaller the standard deviation, the better
the precision.  There are various ways in which the standard deviation may be
used in presenting precision.  One of the most widely accepted ways is to use
precision limits:
          P  =  ±ts

where t is a probability factor (approximately equal to 2.0 for 95 percent
limits of precision) and s is the calculated standard deviation.  The ASTM
Standard for expressing precision (ASTM, 1977) gives other ways of present-
ing precision.
     It may be desired to determine the precision of an average.  Then, pre-
cision of the average is
          P- = ± ts / Jn
           x
where n is the number of measurements in the average.

     The accuracy of a method may be determined initially by a minimum of
5-10, preferably 30, replicate analyses of samples to which known amounts of
reference standards have been added (spiked samples).  The results should be
reported as percent recovery at the final concentration of the spiked sample.
The spiking of actual samples for these determinations allows for a more
realistic measurement of accuracy than the exclusive use of pure reference
standards, although again comparison of the accuracy obtained with spiked
samples and that obtained with reference standards may be of interest in
identifying sources of error.  Analysis of blanks also will be important for
many paramenters where background level may be non-zero and where a blank
correction may be necessary.
     It should be noted that there is some uncertainty (imprecision) in the
calculation of percent recovery.  The precision of the average percent re-
covery may be calculated as above.  Strictly speaking, the percent recovery
measures the bias in the method, and accuracy should be expressed as the bias
plus or minus the precision of the average percent recovery.


                                     36

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 2 ..412  Control of Precision and Accuracy

2.4.2,1  Use of Standard Methods—

     The availability of standard test methods is one of the indicators of
maturity of a scientific discipline.  In industry and in regulatory activi-
 ties the need for standard methods is obvious to assure comparability of
 results and as a basis for adjudication.  In scientific research, the re-
quirement of flexibility has been used as a justification for caution in the
development of rigid standards.  In new disciplines, the development of the
 test methods is a part of the research problem.  However, the extent to
which attention is given to standards development is a measure of the trust-
worthiness of the major scientific results.

     In biological research, the experimental protocol may itself be the
 test, with the animal subject serving as the instrument.  If this view is
accepted, there can be no excuse for delay in moving toward standard proto-
cols.  The requirement of good science, that results can be verified by
other investigators and at other times and places, is a sufficient impera-
tive.

     It is sometimes suggested that standardization and other quality con-
trol activities are appropriate only where routine, meaning repetitive,
measurements are made.  Such an argument can be made logically only when
the research is truly basic.  A novel method of test may be the key to
successful research.  Even the keenest researcher may not be able to write
the rules in advance.  But biological research to which society has committ-
ed itself has moved the experimenter out of the ivory tower and there can
be no valid pretense that the science is not applied science.  The increased
availability of standard methods of test is a requirement for progress.

     In Section 2.6 there is given a Guide to the Preparation of Specifica-
tions and Standards, which suggests, among other things, a format for stand-
ard  methods of test.  An example is given of a standard method of test for
purity of chemicals for use in a bioassay program.  In the various parts of
Section 3, covering different kinds of bioassay, sample bioassay protocols
are given.

     Copies of all methods in use should be collected, preferably in a
loose-leaf binder, and kept in a place readily accessible  to the researcher.
Performance should be closely supervised to assure that all testing is by
approved, standard methods.

2.4.2.2  Maintenance and Calibration of Instruments—
                                t
     Maintenance and calibrationsof instruments are critical to the genera-
tion of good data.  Instruments and apparatus must be maintained in good
working order, calibrations must be performed in an appropriate manner and
with sufficient frequency, and records and documentation of maintenance
and calibration must be adequate.
                                    37

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     Someone in the laboratory should have the responsibility to see that
each of the instruments is properly maintained and calibrated on schedule.
This may or may not be the same person who actually does the maintenance
and calibration.  The important thing is that the responsibility be clearly
assigned.

     For legal and scientific reasons, it is important to keep careful
records of maintenance and calibration of instruments and apparatus.  Gener-
ally, these records should be kept in permanent (bound) notebooks in ink with
each entry signed and dated.  A separate log (or a separate section of a log)
should be assigned to each instrument or piece of apparatus that requires
any sort of periodic calibration or maintenance, whether that activity is
performed by laboratory personnel or by an outside agency under contract.
It is convenient to include all calibration, maintenance, and repair actions
on an instrument in the log, as a complete and accessible record of the con-
dition of that instrument.  This includes traceability of standards to the
National Bureau of Standards or other recognized source.

     Each entry must specify clearly what action was taken when and by whom.
For example, if a new calibration curve was established which will be the
basis for future analyses, either the curve or a reference to a notebook
containing the curve should be included, along with an explanation of how
the curve was established (identification of reference standards, methodol-
ogy) and when the analyst began using the curve.

     The critical factors are the calibration and maintenance procedures and
the frequency and regularity with which they are carried out.  This informa-
tion should appear in the instrument calibration and maintenance logs and
the laboratory quality control manual.

     Calibration recommendations for some of the major instruments are in-
cluded in Table 2.3.  These recommendations are not to be considered as
rigid rules but rather as guidelines in controlling laboratory performance.
It is recognized that optimum procedures may vary somewhat as a function of
instrument manufacturer and model.  Additional materials that could be use-
ful to the scientist are operation and maintenance manuals for the various
instruments.

2.4.2.3  Routine Control of Test Performance—

     After the precision and accuracy of the method are established, the
analyst will need to incorporate replicates, spikes, standards, and blanks,
as appropriate, into the sequence of routine analyses to insure that valid
data are being generated.  The frequency and procedures required for adequate
monitoring of the quality of the data will depend on the method itself.  The
experience of conscientious analysts and statisticians in the field is an
invaluable source in this matter.  For example, one group of chemists
experienced on the Technicon Auto Analyzer usually runs a duplicate, a
spiked sample, and a reference standard every 8 samples in a large series
of similar samples, or one  in each set of samples, whichever  is more
frequent.  A chemist experienced in the analysis of phenols and cyanide

                                    38

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suggests verifying the standard curves each day, that these parameters are
analyzed with a low and a high reference standard and a blank, and running
a duplicate and a spike with each small set of samples.  Gas chromatpgraphy
often requires multiple injections of the sample with and without an intern-
al standard, in addition to spiked samples and a blank, for each sample
analyzed.  These examples are given only to demonstrate how quality control
protocols will vary considerably with the method and the experience of the
analyst.  The nature of the samples (simple or complex mixtures), the con-
dition of the instrument, the importance of the sample, the breadth of the
precision and accuracy control limits, and many other factors may also af-
fect the quality control requirements.

     Because there are no universal guidelines for the frequency and pro-
cedures required in the use of quality control samples, it is very important
that each laboratory develop its own internal guidelines based on sound
statistical methods and experience.  These should be in the form of written,
explicit protocols for each parameter or group of parameters.  Some tech-
niques for quality control of instruments are outlined in Table 2.4.

     It is of primary importance that the analyst and the laboratory have
a proper appreciation of the importance of replicates, spikes, standards,
and blanks in assuring the validity of their analytical data.

     It should be noted that a popular method of monitoring daily perform-
ance has been the use of Quality Control Charts.  Basically, these charts,
constructed separately for each method or parameter, display the control
limits for precision and accuracy, and the actual precision and accuracy
measured from day to day, and provide a continuous visual picture of the
control of data quality for that method or parameter.  Details of control
chart construction will be found in Section 2.10.

                   TABLE 2.3  INSTRUMENT CALIBRATION (U.S. EPA, 1978)
     Instrument
Procedure
                                Frequency
1)  Analytical balances
2)  pH meters

3)  Conductivity meters
4)Nephelometer/
  turbidimeters
                      (a) Before each weighing
                      (b) Monthly
                      (c) Annually
 (a)  Zero
 (b)  Standard weights
 (c)  Full calibration
     and adjustment
 At pH 4, 7, and 10           Dally
(a) Obtain cell constant      Daily
    with potassium chloride
    reference solutions
(b) Construct temperature     Monthly
    curve if measurements'
    are to be made other than
    at 25 ±_ 0.5°C
(a) Check instrument scales   Monthly
    or develop calibration
    curve with formazine stds (f_40NTU)
                                    39

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                          TABLE 2.3 (continued)
     Instrument
     Procedure
Frequency
5) Colorimeters/filter
   photometers
6) UV/visible
7) Infrared spectro-
   photometers
8) Atomic absorption
   spectrophotometers
9) Carbon analyzers
(b)  If manufacturer's stds. are    Annually
    not formazine, check against
    formazine stds. (5_40NTU)
 Curves determined with 5 to 6     Daily
 laboratory-prepared std. solu-
 tions for each parameter in
 cone, range of samples
(a)  Wavelength calibration with    Quarterly
    holmium oxide glass or solu-
    tion, low-pressure mercury
    arc, benzene vapor (UV), or
    hydrogen arc (visible)
(b)  Absorbance vs. concentration   Daily
    curves with 5 to 6 std.
    solutions for each parameter
    at analytical wavelength in
    cone, range of samples
(c)  Full servicing and adjust-     Annually
     ment
(a)  Wavelength calibration with    Daily
    polystyrene or indene
(b)  Absorbance vs. concentra-      Daily
    tion curves with 5 to 6
    std. solutions for each
    parameter at analytical
    wavelength in cone, range
    of samples
(c)  Full servicing and adjust-
    ment
(a)  Response vs. concentration
    curves with 6 to 8 std.
    solutions for each metal
    (std. mixtures are accept-
    able, but with same acid as
    sample to be run) in cone.
    range of samples
(b)  Full servicing and adjust-     Annually
    ment
Curves determined with 5 to 6      Daily
std. solutions in cone, range
of samples

                              (continued)
  Semi-Annually

  Daily
                                    40

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                          TABLE 2.3  (continued)
    Instrument
                             Procedure
Frequency
 10) DO meters
                         Calibrated against modified
                         Winkler method on aerated dis-
                         tilled or tap water

                         Curves determined with 5 to 6
                         std. solutions in cone, range
                         of samples

                         Calibrate in constant tempera-
                         ture baths at two temperatures
                         against precision thermometers
                         certified by NBS

                         (a)  Curves determined with std.
                             solutions for each parameter
                         (b)  Full service and adjustment
                             (esp. colorimeter)
14)  Gas chromatographs   (a)  Retention times and detector
                             response checked with std.
                             solutions
                         (b)  Response curves for each
                             parameter determined with
                             std. solutions
 11) Other selective
    ion electrodes
    and electrometers
 12) Thermometers
13) Technicon auto
    analyzers
15) Radiological
    equipment

16) Sulfur dioxide in
    air sampler/analy-
    zers (pararosani-
    line method)
     Daily
     Daily
     Quarterly
    Each set of
    samples
    Annually

    Daily
                                                               Monthly
17)  Suspended particu-
    lates
    (high-volume sampler
     method)
                         (See Standard Methods,  Sect.  300)


                         (a)  Calibrate flowmeters and  hy-
                             podermic needles against  a
                             wet  test meter
                         (b)  Spectrophotometrie  calibra-
                             tion curve with 5 to 6 std.
                             sulfite-TCM solutions at
                             controlled temperature (+1°C)
                         (c)  Sampling calibration curve
                             with 5  to 6 std. atmospheres
                             from permeation tubes or
                             cylinders
                         (d)  Calibrate associated ther-
                             mometers,  barometers, and
                             spectrophotometer (wave-
                             length )

                         (a)  Calibrate sampler (curve  of true
                             air  flow rate  vs. rotameter or
                             recorder reading) with orifice
                             calibration unit and differential
                             manometer at 6 air  flow rates
                                   41
    Quarterly


    Monthly



    Monthly



    Quarterly




    Monthly
                                                                (continued)

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                           TABLE 2.3 (continued)
    Instrument
    Procedure
Frequency
18) Carbon monoxide
    (non-dispersive IR)
19) Photochemical
    oxidants (ozone)
(b)  Calibrate orifice cali-
    bration unit with posi-
    tive displacement primary
    standard and differential
    manometers
(c)  Calibrate relative humidity
    indicator in the condition-
    ing environment against wet-
    bulb/dry-bulb psychrometer
(d)  Check elapsed time indicator
(e)  Calibrate associated analyt-
    ical  balances,  thermom-
    eters , barometers

(a)  Determine linearity of
    detector response (cali-
    bration curve) with cali-
    bration gases (0, 10, 20,
    40, and 80% of full scale,
    certified to ±2% and checked
    against auditing gases
    certified to +1%)
(b)  Perform zero and span cali-
    brations
(c) Calibrate rotameter and
    sample cell pressure gauge

(a) Calibrate standard KI/I2
    solutions in terms of
    calculated 0_ equivalents
    at 352 nm
(b) Calibrate instrument re-
    sponse with 6 to 8 test
    atmospheres from ozone gener-
    ator,  spanning expected
    range  of sample concen-
    trations (usually 0.05-
    0.5 ppm 0 )
(c) Calibrate flowmeters, ba-
    rometer, thermometer
(d) Calibrate and service
    spectrophotometer
                                                              Annually
                                                              S emi-annually
                                                              Semi-annually
                                                              As needed
Monthly
                                                              Before and
                                                              after each
                                                              s ampling
                                                              period
                                                              Semi-annually
Weekly
                                                              Monthly
                                                              Semi-annually

                                                              As specified
                                     42
                                                                 (continued)

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                           TABLE 2.3  (continued)
     Instrument
     Procedure
                                 Frequency
 20) Hydrocarbons
    (corrected for
    methane)
21) Nitrogen dioxide
    (arsenite 24-hr.
    sampling method)
22) Nitrogen dioxide
    (Griess-Saltzman
    colorimetric,
    continuous)
23) Nitrogen dioxide
    (chemiluminescence,
    continuous)
(a) Determine linearity of      Monthly
    detector response with
    calibration gases (0,
    10, 20, 40, and 80% of
    scale, certified to ±2%)
(b) Perform zero and span
    calibrations
(c) Calibrate flowmeters and
    other associated apparatus

(a) Calibrate flowmeter with
    wet test meter
(b) Calibrate hypodermic
    needle (flow restrictor)
    with flowmeter
(c) Obtain colorimetric cali-
    bration curves with 5 to
    6 std. nitrite solutions

(a) Dynamic calibration with
    std.  atmospheres (e.g.,
    from permeation tubes)
    spanning the range of
    observed concentrations
(b) Static colorimetric cali-   Weekly
    bration with 5 to 6 std.
    nitrite solutions
                                                         Before and after
                                                         each sampling period
                                                         Semi-annually
                                Monthly

                                Each new needle


                                Weekly



                                Monthly
                                Each new cylinder
(a) Calibrate std. NO cylinder
    with ozone generator (pre-
    calibrated by iodometric
    procedure)
(b) Calibrate NO monitor with   Monthly
    std. NO cylinder at several
    concentrations
(c) Calibrate N0_monitors       Monthly
    with std. NO cylinder
    (diluted NO concentrations
    determined with NO moni-
    tor) and calibrated ozone
    generator
(d) Calibrate associated flow-  Semi-Annually
    meters
                                    43
                                                                 (continued)

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                           TABLE 2.3  (Continued)
    Instrument
      Procedure
Frequency
24) Autoclaves and
    sterilizers
(a)  Sterilization effectiveness
    checked (e.g., B.  stearo-
    thermophilus. color indi-
    cator tape for ethylene
    oxide)
(b)  Temperature-recording device
    calibrated
  Daily
                                                              Semi-annually
   TABLE 2.4  TECHNIQUES FOR QUALITY CONTROL OF INSTRUMENTS (ASTM, 1977)
      Control Parameter
                   Control Technique
Instrument operating range
Interferences
Environmental conditions
Associated equipment operation
(cuvettes, volumetric ware,
dilutors, etc.)


Normal system drift

System component functions
Response readout
            Coordinate instrument selection with
            method requirements

            Sample conditioning (drying, sepa-
            rating, mixing, etc.)

            Use of blanks

            Use of spiked samples
            Monitor and control temperature,
            humidity, pressure, and atmospheric
            parameters that can affect system
            response.  Consult manufacturer's
            instructions and method descriptions.

            Proper handling procedures

            Standard procedures for cleaning

            Standardization or calibration

            Zero adjust

            Apply function tests

            Plot response to changing concen-
            trations
            Perform maintenance when indicated

            Use calibration curve, adjust using
            blanks and zero-span controls
                                     44

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2.5 PHYSICAL ENVIRONMENT OF RESEARCH

    The environmental factors in the research laboratory can affect the
quality of sampling and observation.  Good housekeeping provides the proper
setting for a quality control program.  Some effects of poor housekeeping are
related to occupational safety and health, which are important.  Lack of
care also usually goes with poor maintenance which leads to deterioration in
the quality of data.  Some elements of poor housekeeping practices, which
quality-minded management will guard against are given in Table 2.5 (U.S.
EPA, 1973b).

    Laboratory support services require quality control.  Services include
gases, water, electricity and space conditioning.  Some of the parameters of
support services that affect quality, and suggested control techniques are
given in Table 2.6 (U.S. EPA, 1973b).

    Purchasing guides, or specifications, are required for all expendable
materials used by the laboratory.  Purchasing and acceptance specifications
are discussed in Sect. 2.6.  The same considerations apply to purchased
support services.

    The quality of reagent water is a matter deserving special attention.
If the water has been purchased, each batch should be tested for conductivity
before acceptance.  High purity water is generally defined as water having a
conductivity of 2.0 micromhos or greater.  It may be necessary to redistill
water if greater purity is required.  Stills, storage  tanks and piping must
be specified, installed and maintained so as to minimize contamination.
Pretreatment of feed water will improve still operation.  Ion exchange resins
are used to remove calcium and magnesium.  A carbon filter on the feed water
intake will remove organic materials.  Certain needs in biological research
may call for double- or triple-distilled water.

    Also it may be a requirement that the water be ammonia-free, carbon-
dioxide-free, or ion-free.  Ion exchange columns using research grade
cartridges can produce high quality water (ASTM Referee Reagent Grade) with
a maximum of 0.1 mg/1 total matter and maximum conductivity of 0.1 micromho.
                                     45

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                   TABLE 2.5  EFFECTS OF HOUSEKEEPING PRACTICES
                              ON LABORATORY PERFORMANCE (U.S EPA, 1973b)
    Element
Possible Effects
Excess atmospheric or
    accumulated dust
Reagent spillage or
    leaks

Improper maintenance of
    air conditioning and
    heating equipment
Improper use of extension
    cords or overloading
    of circuits

Improper cleaning of glassware
    and reagent containers

Non-systematized storage of
    parts and tools
Failure of electrical contacts and
switches, excessive wear of
mechanical components, excessive
soiling of optical components

Corrosion, hazardous vapors,
electrical hazards, insecure footing

Air conditioning and heating equip-
ment failure, operation outside of
designated limits, equipment damage,
freezing, inking pen failures,
excessive reagent evaporation

Poor voltage control, excessive
circuit failures, electrical
hazard

Reagent contamination
Loss of tools, absence of tools
and parts when required, subsequent
system failure
                                     46

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                   TABLE 2.6  TECHNIQUES FOR QUALITY CONTROL
                              OF LABORATORY SUPPORT SERVICES
                              (U.S.  EPA, 1973b)
Support
Service
Parameters Affect-
ing Quality
Control
Techniques
Laboratory
    gases
Reagent water
Electrical
     service
Ambient
     conditions
Purity specifications -
vary among manufacturers

Variation between lots
                   Atmospheric interferences
Commercial source
variation

Purity requirements
Develop purchasing
guides

Overlap use of old
and new cylinders

Adopt filtering and
drying procedures

Develop purchasing guides -
Batch test for conductivity

Redistillation, heating,
deionization with ion
exchange columns
                   Atmospheric interferences Filtration of exchange air
Generation and storage
equipment

Voltage fluctuations
Temperature


Humidity
Maintenance schedules from
manufacturer recommendations

Battery power

Constant voltage transformers

Separate lines

Motor generator sets

Heating and air conditioning
systems

Humidity controls
                                     47

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2.6  CHEMICALS AND REAGENTS

     The quality control plan should include standard procedures for choos-
ing chemicals, preparing standard solutions, storing and handling chemicals
and reagents, and choosing and handling standard reference materials.  Table
2.7 (U.S.EPA,1973b) lists some of the factors affecting such procedures with
some of the appropriate control techniques.

2.6.1  Purchase Specifications

     Chemical reagents, solvents and gases are available in a range of puri-
ties from technical grade to  ultrapure  grades.  For many purposes, analyt-
ical  reagent grade or pesticide grade will be satisfactory.  Other uses,
such as trace analysis or treatment in biological assay, will require
special grades of purity.  If purity is not specified, it is generally
understood that analytical reagent grade is wanted.  However, the chemical
procurement specification should always state the desired chemical and
physical properties and the purity required.

     For most grades, it will be sufficient to specify grade based on the
manufacturer's published data sheets,and acceptance may be on the basis of
the supplier's certification without sampling and testing.  Pure grades
may have to be specified in detail and, depending on criticality of use,
may have to be sampled and tested before dilution and use.

     At this point, it is pertinent to consider the whole matter of the
preparation of specifications and standards.  Specifications and stand-
ards are required not only for chemicals and reagents but also for pur-
chase of facilities, equipment, and supplies of all kinds; for field and
laboratory operating procedures; for methods of test, including bioassay
protocols; and for quality assurance procedures.  The next sub-section
gives a guide for specification and standard preparation in general.

2.6.1.1  Guide to the Preparation of Specifications and Standards —

     •  Introduction

     This "Guide" provides the basis for the preparation of a system of
specifications and standards in conformance with regulatory requirements
and with current good laboratory practices.  It provides the framework for
a system suitable to health effects research, biological research,  and
environmental research in general.

     •  General Philosophy of Specifications and Standards

          o  Definitions

          A specification is a precise statement,  usually for use in  procure-
     ment, of the requirements for a material, product,  system or service,
     including the procedure by which it can be determined that the require-
     ments have been met within the limits specified in the statement.
                                      48

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     A standard is a document containing a set of conditions to be ful-
filled by an item, process or method based on the consolidated results
of science, technique and experience which is approved by a recognized
authority and usually determined to be acceptable to all to whom it may
apply.

     o  Basic Considerations

     The definitions of specification and standard agree with those
approved by the International Standardization Organization, and are in
accordance with the Federal Standards.  A specification may be a stand-
ard, a part of a standard, or independent of a standard.

     It is understood that the complete specification system shall have
been committed to writing.

     The purpose of specifications and standards in a research program
is to ensure the validity and integrity of the data produced.  Validity
refers to  the scientific faultlessness of the data and integrity
refers to its presentation in unaltered form.  The quality of results
depends on appropriate control and verification procedures in the re-
spective parts of the system.

     o  Categories of Documents

     Materials specifications (or purchase specifications)

     Standard Operating Procedures (Good Laboratory Practices)

     Standard Bioassay Protocols

     Standard Methods of Test, including  Histology and Pathology

     Quality Assurance Procedures

     o  Suggestions for the Preparation of Specifications and Standards

     Requirements, as far as practicable, should be expressed in numeri-
cal terms and must include acceptable levels or limits of permissible
variation.

     The language used should contain the simplest words and phrases
that will convey the intended meaning.  Use "shall" whenever a speci-
fication expresses a provision that is binding; use "should" or "may"
to express non-mandatory provisions.  "Will" may be used to express a
declaration of purpose on the part of the Government or where simple
futurity is to be expressed.

     Measurements shall be expressed in units of the metric system in
accordance with the International System of Units  (SI) as detailed in
the National Bureau of Standards Special Publication 330.  Equivalent
units may be given in parentheses.

                                 49

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     •  Recommended Coding of Specifications

     As a means of identification of specifications a uniform code system
combining letters and numerals is established.

     The first two letters of the Code are reserved to indicate the partic-
ular program.  The next letter will indicate the category of specification
as follows:

          Materials Specification           M

          Operating Procedure               0

          Bioassay Protocol                 P

          Method of Test                    T

          Quality Assurance Procedure       Q

     Succeeding numerals will identify the particular specification uniquely.

     Example:  CBP1 could indicate the carcinogenesis bioassay protocol for
an Acute Toxicity Test.

     •  Recommended Format of Specifications

          o  General outline form should be used.  Each section should be
     numbered in arabic numerals and subsections in decimal notation.
     Active voice should be used.  Tables may be used, for convenience*
     except that the clarity and completeness of the written specification
     shall not be sacrificed for brevity.

          o  Information Common to Headings of All Specifications

               Specification Number

               Type of Specification

               Page Number

               Title

               Approval.  Initials of the person authorized to approve for
                          each organizational unit should appear.

          o  Content of Specifications

          The following section headings shall be included in all documents
     in the system, with the note "Not Applicable," if the section is  not
     required.
                                     50

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          1.  Scope

          2.  Applicable Documents
          3.  Requirements

          4.  Quality Control
          5.  Packaging (Materials specifications only)

          6.  Notes

          7.  Reference Documents
          8.  Appendix

     Scope.  A clear, concise delineation of the extent or range of
technical content shall be given which may be clarified as needed by
naming specific exclusions from coverage.  A subparagraph headed
"Application" may be included to indicate the general field or particu-
lar area of use.

     Applicable Documents.  Government or nongovernment specifications
and standards may be referenced.  Government regulations or codes may
also be referenced if essential.  Only documents identified in Sections
3, 4 and 5 of the specification that are supportive to or clarifying
requirements of those sections shall be listed in Section 2.  Refer-
enced documents shall be currently available.

     Requirements.  All necessary requirements (materials, processes,
systems and performance characteristics) shall be given.  Only those
characteristics should be stated that can be confirmed by reliable
quality criteria or test equipment.

     Quality Control.  This section shall describe all sampling, testing
and analyses to be performed to control specified procedures and super-
visory actions to assure that the results conform to the requirements.

     Packaging.  Packaging is defined as the means of providing protec-
tion to items during shipment, storage, or redistribution operations.

     Notes.  This section shall contain information of a general or
explanatory nature.

     Reference Documents.  Information sources are located  in this section.

     Appendix.  Large data tables or detailed procedures or management
plans may be appended to the specification.  Such material  applies to
references in the body of the specification.

•  Control of Specifications

     o  Preparation and Distribution
                                51

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          This specification system applies to the whole of a particular
     program, and its staff is responsible for the identification of exist-
     ing specifications and for obtaining or preparing new specifications.
     The program will maintain a complete file of specifications and sub-
     contractors will maintain files of all specifications applicable to
     them.

          o  Review and Approval of Specifications

          Approvals are required of the Program Director.  Specifications
     must be accepted by sub-contractor to which they apply.  The Program's
     Quality Control Officer shall review specifications based on the
     following criteria:

               Conformance to coding, categories, and format

               Provision of acceptable limits of variability

               Inclusion or reference to a procedure for verifying
               that specification limits have been met

               Necessary approvals and acceptances.

     Specifications for a Mammalian Bioassay Program include

          o  Standard Bioassay Protocol
          o  Physical Plant and Material Specifications
          o  Good Animal Care Laboratory Practices
          o  Standard Methods of Test
          o  Safety Standards

     Examples of specifications prepared in accordance with the Guide are
given in the following pages:  a physical plant specification, a materials
specification and a standard method of test.

     Examples of bioassay protocols in a format which departs considerably
from the format suggested in the Guide will be found in the parts of Section
3 for specific kinds of bioassay.  See Sect. 2.7.2 and Appendix B for a
complete set of Good Animal Care Laboratory Practices suitable for mammalian
bioassay with rodents.
                                    52

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                TABLE 2.7  GUIDELINES FOR QUALITY CONTROL
                OF CHEMICALS AND REAGENTS (U.S.EPA,  1973b)
Procedure
    Control Parameter
    Control Technique
Choice of
 chemicals
Preparation
 of standard
 solutions
Storage and
 handling
Standard
 reference
 materials
Manufacturer designations

Method purity specifications
Calibrated glassware

Standard reference materials
 (SRM)

Stability

Container composition

Filtering or pretreatment

Environmental sensitivity
Availability

Stability
Develop purchasing guides

Use American Chemical Society
 designations as a base

Develop purification or
 treatment procedures speci-
 fied by method

Purchasing guidelines

Schedules for restandardi-
 zation of solutions
Design a labeling system

Purchase single lot numbers

Rotate stock

Control temperature, light,
 atmospheric exposure

Store in temperature-
 controlled atmosphere

Desiccate when necessary

Replace if instability is
 suspected

Weigh to determine loss or
 degradation
                                     53

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Carcinogen Bioassay Program Specificati
Type:
PHYSICAL PLANT SPECIFICATION
on No. CBM-2
Sheet |Of
1 I'
Subject:
    BARRIER FOR PREVENTION  OF  CONTAMINATION BY  PATHOGENIC MICROORGANISMS
Approved:
Proj.
Q.C.
Lab
Other
Date
                                  1.   SCOPE

    This  specification covers  considerations  for  the  location  and  con-
struction materials   of the barrier  system.   Ideas  on the  room size  and
floor  plan  of  the  barrier  system are also  mentioned.   Equipment areas,
laboratories and quarantine area within the barrier system are described.
A  list of ancillary  equipment  for the barrier system  is  given.   Lastly,
the four  different types of barrier  system are suggested.
    None
     3.1   Location
                            2.   APPLICABLE DOCUMENTS
                                3.   REQUIREMENTS
          3.1.1 Preferably,  the barrier should be remote from other  build-
 ings  or  activities  that could endanger its operation.

          3.1.2 If it is part of a building,  there should be  a maximum
 isolation.   This could be achieved by:

               3.1.2.1  Spearate heating systems.

               3.1.2.2  Installation of devices to prevent backflow
                        through drains.

               3.1.2.3  Containment of water leaks.

               3.1.2.4  Use of differential air pressure to control
                        air flow.

               3.1.2.5  Separate access and egress corridors.

               3.1.2.6  Controlled access by personnel.

     3.2  Construction Materials

          3.2.1  Interior materials should be chosen for durability, longevity,
 and low  maintenance.
                                      5A

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    Carcinogen Bioassay Program
Subject: BARRIER FOR PREVENTION OF CONTAMINATION
BY PATHOGENIC MICROORGANISMS
Date:
Sheet
2
Of
6
         3.2.2  They should be resistant to corrosion, scrubbing, and harsh
chemicals, but they should be easy to clean.

         3.2.3  The entire facility must be protected from climatic con-
 litions, and the building must be secure against such organisms as insects,
wild rodents, and vermin.

    3.3  Room size.

         3.3.1  It is easier to contain a point outbreak of disease if the
animal rooms are small and independent from each other.

         3.3.2  Rooms should not contain more than one animal species.

         3.3.3  Ideally, rooms should not be so large as to contain more
cages than can be serviced by one person.

    3.4  Floor plan.

         3.4.1  The relation of one room to another and one floor to another
will be dictated by the functions (in addition to animal care) of the
facility and by the flow of people, supplies, animals, and so on through
the facility.

         3.4.2  The traffic pattern should avoid backflow from any area to
a cleaner area.

         3.4.3  The animal rooms are to be the most protected area.

    3.5  Equipment areas.

         3.5.1  All mechanical equipment should be located where it can be
serviced without having the service personnel enter the more protected areas
 )f the barrier.

         3.5.2  Piping of any kind should not run directly over animal rooms
 >ut should be located above corridors.

    3.6  Laboratories.

         3.6.1  Areas outside of the animal rooms where animals will be
 landled must also be designed for ease of cleaning and have features  to
 ninimize possible contamination of animals by handling procedures.

         3.6.2  Animals removed to conventional laboratories outside  the
 jarrier should not be brought back into animal rooms.
                                      55

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    Carcinogen Bioassay Program                    Specification No. CBM-2
Subject: BARRIER FOR PREVENTION OF CONTAMINATION
BY PATHOGENIC MICROORGANISMS
Date:
Sheet
3
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6
         3.6.3  Consideration should be given to providing clean labora-
tories within the barrier with direct access to animal rooms.

    3.7  Quarantine Area.

         3.7.1  If animals are brought in from an outside source, or if
animals removed from the barrier are to be returned, a protected area must
be provided where they can be held until their freedom from contaminants
Ls determined.

    3.8  Ancillary Equipment.

         3.8.1  The selection of the ancillary equipment, its placement,
performance monitoring, servicing, and dependability play a major role in
:he success or failure of a barrier system.

         3.8.2  Major movable and nonmovable equipment may be divided into
the following categories:

              3.8.2.1  HVAC (heating, ventilating, and air conditioning):
Air-handling equipment, refrigeration compression or steam absorption
 quipment, humidifiers, filtration systems, ductwork and air diffusers,
leat source, controls and alarm systems.

              3.8.2.2  Utilities (types):  Electric service and emergency
generators, high-pressure, steam source, water supply (potable, chlorinated,
acidified, demineralized, UV-sterilized, filtered).

              3.8.2.3  Sterilizing equipment (types):  High-vacuum, double
door autoclave system, ethylene oxide, ultraviolet equipment, ionizing
radiation source.

              3.8.2.A  Mechanical washing equipment (types):  Rack washer,
tunnel washer, batch washer, bottle washer.

              3.8.2.5  Water-dispensing equipment:  Automatic distribution,
chlorinators, filters, demineralizers, ultraviolet sterilizers.

              3.8.2.6  Waste disposal:  Incinerators, vacuum systems,
mechanical disposal.

    3.9  Classification of Barrier Systems Based on Method of Contamination
Control.
                                      56

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Carcinogen Bioassay Program Specification No.
Subject: BARRIER FOR PREVENTION OF CONTAMINATION
BY PATHOGENIC MICROORGANISMS
Date:
Sheet
4
CBM-2
Of
6
         3.9.1  The major operational variables in a barrier system are
quality,  quantity,  and source of animals;  frequency and method of intro-
ducing animals through the barrier;  processing of materials through the
barrier;  entry of animal technicians into  the barrier;  method of housing
and handling animals;  the environmental systems,  with special emphasis on
the air-handling systems; and monitoring practices.

         Type 1:   Maximum-security barrier

              1.   Animal source — defined microbially associated animals.

              2.   Animals are maintained in isolation and then introduced
via a port system into the barrier.

              3.   Sterile materials, Including cages, food, bedding, and
other supplies enter the barrier without contamination.

              4.   All personnel entering the barrier must strip, shower,
or pass through an air wash,  wear sterilized uniforms,  wear face mask,
gloves, and hair and shoe covers.

              5.   All animals are transferred by forceps previously dis-
infected; manual handling is  kept to a minimum.

              6.   Air supply  is HEPA filtered (99.97 percent effective at
0.3 micron particle retention).  Air recirculation is permitted if properly
monitored.

         Type II:  High-security barrier

              1.   Animal source - barrier-maintained animals.

              2.   Animals are shipped in filter boxes and introduced via
a secure port system (quarantine within the barrier is optional).

              3.   Materials - same as Type I.

              4.   Personnel - same as Type I.

              5.   Animal Care - same as Type I.

              6.   Air supply  is filtered (95 percent effective at 0.3 y).
   air recirculation is permitted unless HEPA filtered.
                                      57

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    Carcinogen Bioassay Program                       Specification No. CBM-2
Subject: BARRIER FOR PREVENTION OF CONTAMINATION
BY PATHOGENIC MICROORGANISMS
Date:
Sheet
5
Of
6
         Type III:  Moderate-security barrier

              1.  Animals are obtained from a reputable breeder and des-
ignated as barrier or monitored animals.  Monitoring results are available
for review in order to select suitable animals for research projects.

              2.  Animal entry - same as Type II, but each shipment should
be placed in room containing animals from only one vendor.

              3.  Materials are either sterilized or  sanitized or are
heat-treated to kill all pathogenic vegetative microbial forms.  If cages
are sanitized instead of autoclaved, water temperature sensors that shut
off the washing machine (less than 108°F) are recommended.

              4.  Personnel - same as Type I, but use of face masks and
gloves may be modified.

              5.  Animal care - same as Type I or modified to include hand
contact.

              6.  Air supply filtration is rated at 85 percent efficiency
or better for 0.3 y particle retention.

         Type IV:  Minimal-security barrier

              1.  Source of animals - same as for Type III, except that
these are usually monitored animals held within a barrier.  The supply
colony may therefore have antibodies to known viral pathogens, and certain
bacterial agents may be present.  Knowledge of monitoring results is criti-
cal for selection and proper use of these animals.

              2.  Animals may be introduced via exit corridors, minimizing
exposure. Containers do not enter rooms.  Animals may be quarantined out-
side barrier then introduced via transport cages.

              •3.  Materials - same as Type III.

              4.  Technicians enter through personnel lock, but security
measures less stringent than Type III

              5.  Investigators abide by rules for animal techincians or
have an option in some areas of the barrier to enter their own animal
rooms from the exit corridor after donning disposable shoe covers and
clean laboratory coats and then washing hands and using disposable gloves.
They cannot enter other animal rooms or enter clean corridors.
                                      58

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    Carcinogen Bioassay Program                       Specification No. CBM-2
Subject: BARRIER FOR PREVENTION OF CONTAMINATION
BY PATHOGENIC MICROORGANISMS
Date:
Sheet
6
Of
6
         6.  Animal handling - generally the same as Type III.

         7.  Air supply - same as Type III.

                           4.  QUALITY CONTROL

    4.1  Methods used in monitoring must include a thorough visual exami-
nation of the overall barrier system and its operating components, par-
ticularly of personnel involved in animal husbandry, cage sanitation,
machine maintenance, and decontamination.

    4.2.  Monitoring procedures for Types I  and II barrier systems should
include statistically significant sampling by microbiologic, histopathologic,
and physical methods.

    4.3  Monitoring procedures for Type III  barrier system are the same as
Type I & II, but depth and breadth of monitoring practices are reduced.

    4.4  Monitoring procedures for Type IV   barrier system are the same as
lype III,  but may be further reduced.   Level of monitoring must be adequate
for the purpose of the experiment.

    4.5  Perform serology on personnel for the presence of antibodies to
animal viruses.

                               5.   PACKAGING

    Not applicable here.

                                6.  NOTES

    6.1 This  specification  is  taken from:   Long-Term Holding of Laboratory
Rodents,  ILAR News,  Volume  XIX, Number 4, 1976,  L9--L12.

    6.2 Calling a Type I barrier  "maximum security" does  not presuppose
that  contamination will not occur.  Actual  quality of the animals in such
a  system  should be known and duly recorded.
                                     59

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Subject:
Approved:
Proj.
Q.C.
Lab
Other
Date
    Carcinogen Bioassay Program
Specification No. CBM-17
Type: MATERIALS SPECIFICATION
Sheet
l
Of
8
        CHEMICALS  FOR  TESTING IN THE  CARCINOGEN BIOASSAY PROGRAM
                                   1.   SCOPE

    This  specification covers  chemicals  to be tested for carcinogenic
potential in the  Carcinogen Bioassay  Program.

                            2.   APPLICABLE DOCUMENTS

    2.1   Code of  Federal Regulations, Title 42,  Section 72.25,  1972.

    2.2   Code of  Federal Regulations, Title 49,  Section 173,  1973.

                               3.   REQUIREMENTS

    3.1   All samples of chemicals to  be  tested for carcinogenic potential
in the Carcinogen Bioassay Program shall be collected by the  supplier in
a manner  that insures that the sample is representative of the  entire batch
or lot.

    3.2   Chemicals to be tested will  be  specified  and supplied  to the
Analytical Subcontractor and Bioassay Laboratory by Program Management
 (6.1,  6.2, 6.3).

    3.3   Pure reference standards to  be  used in all relative  purity assays
as well  as for comparison of different lots of chemicals shall  be obtained
from the.National  Cancer Institute, U.S.  Pharmacopeia, National  Formulary,
commercial sources, or shall be prepared by the Analytical Subcontractor
 (6.3).

     3.4   The homogeneity, chemical identity, impurity content,  stability,
and storage parameters of each test chemical shall be determined prior to
 its bioassay by the Analytical Subcontractor.  Results shall  be given to
 the bioassay laboratory as well as to Program Management (6.1,  6.2, 6.3).

     3.5   Identification and quantification of impurities as well as puri-
 fication of the test chemical may be necessary in some instances (6.1, 6.3)

     3.6  Homogenization of test chemical (6.3)

          3.6.1  Samples of the test chemical shall be ground  in a Fitz
                                      60

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Subject: CHEMICALS FOR TESTING IN THE CARCINOGEN
BIOASSAY PROGRAM
Date:
Sheet
2
Of
8
Mill, homogenized in a Day Blender, and then analyzed for homogeneity by
the Analytical Subcontractor.   Samples shall be taken at three levels in
the blender for analysis.

        3.6.2  The entire  batch of chemicals to be used by the bioassay
laboratory shall be ground and homogenized by the Analytical Subcontractor.

   3.7  Identification (6.3)

        3.7.1  Single compounds

             Two or more of the following tests shall be used depending on
the amount of sample available, nature of the compound, and the number of
techniques necessary to identify the compound:

             3.7.1.1  Spectral data

                      o Infrared
                      o Ultraviolet
                      o Visible
                      o Nuclear Magnetic Resonance
                      o Mass  Spectroscopy - when necessary to
                                             clarify structural data.

             3.7.1.2  Physical constants

                      o Melting Point
                      o Boiling Point
                      o Refractive Index
                      o Optical Rotation
                      o Elemental Analysis

             3.7.1.3  Chromatography

                      o Thin-Layer - all but highly volatile cmpds
                      o Vapor-Phase - highly volatile compounds
                      o High-Pressure Liquid - non-volatile polar
                                                compounds
                      o Gel Permeation Mtds. - non-volatile polar
                                                compounds

   3.8  Assay (6.3)

        3.8.1  Single Compounds

             3.8.1.1  Assay methods for the test chemical shall be deter-
                                     61

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   Carcinogen Bioassay Program                       Specification No. CBM-17
Subject:
CHEMICALS FOR TESTING IN THE
CARCINOGEN BIOASSAY PROGRAM
Date:


Sheet

3
Of

8
mined by the Analytical Subcontractor on the basis of chemical nature of the
compound and the procedure by which it was synthesized.

             3.8.1.2  Two or more of the following methods shall be used for
each chemical depending on the amount of sample available, nature of the
compound, and number of procedures necessary to determine the level of purity

                  3.8.1.2.1  Elemental Analysis

                  3.8.1.2.2  Chromatography - as  for 3.7.1.3

                  3.8.1.2.3  Spectroscopy

                      o  Emission
                      o  Visible
                      o  Ultraviolet
                      o  Infrared
                      o  Fluorescence
                      o  Nuclear Magnetic Resonance
         	  » .  .. _,.o. .Electron Spin,. Resonance           .       ,       .. „
                      o  Mass Spectroscopy      	

                  3.8.1.2.4  Titrimetry and Electroanalysis

                      o  Colorimetry
                      o  Potentiometry - Compounds with reactive  groups,
                                         e.g., amines,  acids, oxidizable,
                                         reducible groups, etc.
                      o  Polarography  - Reducible compounds
                      o  Voltammetry   - Oxidizable  compounds
                      o  Coulometry
                      o  Amperometry

                  3.8.1.2.5  Absolute Purity Analysis - Reference standards
                                                         and compounds where
                                                         high purity  is
                                                         critical
                      o  Differential Scanning Colorimetry
                      o  Phase  Solubility

         3.8.2 Mixtures  (6.3)

              3.8.2.1 Isolation of Components

                  At least  two  of  the  following methods shall be  used:
                                      62

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   Carcinogen Bioassay Program                     Specification No. CBM-17
Subject: CHEMICALS FOR TESTING IN THE
        CARCINOGEN BIOASSAY PROGRAM
Date:
Sheet
    4
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                     8
                      o  Crystallization
                      o  Preparative Chromatography
                           o  Thin-Layer Chromatography
                           o  Column Chromatography - Preliminary to high
                                                      pressure liquid or
                                                      vapor-phase for
                                                      solid compounds
                           o  Vapor-Phase Chromatography - Volatile compounds
                           o  High-Pressure Liquid Chromatography
                      o  Spinning Band Distillation - Volatile compounds
                      o  Zone Refining - Solid compounds
                      o  Sublimation   - Solid compounds

             3.8.2.2  Identification of Components - As for 3.7

             3.8.2.3  Quantification of Components - As for 3.8

   3.9  Reanalysis (6.2)

        A sample of the bulk test chemical shall be analyzed for purity
at various intervals by the bioassay laboratory, or by a subcontractor
in close proximity to the laboratory so that the analytical results are
available within one week.  Analytical methods to be used will be provided
by the Analytical Subcontractor.

        3.9.1  Each chemical lot shall be reanalyzed for purity at four-
month intervals from receipt of the lot through the subchronic test.

        3.9.2  Each batch of chemical to be used for the chronic test
shall be analyzed for purity two weeks prior to initiation of the test,
during the test at three, six, twelve, and eighteen months, and within
two weeks after sacrifice of the last treatment group.

        3.9.3  If a new lot of chemical must be used after beginning of
the chronic test, it shall be analyzed immediately, and thereafter at
the same times the initial batch would have been analyzed.

        3.9.4  Any significant change in purity or appearance of the
test chemical shall be reported to Program Management immediately via
telephone by the Principal Investigator (6.2).

   3.10  Stability and Storage

        3.10.1  Stability (bulk and solution) and storage parameters for
each test chemical - with respect to temperature, light, air, and moisture  -
                                     63

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   Carcinogen Bioassay Program                   Specification No. CBM-17
Subject: CHEMICALS FOR TESTING IN THE
CARCINOGEN BIOASSAY PROGRAM
Date:
Sheet
5
Of
8
shall be determined prior to its bioassay by the Analytical Subcontractor
(6.1, 6.2, 6.3).

        3.10.2  Light-sensitive chemicals shall be stored in the dark
in amber bottles.  All work with such chemicals shall be performed in a
darkened room with filters to exclude ultraviolet light.

        3.10.3  Bulk stability shall be determined at 0°C, 25°C, and 60°C
for periods up to two months; and decomposition shall be followed by
analytical techniques (6.1, 6.2, 6.3).

        3.10.4  Each test chemical shall be handled and stored by the
bioassay laboratory in accordance with directions provided by the Analytical
Subcontractor (6.2, 6.3).

   3.11  Purification (6.3)

        3.11.1  Chemicals requiring purification prior to the bioassay
shall be subjected to treatment appropriate for the chemical nature of the
mixture and required purity of the test chemical.

        3.11.2  The following techniques are to be used singly or in
combination:

             o  Crystallization
             o  Preparative Chromatography
                  o  Thin-Layer - For all but highly volatile compounds
                  o  Column - Preliminary method for solid compounds
                  o  Vapor Phase - Volatile products
                  o  High-Pressure Liquid - Non-volatile polar compounds
                  o  Spinning Band Distillation - Volatile products
                  o  Zone Refining - Solids
                  o  Sublimation - Solids

        3.11.3  Following purification, the test chemical shall be analyzed
as indicated in 3.8.

   3.12  Disposal of Residual Chemicals (6.2)

        3.12.1  All test chemicals shall be retained by the bioassay
laboratory until directed by Program Management to ship the materials to
the Analytical Subcontractor.

        3.12.2  All chemicals shall be packaged and shipped in accordance
with  (5).
                                      64

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    Carcinogen Bioassay Program                     Specification No. CBM-17
Subject, cHgHjc^s FOR IESXHJG IN THE
CARCINOGEN BIOASSAY PROGRAM
4. Quality Control

Date:
Sheet
6
Of
8

    4.1  Bioassay tests shall be performed only with chemicals which meet
 project identity, purity, and stability standards as indicated by the
 Analytical Subcontractor's testing results.

1    4.2  The supplier shall certify that all samples submitted to Program
 Management were collected in accordance with project specifications.

    4.3  Sample Storage, Labeling and Records

         4.3.1  All samples shall be logged in upon receipt with the
 following information:  log number, identification of material, purchase
 order number, manufacturer, date.

         4.3.2  Log number and shelf-life expiration date shall be added to
 the manufacturer's label on all containers.

         4.3.3  The Quality Control Supervisor shall make certain that all
 test chemical samples are stored in accordance with recommendations of
 the manufacturer and Analytical Subcontractor.

I    4.4  Identification and Quantitation

         4.4.1  Samples which do not meet all project identification
 criteria shall be considered unacceptable for bioassay testing.

         4.4.2  Quantitative assays in which reference standard results
 differ by more than 10% from the certified value shall be considered
 invalid and must be repeated.

         4.4.3  Samples shall be rejected if:

              4.4.3.1  Percentage of main ingredient differs from project
 specifications by 10 or more percent.

              4.4.3.2  Impurities, other than those indicated acceptable
• in project specifications, are found.

              4.4.3.3  Any contaminant exceeds the maximum acceptable
 concentration according to project specifications.

":             4.4.3.4  The Analytical Subcontractor deems that the sample
 Scan be satisfactorily purified to meet project specifications.  The sample
  ly be accepted provisionally under these conditions.
                                      65

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   Carcinogen Bioassav Proeram                   Snecification No. CBM—17
Subject:
CHEMICALS FOR TESTING IN THE
CARCINOGEN BIOASSAY PROGRAM
Date:


Sheet

7
Of

8
        4.4.4  All assays of purified samples, stability tests and
reanalyses shall be subjected to the same controls indicated in 4.4.1 -
4.4.3 above.

        4.4.5  All identification and quantitation review results and
actions shall be recorded in the Quality Control Record Book and signed
by responsible personnel.

   4.5  Storage Control

        4.5.1  Storage areas for test chemicals shall be equipped with
automatic temperature and humidity regulators connected to an automatic
alarm system.

        4.5.2  The Quality Control Supervisor shall make certain that all
environmental storage parameters (3.10.1) are checked periodically and that
any indicated adjustments are made promptly.

        4.5.3  All outdated test chemicals shall be withdrawn and disposed
of as indicated by Program Management (3.12).

   4.6  Equipment Control

        4.6.1  All equipment shall be inspected at intervals recommended by
the manufacturer.  Cleaning and all other stipulated maintenance operations
shall be performed as scheduled.  Defects shall be repaired properly.

        4.6.2  Precision instruments shall be recalibrated at intervals  and
by procedures, recommended by the manufacturer.

        4.6.3  All inspections, maintenance operations, and recalibrations
shall be recorded in the Quality Control Record Book and signed by the
responsible personnel.

   4.7  Reagents Control

        4.7.1  Packing slips accompanying all reagent shipments shall be
examined for conformance with project specifications.  Reagents which
differ significantly from project specifications shall be rejected.

        4.7.2  All reagents shall be performance tested upon receipt
and at stated intervals during storage.  Reagents giving substandard
performance shall be returned to the supplier or discarded.
                                      66

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   Carcinogen Bioassay Program
Specification No. CBM-17
Subject:
CHEMICALS FOR TESTING IN THE
CARCINOGEN BIOASSAY PROGRAM
Date:


Sheet

8
Of

8
        4.7.3  All results and actions involved in reagents control shall
be recorded in the Quality Control Record Book and signed by responsible
personnel.

                               5.  Packaging

   5.1  Stable carcinogens shall be packaged and shipped in accordance with
regulations of the Department of Health, Education, and Welfare for the
transportation of etiological agents (2.1).

   5.2  Unstable chemical carcinogens (corrosive, explosive, flammable)
shall be packaged and shipped according to Department of Transportation
regulations (2.2).

                          6.   Reference Documents

   6.1  Guidelines for Carcinogen Bioassay in Small Rodents, NCI-CG-TR-1,
Sontag, J.M., N.P. Page,  and U. Saffioti, National Cancer Institute, DREW,
Bethesda, Maryland, February 1976.
                                    67

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TYPO' STANDARD METHODS
OF TEST
Sheet
1
Of
2
Approved:
Proj.
Q.C.
Lab
Other
Date
    Carcinogen Bioassay Program
Specification No. CBT-1
Subject:
 PURITY TESTS ON CHEMICALS FOR STUDY IN THE NCI CARCINOGEN BIOASSAY PROGRAM
                                   1.   SCOPE

    This specification covers  tests  for  purity  on  chemicals  selected  for
 study  in  the  Carcinogen Bioassay  Program.
    None
                           2.  APPLICABLE  DOCUMENTS
                               3.   REQUIREMENTS
     3.1   Each  chemical  to be  studied  in the Carcinogen Bioassay Program
  shall be tested  for  purity prior  to  its bioassay by the  program  management
  analytical  subcontractor.

     3.2   Purity tests shall be  designed to:

          3.2.1 Confirm identity of the test chemical.

          3.2.2 Determine concentration of test agent in  bioassay batch.

          3.2.3 Characterize  each  contaminant encountered physically
                 (e.g.,  chromatographic behavior).

          3.2.4 Identify major  or  critical contaminants and,  in some  cases,
                determine percentage  of each, if requested by program
                management  (2.1).

     3.3   Purification of test chemical may be necessary in some cases.

     3.4   Chemicals will not be  released for bioassay until analytical
  results indicate that  the chemical is of sufficient purity.

     3.5   The bioassay test laboratory shall reanalyze the chronic test
  chemical batch  for purity two  weeks  prior to the start of test and at  three,
  six, twelve,  and eighteen months  during the bioassay, as  well as  within two
  weeks after sacrifice  of the last treated group.  The analytical methods
  will be supplied by  the analytical subcontractor.

     3.6  If a new batch of  chemical must be used after initiation of  the
                                     68

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   Carcinogen  Bioassay Program                Specification No.  CBT-1
Subject: PURITY TESTS ON CHEMICALS FOR STUDY
IN THE CARCINOGEN BIOASSAY PROGRAM
Date:
Sheet
2
Of
2
•HMM^BHH
 chronic test, its purity shall be tested immediately upon receipt and  there-
 after at the same times indicated for the initial batch.

     3.7  Purity analysis results shall be reported to the Principal Investi-
 gator immediately and no later than four weeks to program management.
 Reports shall include methodology and critical raw data  (spectra, chroma-
 tographic traces), analysis and interpretation of the data, and conclusions.
 The  report shall be signed by the responsible chemist.

     3.8  Any significant changes in purity of the test  chemical during  the
 bioassay shall be  reported immediately to program management via telephone
 by the Principal Investigator.

                            4.  QUALITY CONTROL

     4.1  All analytical' instruments used in purity tests on chemicals to  be
 studied in the Carcinogen Bioassay Program shall be recalibrated monthly.
 All  recalibration data shall be recorded in a bound notebook, dated, and
 signed by personnel involved.

     4.2  Standard reference samples of known purity supplied by the manu-
 facturer shall be run in parallel with test chemicals in all purity tests.

                               5.  PACKAGING

     Not Applicable

                                 6.  NOTES

     6.1  Two methods generally will be used in reanalysis for purity  of the
 test chemical.  The methods shall be pertinent to the chemical and its
 suspected degradation products.  The methods also should be complementary
 and  as simple as possible.  The purity of a volatile liquid, for example,
 might be checked by gas-liquid chromatography and a spectroscopic technique.

                           7-   REFERENCE DOCUMENTS

     7.1  Guidelines for Carcinogen Bioassay in Small Rodents,  NCI-CG-TR,
Sontag,  JM.,  N.  P.  Page,  and U.  Saffiotti,  National Cancer Institute,
National Institutes of Health,  Bethesda,  Md.,  1976.
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2.6.2  Acceptance Specifications

     The  acceptance  specification,  the  next  important  part  of  the  labora-
 tory's  quality control system,  should spell  out the sampling to be done
 on  receipt  of  the  chemical,  the analyses to  be performed, and  should  state
 the acceptance criteria.   If further  purification is required  before  use
 in  a bioassay, the purification steps should be specified.

     A  sample  should be representative  of the lot as received.  The general
 principles  of  sampling are covered  in Section 2.3 in the context of the
 larger  task of the laboratory to perform sampling and  measurement  at  all
 stages  of research.

     The  laboratory  operating protocol  should contain  specifics regarding
 the analyses to be performed at the materials acceptance stage. These will
 include:  identity of the material  (qualitative analysis),  purity, identity
 of  impurities, percent of each impurity (quantitative  analysis), and  gen-
 eral nature of unidentified impurities.  Also, it is important that pos-
 sible contaminants,  if they could have  an adverse effect on the experiment,
 be  shown  to be absent.  In identifying  the impurities  all should be char-
 acterized  physically, as by chromatographic behavior, and  the major  ones
 should  be directly identified.

     By acceptance criteria are meant the rules for accepting  or rejecting
 a lot for failure  to meet specification.  In general these  criteria are
 expressed as plus  and minus ranges  about the nominal quality beyond which
 results  are to be judged inacceptable.  These plus and minus  limits  are
 statistically  calculated confidence limits obtained from repetitive measure-
 ments of  the same  sample.

     Filter media  can be classified as  reagents (U.S.  EPA,  1973b)   The pur-
 chase specification  should include  requirements for flow characteristics,
 surface uniformity,  occurrence of pinholes,  pH, ion blanks, and light re-
 flectance or transmittance.

      Incoming  lots should be sampled and tested for measurable character-
 istics.  Attributes  sampling (for example for pin hole leaks in glass
 fiber filters) may not be describable because each filter should be examined
 before  use  in  the  field.

2.6.3  Control of Chemicals and Reagents

      The  purchase  specification or  purchase order should instruct  vendors
 to  mark individual containers and packing slips with name of material,
 vendor's  name  and  address, vendor's lot number, quantity, and  material
 specification  number and date.

      Upon receipt, the package marking  or packing slip should  be checked
 against the purchase order.  Discrepancies will subject the lot to re-
 jection.   If it is desired to check the validity of the certification, or
 if  intended use requires acceptance sampling and testing, it is done  at
 this time.   The material is then logged in.   The log sheet  should  have

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the following columns:  Assigned log number, identification of the material,
purchase order number, name of vendor, date, and disposition (accepted or
rejected).  The label of each container should be marked with the log number
and the shelf-life expiration date.  Shelf-life, particularly of biological
reagents, is usually determined by the vendor and included on the container
label.  The inventory of chemicals and reagentjs >should be checked monthly to
identify materials approaching the shelf-life expiration date.

     The disposition record may be used to establish trends in vendor per-
formance and may indicate a need to clarify specifications or change vendors.
If purity tests are made, the record of these tests may be charted providing
another opportunity to keep an eye on quality variations.  A check on qual-
ity, strength, concentration and composition of chemicals and reagents is
usually made as part of the analytical procedure as a precaution against
omissions in the acceptance procedure.

     Storage of chemicals and reagents should be under conditions to mini-
mize deterioration with time.  A first in, first out Inventory policy should
be applied.

     Reagents must be prepared and standardized with utmost care.  Written
procedures should be available in the laboratory.

     Standard solutions will require occasional restandardization.  Storage
and standardization requirements for several standard solutions are given
in Table 2.8 (U.S. EPA, 1973b).

     Labels on standard solution bottles should include chemicals used,
manufacturers, lot numbers, date of preparation, date of next standardi-
zation, standardization conditions of analysis (temperature, pressure and
humidity).

     Standard reference materials are available for many chemicals from
the National Bureau of Standards.  The availability of primary standards,
particularly of biological materials may be limited and commercial manu-
facturers must be depended upon.  Standard reference materials are used for
standardizing solutions, calibrating equipment and monitoring precision
and accuracy of measurement methods.  Supplier's recommended storage and
handling procedures should be followed.
                                     71

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              TABLE 2.8  RESTANDARDIZATION REQUIREMENTS
                         (U.S. EPA, 1973b)
          Solution
Storage       Frequency of
Requirements  Restandardization
0.02-IN  Sodium hydroxide

0.02-IN  Hydrochloric acid

0.02-IN  Sulfuric acid

0.1N     Iodine



0.11J     Sodium thiosulfate

0.1N     Ammonium thiocyanate

0.1N     Potassium bichromate

0.1N     Silver nitrate

0.1N     Potassium permanganate
Polyolefin

Glass

Glass

Amber glass
Refrigerate


Glass

Glass

Glass

Amber glass

Amber glass
Monthly

Monthly

Monthly

Weekly (open bottles)
Monthly (sealed
bottles)

Weekly

Monthly

Monthly

Monthly

Weekly
                                      72

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2.7 CONTROL OF TEST SUBJECTS


2.7.1  Control of Animal Breeding

    Quality control must begin in the breeding and production colonies.
Since in most instances, biological research laboratories purchase animals
from outside suppliers,  it is important that quality control requirements
be spelled out in purchase contracts.  The detail required in contracts
increases if the supplier does not have a good reputation for quality or if
he cannot produce evidence that he maintains an adequate quality control
program.  It is even more important that quality requirements be very
specific if the laboratory is contracting for purchase of animals (such as
primates) caught in the  wild.

    All the requirements for Good Animal Care Laboratory Practices (see
following Section and Appendix B) apply and in addition to requiring
conformance to the BLP's, the contract may specify the following taken from
a contract for supply of Sherman stock rats used by the Health Effects
Research Laboratory,  Research Triangle Park:

    •   The Contractor shall maintain a production colony under barrier
    conditions in accordance with standard Industry practices (Reference:
    Defining the Laboratory Animal, National Academy of Sciences,
    Washington, D.C., 1971).

    •   The Contractor shall re-certify the continued absence of known
    pathogens in the production colony every six (6) months for the
    duration of this contract.  Such certification shall include as a
    minimum, lists of tests used and results for the following pathogens:
    viral, PVM, Reo 3, GDVII, KRV, H-l, Use, Adeno, MHV, LCM, RCV, Sendai,
    bacterial, mycoplasma pulmonis, bordetella bronchiseptica, pseudomonal
    aeruginosa, salmonella typhimurium, coryne bacterium kutsheri,
    streptobacillus moniliformis, bacillus piliformis, and pasteurella
    pneumontropica.  In  addition, animals shall be free of arthropod and
    helminth parasites known to infect this species (rats).

    •    The Contractor  shall re-derive replacement breeding stock as often
    as necessary to maintain the quality of animals specified in this
    contract.

    •    The Contractor  shall group-house the holding stock animals with
    three to five animals per cage.  All such animals shall be held in
    stock until shipment is requested by the Project Officer or his
    designated representative.  Animals over 90 days of age shall be
    disposed of by, and  at the discretion of the Contractor.  All animals
    shall be housed in existing Contractor-owned and-operated facilities.
    All testing of animals, to ascertain their quality, shall be done by
    Contractor personnel in the Contractor's own laboratories.
                                      73-

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    The National Academy of Sciences published a series of Procurement
Specifications (Contract Clauses) for experimental animals including:

    •  Conditioned Random-Source Dogs, 1968
    •  Conditioned Random-Source Cats, 1968
    •  Kennel-Produced Dogs, 1969
    •  Colony-Produced Cats, 1969
    •  Defined Laboratory Rodents and Rabbits, 1973
    •  Defined Wild Caught Old World Monkeys.

2.7.2  Good Animal Care Laboratory Practices

    The basic references for good animal care are U.S. DHEW (1974) and
Sontag et al. (1976).  In addition, the FDA Regulations (FDA, 1976) have
had a substantial impact on thinking about improvement in non-clinical
laboratories.

    A complete set of Good Animal Care Laboratory Practices suitable for
mammalian bioassay with rodents is given in Appendix B.
                                      74

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2.8  CONTROL OF PERFORMANCE OF EXPERIMENTS

2.8.1  Quality Control Charts

     The control chart is a graphic means of analyzing data and of con-
trolling the consistency of results over time.  The basic concept on which
the control chart is based is that the random variations to which all
measurements are subject occur over short periods of time; on the other
hand, special causes of variation, for which an assignable cause may be
found, occur over relatively longer periods of time.  Therefore, control
limits are calculated from the average variation within •••11 Mt» •*
subgroups of data collected essentially at the same time.  The limits are
used to control the variation of subgroup averages over time.  This is
possible because the variance of an average is related to the variance of
the individual measurements inversely as the number of measurements
averaged:  ~              2
          s  (average) * s  (Individuals)/ n,
          and therefore, s_ » s / Jn .
                          x
If the control limits are exceeded, a signal is given that a non-random
event has occurred.

     This gives to control limits an entirely different significance than
that of confidence limits as calculated using Eq. 2.2.3.  Confidence limits
are calculated from the whole set of data and include both short-term random
variation and any longer-term nonrandom variation that may have occurred
while the data were being collected.  Confidence limits are calculated as
though all variation was random but, since an internal check of randomness
may not have been made, this may not be the case.  Usually, the variance
calculated from the whole of a set of data is larger than the variance cal-
culated by control chart techniques using the same data but arranging them
in subgroups.  Charts on which confidence limits were plotted would be use-
less for control.

     If there are no special assignable causes of variation in a set of data
the variance in the long-term should not be significantly different from the
average variance within short-term subsets of the data.  Then the measure-
ment system is said to be in a state of control.  Only random causes affect
the variance and there are no perturbations.  In the controlled state, con-
fidence limits calculated from the whole set of data should be very close
to the control limits.

     Control charts are used to prevent persistence of assignable causes of
variation, such as operator error, instrument drift, changes in reagents, or
environmental effects, by providing a visual signal when something non-random
has occurred.  If a point goes out of control (is outside the control limits)
when plotted on the control chart, action should be taken,to identify and
correct the cause.  The limits are placed (usually at plus and minus three
standard deviations from the average of the measurements) so that it is very
unlikely that a departure from the limits could have been caused by chance
alone.  Therefore, it is worth while to look for the cause of trouble every
time the measurement process goes out of control.  As originally proposed by

                                     75

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Shervhart (1931), the control chart was intended for economic control, i.e.,
effort would be spent on trying to identify assignable causes of variation
only infrequently when actually variation was only random.

     The control chart method can be used to analyze any set of data, even
small sets usually associated with biological experimentation.  It takes
larger sets of data  (small sets gathered over a period of time) to make the
control chart work well for control or for improvement of an experimental
procedure.

     One of the advantages of the control chart, which makes it attractive
for analysis as well as control of data, is that the variance on which the
limits are based is calculated using the range  (difference between the
largest and smallest number in a small set) rather than the mean square vari-
ation.  This lessens the calculation load because the arithmetic is simpler.
In addition, the control chart calculation provides a within-group/between
group comparison of variation which is easier than the formal analysis for
variance.  Thus, single factor experiments (the kind most frequently met
with in biological research) could be analyzed using the control chart tech-
nique rather than by the methods illustrated in Section 2.2.

     The selection of the small sets, or subgroups, of the data must be made
on a rational basis.  For example, it is rational to try to control measure-
ment systems by making replicate tests (two or more) on standard samples on
a periodic basis.  The control limits are based on the average variance
within the replicate subgroups and the averages of successive replications
are plotted.  The rationale is that it is worth trying to control the test
over a period of time (differences between the averages) as closely as
possible to control  the differences within the replicate subgroups.

     A generalized control chart for averages of small subgroups of data is
given in Figure 2.2.

     The central line on the chart is the grand average of all the available
data.  A minimum of  10 subgroups of data should be available before plotting
of a control chart is attempted.  It is necessary to have about 30 subgroups
before the limits can be adopted as standard control limits.

     Three standard  deviation limits  (3-sigma limits) are generally used.
The formula for the  control limits for a control chart for subgroup averages
is:

          X + A R

where X is the grand average of all the data, R is the average range of
the subgroups, and A_ is a factor for 3-sigma limits for subgroups of a given
size, available in any quality control text book.
                                     76

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IX
                    UPPER CONTROL LIMIT
                               -OBSERVED VALUES OF X
                                       S   \
/N.   S     CENTRAL LINE
                              V
            V-
                    LOWER CONTROL LIMIT
                     123456789  1O
                           SUBGROUP (SAMPLE) NUMBER

          Figure 2.2  Generalized control chart for averages.

     It is also possible to plot a control chart for ranges to  control the
variability within the subgroups (i.e., the difference among replicates).
The formula for limits for ranges are:
          Lower limit:  D-R
          Upper limit:  D.R

where R is the average range of the subgroups and D_ and D, are factors for
3-sigma limits.  These limits are plotted beLow and above  a central line
plotted at R.  They are non-symmetric about R.

     The horizontal scale on the chart is the subgroup number.   The vertical
scale is a measurement scale.

     Averages of subgroups of the data are plotted, usually in  time sequence,
so that the occurrence of a point out of control may be identified by the
time it occurred.

     A convenient format for recording of data and calculations follows:
Observations Average
Subgroup No 1 2 3 4 5 X
Range
R
-1
2
3
•
•
•
etc.
                                Totals
                      s±    —
                  SS-  X_ = IX / No. subgroups
                  SS*  R = ZR / No. subgroups

                                     77

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     The central line for an average chart is SS  .
     The c_entral line for a range chart is SS2.
     The X's from the body of the table are plotted on an average chart.
     The R's from the body of the table are plotted on a range chart.
     The number of observations per subgroup (subgroup size = n) determines
the values of A , D,, and D..  The subgroup size is usually small.  Experi-
ence has shown that subgroup  sizes from 2 to 5 are most used.

     A partial table for control chart factors follows (ASTM, 1976):

Subgroup Size (n)         A^          D.          D,           2


        2                1.880         0         3.267       1.128

        3  '              1.023         0         2.575       1.693
        4                0.729         0         2.282       2.059

        5                0.577         0         2.115       2.326

        6                0.483         0         2.004       2.534

        7                0.419       0.076       1.924       2.704
        8                0.373       0.136       1.864       2.847
        9                0.337       0.184       1.816       2.970

       10                0.308       0.223       1.777       3.078

     Values for the factor, d~, are given above because this factor is use-
ful in estimating the standard deviation from the range as follows:

          8 = R/d2

Using this relationship, the precision of a method can be calculated from
the average range of successive replicate determinations on a standard
material, as:
          P = + t I/d2

2.8.2  Assessing Laboratory Performance

2.8.2.1  Precision—

     For control of precision of results, replicate measurements on a stand-
ard  material are made periodically (e.g., daily) by the operator.  When ten
sets of replicates are available, tentative control limits for averages
(and for ranges, if desired) are calculated and an average chart is con-
structed as explained above.  The ten averages are plotted on this chart.
The limits are extended over more daily periods and an additional point is
put on the chart daily, as the tests are completed.  A point out of control
means that something unusual has occurred and that it is worthwhile to look
for a cause.  The cause may be an operator error, a change in reagents,
instrument malfunction, a change in the environment, or some other identi-
fiable change in the procedures.  If precision is to be maintained,

                                     78

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corrective actions must be taken.

     When the average chart is in control,  the control limits may be used to
express the precision of the average of replicates:

          P_ " ± A2*
           x
The precision of the method is usually expressed in terms of individual
measurements, so if the average control chart is in control,

          P = + A2R Jn

where n is 2, for duplicate daily determinations.

Relatively infrequently, something can happen to the replication of measure-
ment causing the range to be larger than usual.  If the operator is new to
the method, it may be desirable to plot the ranges of the replicates, at
least until it is evident that his skill is sufficient to warrant dropping
the range chart.

2.8.2.2  Accuracy—

     Accuracy may be controlled using spiked samples.  Percent recovery is
determined on two or more samples at periodic intervals, say daily.  Control
charts for averages (and perhaps ranges) are set up, as for control of pre-
cision.

     Action should be taken daily (or whenever percent recovery is deter-
mined) to keep this chart in control.  When the accuracy control chart is in
control, the control limits may be used to develop a statement about accuracy
of the method.  As defined in Section 1, accuracy is made up of the bias  (or
constant error) of the average of a number of measurements from the known
amount added to the spiked samples plus uncertainty of the average.  There-
fore, accuracy is better pinned down when  the number of measurements in  the
average is larger.  We would express it using all the information available
to us.  The limits on the average chart apply to averages of the periodic
replicate determinations of percent recovery.

     Using the control limits of a measurement process that is in control,
accuracy of the method is expressed by:
where n is 2 for duplicate periodic determinations and N is the total number
of measurements at hand and used to calculate R.

     It is possible, in both the precision and accuracy control chart pro-
cedure, to use subgroups of varying size.  This complicates the calculations
but it can be handled using methods available in quality control texts
(Juran, 1974; Duncan, 1965; Grant and Leavenworth, 1972).

     For further application of control charts in the environment and
related areas see U.S. EPA, 1972, 1973b; NIOSH (undated).

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2.9  INTERLABORATORY TESTING

     After a laboratory has brought its measurements under control, it is
desirable to extend the efforts at improving laboratory performance to
checking with other laboratories to see what can be done to improve agree-
ment of results between laboratories.

     This is usually done using standard samples.  These standard samples
are often prepared by a reference laboratory and distributed according to
plan among participating laboratories.  Large organizations, such as EPA,
may use one of its laboratories as the reference laboratory and send samples
to all in-house and contractor laboratories.

     Replicate results from each of a number of laboratories may be analyzed
using analysis of variance (single factor model).  Also, range control
charts can be used to compare the variance within the laboratories.  If the
within-laboratory ranges are in control, an average control chart can be
used to plot laboratory averages using the grand average of all laboratories
as the center line.  Limits would be based on the average within laboratory
ranges and the points plotted would be the laboratory averages.

     Correction of points out of control on the range charts would be the
responsibility of the individual laboratory because they represent internal
laboratory problems.

     If the average chart is out of control the reason may be different
instrumentation in the different labs, different degrees of environmental
control (i.e., temperature, humidity, etc. ), or differences in methods or
in the closeness with which standard methods are followed.  Moreover, some
labs may be out of control on the high side and some on the low.  Bringing
the laboratory community into line requires collaborative effort.  Some
causes of failure to compare well with the average may be correctable and
some not.  However, experience has proved that it is well worthwhile  to do
this kind of proficiency testing because many problems require comparison of
results from more than one laboratory.  These comparisons cannot be made
with confidence unless the laboratories involved have internal control and
there is some consistency in testing the same thing in different laborator-
ies.

     When an interlaboratory testing program results in evidence of control
between laboratories, some kind of a calculation can be made of the pre-
cision and accuracy of measurement methods in the making of interlaboratory
comparisons.  Until such is the case there must remain some doubt about
apparent differences between laboratories.
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2.10  DATA HANDLING AND REPORTS

     The quality of the output of research, the data and the reports, is
what the whole quality assurance activity is about.  By controlling all the
elements of work, assurance is given that the results are valid and scien-
tifically defensible.  At the data-handling stage, research management must
take steps to preserve the integrity of the results achieved.

     This begins with the planning for data collection using formats or
forms which are clear, complete, and designed to limit human errors of entry,
transcription and use.  Some examples of data forms in use are illustrated
in the Sections dealing with the various areas of research.  Much data is
still entered manually so that these forms should be helpful.  Increasingly,
data are collected and organized in automated systems.  These systems are
usually designed for a particular purpose in a unique laboratory situation.
The importance of good programming for such a system cannot be over-
emphasized.

     The forms for manual data entry may be loose-leaf.  However, for both
field and laboratory research programs it is highly desirable that the data
be recorded, at least originally, in bound notebooks.  It is good practice
to require strict adherence to the established custom of having the entries
in the notebook signed by the person taking the data and witnessed periodi-
cally,  preferably daily, by the supervisor.  If the experimenter is the
senior individual in the laboratory it is good practice to have the entries
witnessed by an associate.  Although this may appear to be a stricture on
the research task, it is extremely important if the work is later likely to
be subjected to any kind of litigation.

     It is desirable to have all records under control (a sort of "Chain
of custody" of records) which means that notebooks should be numbered,
issued centrally, and returned to a designated repository when filled or at
the end of a project.  There should be written instructions on the retention
period for records and how they shall be filed and stored.

     In some very large research projects, responsibility for design, con-
duct, analysis and reporting of the work may be fragmented among sponsors,
contractors, and subcontractors.  The problems of maintaining validity and
integrity of data may be amplified under such conditions but it is not the
intent of the Guidelines to address the managerial problems encountered.
Much research is still done by smaller laboratories, or groups of laborator-
ies, or by individual researchers.  The responsibility for report prepar-
ation is localized.  Formal reports should be required.  These reports
should be subjected to review within the laboratory.  If the work is to be
published, the major journals require further review by peers in the same
area of research.  One of the major requirements of good scientific work is
that it should be verifiable.  This requires that all the pertinent data
must be reported and that methodology should be well enough described so
that the experiment could be reconstructed independently.
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2.11 REFERENCES
ASTM. 1974.  Book of ASTM Standards, Part  23, Water; Atmospheric Analysis.
     American Society for Testing and Materials, Philadelphia, Pa.

ASTM. 1976.  Manual on Quality Control of  Materials.   STP 15-D.  American
     Society for Testing and Materials,  Philadelphia,  Pa.

ASTM. 1977.  Precision and Accuracy  as Applied  to Measurement of a Property
     of a Material. E 177-71.  Recommended Practice  for Use  of the Terms.
     Annual Book of ASTM Standards,  Part 41.  American Society for Testing
     and Materials, Philadelphia, Pa.

Armitage, P. 1971.  Statistical Methods  in Medical Research.  John Wiley and
     Sons, New York, N.Y.

Bennett, C. A., and N. L. Franklin.  1954.   Statistical Analysis in Chemistry
     and the Chemical Industry.  John Wiley and Sons,  New York, N.Y.

Bicking, C. A. 1968.  Sampling.  In:  The  Encyclopedia of Chemical Techno-
     logy.  2nd Edition, Volume 17.  Interscience Publishers, pp. 744-762.

Bicking, C. A. 1976.  The Sampling of Biological Materials.  In:  Proceed-
     ings of a Seminar on Sampling of Bulk Materials,  European Organization
     for Quality Control.  STAQUAREL 1976,  Prague, Czechoslovakia.

Bicking, C. A., and F. M. Gryna, Jr. 1974.  Process  Control  by Statistical
     Methods.  In:  Quality Control  Handbook.   3rd Edition.  J. M. Juran,
     F. M. Gryna, Jr., and R. S. Bingham,  Jr. (eds.).  McGraw-Hill Book Co.,
     New York, N.Y.

Bliss, C. I., and R. A. Fisher. 1953.  Fitting  the negative  binomial
     distribution to biological data.  Biometrics 9:   176-200.

Cochran, W. A., and G. M. Cox. 1950.  Experimental Designs.  John Wiley
     and Sons, New York, N.Y.

Cox, D. R. 1970.  Analysis of Binary Data.  Methuen  and Co., Ltd., London,
     England.

Cox, D. R. 1972.  Regression models  for  life tables.   J. Roy. Statist. Soc.,
     Ser. B 34:  187-220.

Duncan, J.  1965.  Quality Control and Industrial Statistics.  3rd Edition.
     Richard D. Irwin, Inc., Homewood, 111.

Eisenhart, C. 1947.  The assumptions underlying the  analysis of variance.
     Biometrics 3:  1-21.
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Finncy, D. J. 1964.  Statistical Methods in Biological Assay.  2nd Edition.
     Hafner Publishing Co., New York, N.Y.

Fisher, R. A. 1925.  Theory of statistical estimation.  Proc. Cambridge
     Phil. Soc. 22:  700-725.

Fisher, R. A. 1947.  The Design of Experiments.  4th Edition.  Oliver and
     Boyd, Edinburgh, England.

Fisher, R. A., and F. Yates.  1949.  Statistical Tables for Biological,
     Agricultural and Medical Research.  3rd Edition.  Hafner Publishing
     Co., New York, N.Y.

FDA.  1976.  Proposed regulations for good laboratory practices.  Food and
     Drug Administration, Fed. Regist. 41  (225) :   51206-51230.  November
     19, 1976.

Geldreich, E. E.  1975.  Handbook for Evaluating Water Bacteriological
     Laboratories.  2nd Edition.  U.S. Environmental Protection Agency, EPA-
     670/9-75-006.

Grant, E. L., and R. S. Leavenworth.  1972.  Statistical Quality Control.
     4th Edition.  McGraw-Hill Book Co., Inc., New York, N.Y.

Juran, J. M.  1974.  Quality Control Handbook.  3rd Edition.  J. M. Juran,
     F. M. Gryna, Jr., and R. S. Bingham, Jr. (eds.).  McGraw-Hill Book Co.,
     Inc., New York, N.Y.

Kaplan, E. L., and P. Meier.  1958.  Nonparametric estimation from incomplete
     observations.  J. Amer. Statist. Ass.  53:457-481.

Kempthorne, 0.  1952.  The Design and Analysis of Experiments.  John Wiley
     and Sons, New York, N.Y.

Miller, R. G., Jr. 1966.  Simultaneous Statistical Inference.  McGraw-Hill
     Book Co., Inc., New York, N.Y.

National Bureau of Standards.  1977.  The International System of Units
     (SI).  NBS Special Publication 330.  U.S. Department of Commerce.

NIOSH.  Industrial Hygiene Service Laboratory Quality Control Manual,
     Technical Report No. 76.  National Institute for Occupational Safety
     and Health (NIOSH), Division of Laboratories and Criteria Development,
     Cincinnati, Ohio.

Rand, M. C., A. E. Greenberg, and M. J. Taras (eds.). 1975.  Standard
     Methods for the Examination of Water and Wastewater.  14th Edition.
     American Public Health Association, American Water Works Association,
     and Water Pollution Control Federation.  Washington, D.C.

Shervhart, W. A. 1931.  Economic Control of the Quality of Manufactured
     Products.  Van Nostrant, New York, N.Y.

                                     83

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Slack, K. V., R. C. Averett, P. E. Grewson, and R. G. Lipscomb.  1973.
     Methods for Collection and Analysis of Aquatic Biological and
     Microbiological Samples.  Book 5.  U.S. Department of the Interior,
     Washington, D.C.

Sokal, R. R., and F. J. Rohlf.  1969.  Biometry, The Principles and Practice
     of Statistics in Biological Research.  W. H. Freeman and Co., San
     Francisco, Calif.

Sontag, J. M., N. P. Page, and U. Saffiotti.  1976.  Guidelines for
     Carcinogen Bioassay in Small Rodents.  U.S. Department of Health,
     Education and Welfare, NIH 76-801.

Tarone, R. E. 1975.  Tests for trend in life-table analysis.  Biometrika
     62:679-682.

U.S. DHEW.  1974.  Guide for the Care and Use of Laboratory Animals.  U.S.
     Department of Health, Education and Welfare, NIH 74-23.

U.S. EPA.  1972.  Handbook for Analytical Quality Control in Water and
     Wastewater Laboratories.  U.S. Environmental Protection Agency,
     Analytical Quality Control Laboratory, Cincinnati, Ohio.

U.S. EPA.  1973a.  Biological Field and Laboratory Methods for Measuring the
     Quality of Surface Waters and Effluents.  C. I. Weber  (ed.)-  U.S.
     Environmental Protection Agency, EPA-670/4-73-001, Cincinnati, Ohio.

U.S. EPA.  1973b.  Quality Control Practices in Processing Air Pollution
     Samples.  U.S. Environmental Protection Agency, APTD-1132, Research
     Triangle Park, N.C.

U.S. EPA.  1974a.  Pesticide Residue Analysis in Water:  Training Manual.
     U.S. Environmental Protection Agency, EPA-430/10-74-012.

U.S. EPA.  1974b.  Methods for Chemical Analysis of Water and Wastes.  U.S.
     Environmental Protection Agency, EPA-625/6-74-003, Cincinnati, Ohio.

U.S. EPA.  1975.  Model State Water Monitoring Program.  U.S. Environmental
     Protection Agency, EPA-440/9-74-002.

U.S. EPA.  1976.  Handbook for Sampling and Sample Preservation of Water and
     Wastewater.  U.S. Environmental Protection Agency, EPA-600/4-76-049,
     Cincinnati, Ohio.

U.S. EPA.  1978.  Procedure for the Evaluation of Environmental Monitoring
     Laboratories.  U.S. Environmental Protection Agency, EPA-600/4-78-017,
     Cincinnati, Ohio.

U.S.  Geological  Survey.  1973.  Methods for Collection and Analysis of
     Aquatic Biological and Microbiological Samples.  Techniques for Water
     Resource  Investigations of the USGS, Book 5, Chapter A4.  U.S. Govern-
      ment Printing  Office, Washington, D.C.

                                     84

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Yates, F.  1937.  Design of Analysis of Factorial Experiments.  Int. Bur.
     Soil Sci., Harpenden, England.
                                     85

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                                 SECTION 3

                 QUALITY ASSURANCE IN BIOLOGICAL RESEARCH

3.1  LABORATORY MANAGEMENT

     The character of laboratory management has a strong effect on the qual-
ity of work produced.  This is true no matter what kind of laboratory it is.
For groups of closely related laboratories, such as biological research
laboratories, management requirements in various fields of research differ
only in detail.  After the general aspects of good management have been dis-
cussed, the details can be covered field by field.

     One of the important concepts of statistical quality control is that
the causes of quality problems may be categorized in two ways.  One class
of causes is that which is within the ability of the individual worker to
prevent or correct.  The second class is that which is within the capability
or authority of management only to handle.  Data analysis may be structured
so as to assist in separating and identifying these two classes of causes of
quality problems.

     The first class of causes is called "special" causes; the second class,
"common" or "environmental" causes.  See Blcking and Deming   (1971) for a
discussion of the use of this concept in industry.  Availability of appro-
priate data for analysis of this type may not yet be characteristic of most
biological research laboratories.

     Even before the analysis of data, however, there are certain areas
easily identified as being of concern to management.  See Table 3.1.1.

3.1.1  On-site Evaluation/Accreditation

     The purposes of on-slte evaluation include the use of results as a
management tool for improving performance of the laboratory and, on the
more formal side, for accreditation.  Evaluation may be used as a prelude
to including a laboratory in a study program or to employing the laboratory
on a contract basis.  In particular instances requiring evaluation, accredi-
tation by a recognized organization is usually accepted as evidence of the
laboratory's capabilities without further evaluation.  Accreditation systems
include provisions for periodic renewal of the accreditation status.

     The evaluation may be conducted by a peer scientist group using more or
less formalized check lists, or it may Involve a sophisticated rating system.
Self-evaluation may be involved, or evaluation by a governmental or independ-
ent authority.


                                    86

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     TABLE 3.1.1  ELEMENTS OF LABORATORY MANAGEMENT AND QUALITY CONTROL
Management Element
Quality Control
Facilities

  •  Building
  •  Services
  •  Equipment

Personnel

  •  Project director
  •  Project personnel
  •  Support personnel

Test subjects/materials

Standard procedures

  •  Bioassay protocol

  •  Conduct of experiment

     o  Observations/test methods
     o  Good laboratory practices
     o  Supervision
     o  Quality control

  •  Audit

Record keeping

Data analysis/reporting
•  On-site evaluation accredi-
    tation
•  Certification
•  Sampling and testing
•  Design review/statistical
    consultation
•  Standard test
•  Standard procedures
•  Quality policy
•  Defined program
•  Witnessed log books

•  Statistical treatment
     The resources for evaluation available to the biological research
community are varied, if not complete.  More attention to evaluation has
probably been given in clinical laboratories than in nonclinical  (animal
research) laboratories.  While much can be learned from experience in eval-
uating clinical laboratories the clinical aspects are only a small part of
evaluation of biological laboratories In general.

3.1.1.1  General Criteria for Laboratory Evaluation—

     General criteria for laboratory evaluation have been promulgated by  the
government and by standardization agencies.  In 1974, OSHA conducted hear-
ings on proposed criteria for laboratory accreditation  (OSHA, 1974).
                                     87

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Subsequently the responsibility for developing criteria was transferred from
the Labor Department to the Commerce Department, and the National Bureau of
Standards developed a plan for centralized administration of laboratory
certification.  This was published in the Federal Register of February 25,
1976 (Office of the Secretary of Commerce, 1976).  It provides for incorpora-
tion of existing certification/accreditation programs under a national
umbrella and for the establishment of new programs in uncovered areas of
science or technology by the professional societies or other organizations.
It may eventually include a national certification program for biological
laboratories (nonclinical laboratories).

     The American National Standards Institute (1971) has adopted laboratory
qualification guidelines for use in its certification programs.  Also,
Committee E-36 of the American Society for Testing and Materials (1977)
approved a Standard Practice for General Criteria for Use in Evaluation of
Testing and/or Inspection Agencies.

     Important evaluation programs in nonclinical laboratories include:

          •  FDA Good Laboratory Practice Pilot Program, FY77
          •  American Association for Accreditation of Laboratory
             Animal Care Procedure
          •  EPA Procedure for Evaluating Water Bacteriological
             Laboratories, 1975 (plus state programs)

3.1.1.2  FDA Good Laboratory Practice Pilot Program—

     FDA investigations had shown evidence of significant quality control
problems in some nonclinical laboratories (FDA, 1976a).  Such problems
included, but were not limited to:

          •  poorly conceived, carelessly executed, inaccurately
             analyzed or reported experiments
          •  lack of awareness on the part of technical personnel
             of the importance of protocol adherence
          •  inaccurate observations, record keeping and record
             transcription
          •  failure of management to assure critical review of
             data or proper supervision of personnel
          •  use of poorly qualified or poorly trained personnel
          •  disregard for proper laboratory, animal care, and
             data management procedures
          •  failure to monitor studies performed in whole or in
             part by contract laboratories
          •  lack of verification of the accuracy and completeness
             of scientific data
          •  deliberate falsification of data by management and/
             or laboratory personnel
                                     88

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     These findings led to the issuance of the Compliance Program Guidance
Manual (FDA, 1976a) and the Proposed Regulations for Government Laboratory
Practice (FDA, 1976b).  The Compliance Program directs a pilot effort of
inspections of nonclinical laboratories.  It is designed to ensure the qual-
ity and integrity of the bioresearch data which support the safety of
Agency-regulated products.

     The program includes completion of a nonclinical Laboratory Inspection
Report for each laboratory visited and a Test System Study Report for one or
more studies being conducted in the laboratory.


3.1.1.3  Accreditation of Animal Care Laboratories—

     Laboratories caring for and using experimental animals may be accredited
by the American Association for Accreditation for Laboratory Animal Care
(AAALAC).  AAALAC uses the Guide for the Care and Use of Laboratory Animals,
DREW Publication No.  (NIH) 74-23 (National Research Council, 1972) as its
primary reference for determining eligibility for accreditation.  These
recommendations have been further refined and more rigid or specific stan-
dards applied when necessary for carcinogen bioassay in Guidelines for
Carcinogen Bioassay in Small Rodents (National Cancer Institute, 1976.)
Animal care is discussed in detail in another section.

     Facets of the laboratory animal care program which are evaluated by
AAALAC include:

          •  Laboratory Animal Management

             o  Housing and care
             o  Sanitation practices
             o  Feeding, watering, and identification of laboratory
                animals
             o  Provisions for emergency care

          •  Laboratory Animal Quality and Health

             o  Adequate veterinary care
             o  Quarantine and isolation of animals
             o  Separation by species
             o  Diagnosis, treatment, and control of animal diseases

          •  Personnel

             o  Professional personnel
             o  Animal care personnel
             o  Personal hygiene and personnel health program
                                     89

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          •  Use of Laboratory Animals

             o  Monitoring the use and care of animals
             o  Anesthesia and analgesia
             o  Surgery and postsurgical  care
             o  Euthanasia

     Institutions seeking to participate  in the accreditation program may
obtain an application from the AAALAC office at 2317 West Jefferson Street,
Joliet, Illinois  60435.  A site visit is subsequently made by two repre-
sentatives of the AAALAC Council on Accreditation.  Individuals who have
extensive expertise and experience in laboratory animal science are selected
as consultants for the accreditation program.  The site visitors make a
complete and thorough review of all aspects of the animal care program
carried out at the institution being evaluated.  A detailed report is sub-
mitted to the Council on Accreditation, and after thorough review, recommen-
dations of the Council are forwarded to the Board of Trustees for action.
Following this, a detailed report is sent to the applicant institution out-
lining the decision taken and providing a detailed analysis of the program.
Every effort is made to provide a thorough and comprehensive review of all
programs under evaluation.  In essence, the entire program closely follows
the review processes which have been developed by granting agencies for
evaluating the merits of grant applications.

     Through the accreditation program, institutions have been able to
document their deficiencies and respond to them.  These deficiencies vary,
but in 1973 an analysis of the deficiencies encountered in the AAALAC pro-
gram which were serious enough to warrant not accrediting the institution
was made.  Examples of deficiencies listed in the order of prevalence are:
     Improper sanltization
     Caging of insufficient size or design
     Improper quarantine and isolation
      program
     Improper environmental control
     Improper sanitary waste disposal
     Personnel deficiencies
     Inadequate physical plant.conditions
     Inadequate control of animal ftfieases
     Inadequate personnel health program
     Feeding and watering deficiencies
     Inadequate emergency procedures
Inadequate animal surgery
 and postsurgical care
 program
Inadequate storage space
Inadequate vermin control
 program
Overcrowding of animals
Administrative problems
Inadequate illumination
Inadequate identification
 procedures
Inadequate euthanasia
 practices
     Approximately  70% of the institutions that did not gain accreditation
 after  the  first  site visit ultimately improved their animal care program to
 an  accreditable  level.

 3.1.1.4 Evaluation of Water Bacteriological Laboratories—

     The Municipal  Environmental Research Laboratory of EPA, at Cincinnati,
 has published a  Handbook for Evaluating Water Bacteriological Laboratories
                                     90

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 (U.S. Environmental Protection Agency, 1975).  Here again, many of  the
 sections are applicable to many kinds of laboratories.  Of particular
 interest are the guidelines, or check lists, which accompany each chapter.

     An example, the Guidelines on Laboratory Management, is reproduced  in
 Figure 3.1.1.
Laboratory Records

Results assembled and available for inspection
Data processed rapidly through laboratory and engineering
     sections
Adequate data retention, efficient filing system, and prompt
     channeling of report copies
Number of tests per year
     MPN Test - Type of sample	
          Confirmed (+)	(-)	(Total),
          Completed (+)	(-)	(Total)]
     MF Test - Type of Sample	
          Direct Count (+)	(-)	(Total)
          Verified Count (+)	(-)	(Total)"
Personnel

Adequately trained or supervised for bacteriological examination
     of water	
Personnel involved:
     Professional staff (total)	
     Sub-professional support (total)
     Clerical assistance (total)	
Reference Material

Copy of Standard Methods (current edition) available in the
     laboratory
State or Federal manuals on bacteriological procedures available
     for staff use
Scientific journals in water research accessible

Laboratory Facilities

Laboratory room spaced and bench-top area adequate for needs
     during peak work periods
Prep room space adequate and located near laboratory
Sufficient cabinet space for media, chemicals, glassware,
     and equipment storage
Facilities clean, with adequate lighting and ventilation, and
     reasonably free from dust and drafts
                                                                  (Continued)
            Figure 3.1.1  Guidelines on Laboratory Management.


                                     91

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Office space and equipment available for processing water
     examination reports and mailing sample bottles

Laboratory Safety

Personnel and carts permitted mobility without obstructions
     that cause accidents
Adequately functioning autoclaves and stills, with periodic
     inspection and maintenance
Electrical service conforms to local, state or National
     Electrical Codes
All electrical equipment grounded through three-wire system
     or separate ground to cold water pipe
Foam-type and carbon dioxide fire extinguishers accessible
Fire exits from laboratory clear at all times
Emergency (deluge) shower accessible and functional
Safety features such as pipet waste jars with disinfectant,
     centrifuge shield, splatter guard, and blender covers
     employed to avoid bacterial aerosols
Approved practices for handling and disposing of radio-
     active chemicals used in special bacteriological
     procedures
First aid supplies available and not out-dated
Personnel trained to safely handle steam, flames, chemicals,
     pathogens, etc.
Personnel indoctrinated in first aid emergency procedures,
     fire control, etc.
Broken glass, sharp needles, etc., properly handled and
     disposed of
                           Figure  3.1.1   Continued
      Other sections  of  the  Handbook contain guidelines  on  specific  labora-
 tory activities,  as  follows:

              Sampling and monitoring response
              Laboratory apparatus
              Glassware, metal utensils and plastic  items
              Laboratory materials  preparation
              Culture media  specifications
              Multiple tube  coliform procedures
              Membrane filter  coliform procedures
              Supplementary  bacteriological methods
              Reports

      A number of states conduct laboratory certification programs:

           •  Connecticut State Department of Health,  Laboratory
              Standards  Section, approves water  laboratories
           •  New York State Department of Health, Division of
              Laboratories and Research Programs,  approves

                                      92

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             laboratories analyzing potable water
          •  North Carolina State Department of Natural and
             Economic Resources certifies air and water
             analytical laboratories
          •  Oklahoma State Water Resources Board certifies
             water and wastewater laboratories.
          •  California State Department of Health, Water
             Resources Board licenses water laboratories

3.1.1.5   Accreditation of Industrial Hygiene Laboratories—

     The American Industrial Hygiene Association in association with the
Health Physics Society accredits laboratories based on criteria under the
following headings:

             Laboratory direction
             Laboratory supervision
             Laboratory personnel
             Proficiency testing
             Quality control and equipment
             Facilities
             Records

     The proficiency testing is carried out by NIOSH under their PAT
(Proficiency Analytical Testing) Program.  Satisfactory performance is
based on a statistical estimation of whether the results obtained are
probably representative of analytical competence.

     Quality Control procedures considered essential include:

          •  Routinely Introduced samples of known content
             along with samples submitted for analysis
          •  Routine checking, calibrating and maintaining
             adequate performance of equipment and instruments
          •  Routine checking of procedures and reagents
          •  Good housekeeping, cleanliness, and general
             orderliness of premises

3.1.1.6  Programs of Clinical Laboratories—

     In the clinical laboratory area, The College of American Pathologists
conducts a Laboratory Inspection and Accreditation program that has many
interesting aspects.  Each laboratory seeking accreditation must be enrolled
in the CAP Proficiency Testing Program (Surveys).  Accreditation is renew-
able every two years.  A computer-processed check list is provided for self-
evaluation in the interim year.  Other services offered by CAP include a
Quality Assurance Service, computerized tabulations, plots, and analyses of
a laboratory's daily quality control data; a Proficiency Evaluation Program
(PEP) with self-evaluation testing kits for physicians; and a Product
Evaluation Program for suppliers of laboratory products.
                                     93

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     The Center for Disease Control conducts a laboratory licensing program
under the Public Health Service Act as amended in 42 USC 20 et seq.  Partic-
ipation in a proficiency testing program is required of all laboratories
covered under the act.  See Center for Disease Control, 1975 for a descrip-
tion of the proficiency testing program.  All laboratories having acceptable
results in the program are classed as "licensed" laboratories.  The follow-
ing areas of testing are covered:

          •  Microbiology and serology
          •  Clinical chemistry
          •  Hematology
          •  Immunohematology
          •  Radiobioassay

     In December 1973, the Technical Analysis Division of the Institute for
Applied Technology at the National Bureau of Standards published a study of
results of the CDC Proficiency Testing Program  (National Bureau of Standards,
1973).  This is an interesting assessment of the value of proficiency test-
ing.

3.1.1.7  Significance of On-site Evaluations—

     Acceptable ratings as a result of on-site evaluation usually infer
capability of the laboratory for doing a satisfactory job.  The rating it-
self, or even resulting, certification or accreditation, does hot necessarily
mean that performance by the laboratory will be everything that could be
desired.  That is the reason why most programs for  laboratory evaluation
add a requirement for testing of split samples  (proficiency testing).
Successful identification of samples in a collaborative proficiency testing
system increases the confidence that can be placed  in a laboratory's work.

     Some of the evaluation systems described are administration rather than
operation oriented.  For example, the FDA system is keyed to the Proposed
Regulations for Good Laboratory Practices.  Although casually referred to
as GLP's, these are regulations only and do not contain explicit procedures
for conduct of experiments or making of observations or tests.  The NCI
Guidelines  (NCI, 1976) on which most animal research laboratory evaluation
is based are looked upon properly as guidelines and not standards.  The EPA
procedures for monitoring laboratories and for water bacteriological labo-
ratories are much more explicit as to equipment requirements, test methods,
and operating procedures.  Before the quality of a  laboratory's results can
be improved, much more direction must be given to it in the form of material
specifications, standard test methods, good techniques of experiment design,
standard operating procedures and quality control techniques.  It is identi-
fication of and characterization of the effectiveness of such specific
operating procedures  that really makes on-site evaluation significant in
improving  laboratory  operations.

 3.1.2  Laboratory Personnel

      The study director  and principal personnel to  be associated with a
 study should be identified prior to the start.  This provides an opportunity

                                     94

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for review of appropriateness of the staff.  Obviously, the study director
should be an expert in the area of the study.  He, or other personnel in
supervisory positions should be well qualified by academic training and
experience.  The specific qualifications should be spelled out in job de-
scriptions. Where experience is lacking, in-house training, perhaps on a
continuous basis, is desirable.

     For some disciplines involved in biological research, certification
programs are available.  For example, veterinarians who are needed to con-
duct broad-based laboratory animal preventive medicine programs are certi-
fied by the American College of Laboratory Animal Medicine.  This program
would include screening representative numbers of animals, microbiologically
and virologically, gross and microscopic evaluation of necropsy specimens,
and other tests.  Depending upon the animal species, one or more of the
above should be accomplished as often as necessary to ensure that only ani-
mals of the required quality are placed on experiments.

     Quality assurance also includes staffing animal care facilities with
properly trained personnel.  The American Association for Laboratory Animal
Science has established national testing standards and there are three skill
levels currently recognized.  Training programs may be geared to certifying
technicians under this program.

3.1.3  Biological Sampling and Testing

3.1.3.1  Biological Tests

     Some of the common techniques of analysis In biological research, with
particular reference to the water environment, are given in Table 3.1.2.

     These techniques are described briefly as follows:

          •  Count and identification - A useful test to determine overall
             the health of species in an ecosystem by providing data on
             standing crop and community structure
          •  Weight/length - The growth rate of a community is determined
             and compared to previous studies to indicate a change in
             environmental quality
          •  Flesh tainting - A test of palatability to determine if
             sublethal chemical doses have Imparted an unpleasant taste
             to fish or shellfish flesh
          •  Acetylcholinesterase - An indirect test of the previous
             effect of organophosphate pesticides on the central nervous
             system of fish in a water system
          •  Tissue analysis - A qualitative or quantitative test of
             concentration of histological effects of various materials
             including metals and pesticides in flesh
          •  Stomach contents - An analysis of this will Indicate the
             type and amount of feeding done by an organism prior to
             collection
          •  Wet, dry and ashfree weight - These tests are used to make
             quantitative tests of the standing crop of a population

                                     95

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             TABLE 3.1.2  PARAMETERS OF BIOLOGICAL COMMUNITIES
                 MOST COMMONLY ANALYZED   (U.S. EPA, 1976)
   Community
    Parameter
         Units
Plankton
Periphyton
Macrophyton
Fish
Counts

Chlorophyll a
Biomass (ash-free,
 dry weight)

Counts
Chlorophyll a
Biomass (ash-free
 weight)
Autotrophic index
Areal coverage

Biomass (ash-free
 weight)
Macroinvertebrate  Counts
Biomass
Toxic substances

Toxic substances
Counts
                    Biomass  (wet weight)
                    Condition
Numbers/ml by genus and/or
 species
mg/m^
mg/m ^
Number/mm2
mg/m2

mg/m2
Ash-free weight (mg/m2)
 Chlorophyll a (mg/m2)

Maps by species and species
 associations

g/m2

Grab - number/in2
Substrate - number/sampler
g/m2
mg/kg

mg/kg
Number/unit of effort, expressed
 as per shocker hour or per 100
 feet of a 24-hour net set
Same as counts
        105 x weight in grams
                                         K(TL)
                                                  L3  (length in mm)
           •   Chlorophyll  a  - An  estimate  of  the algal  biomass  is  obtained
              which roughly  indicates  the  standing  crop
           •   ATP determinations  -  ATP tests  measure  the total  viable
              plankton biomass
           •   Diatom species proportional  count  - This  test indicates the
              health of a  diatom  community by comparing the results
              through the  use of  a  diversity  index

 3.1.3.2  Sample Preservation and Handling—

      Sample preservation  is distinctive for  each area  of biological research
 and for each parameter to be measured.  When a  chemical preservative is used,
 extreme agitation may be  necessary to disperse  the chemical preservative
                                      96

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throughout the sample.  If the preservative cannot be dispersed, refriger-
ation or freezing may be an appropriate alternative.

     Various preservatives exist to maintain species in the desired condi-
tion.  Advantages and disadvantages of various preservatives are given in
Table 3.1.3.

     Preservation and handling procedures are given in Table 3.1.4 for:

             Benthic Macroinvertebrates
             Fish
             Macrophytes and Macroalgae
             Periphyton
             Periplankton
             Zooplankton

3.1.4  Preparation of Study Protocols

     A general outline for a bioassay protocol is as follows:

          •  Purpose of study
          •  Design of experiment
          •  Conduct of experiment
          •  Observations and tests
          •  Records and reports

3.1.4.1  Purpose of Study—

     There should be a brief, direct statement of the purposes of the study.
For example, the purpose of a chronic feeding study using rats might be:

          •  Effects of test material on the reproduction
             process in rats

             o  Fertility
             o  Maintenance of offspring
             o  Postpartum effect
             o  Weaning period

          •  Chronic toxicity of test materials

          •  Carcinogenesis due to exposure during organogenesis,
             fetal development, location, and throughout life.
                                     97

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             TABLE 3.1.3  COMPARISON OF CHEMICAL PRESERVATIVES
      FOR BIOLOGICAL PARAMETERS  (SLACK ET AL., 1973; U.S. EPA, 1976)
    Chemical
    Advantage
   Disadvantage
General Preservation

1. Formalin
    (5-10% for-
    maldehyde)

2. 70% ethanol
3. 40% isopropanol
4. Oxyquinoline  (2%
    solution)(8-hydroxy-
    quinoline sulfate)
5. Merthiolate
    solution
6. Glycerin  (added
    with 1,  2 or  3)

7. Copper  sulfate
 8. Detergent
Kills species;
 infinite holding
 period
Safer and easier for
 analyst to use; same
 advantages as formalin

Safer and easier for
 analyst to use; can
 be added as solid
 in premeasured pack-
 ets; same advantages
 as formalin

Morphology and color
 of algae are retained;
 distinguish between
 zoo- and phytoplankton

Prevents tissues
 from drying

Retains bluegreen
 color of algae

Lowers surface tension
 to prevent clumping
 or clinging to con-
 tainer walls
Objectionable odor,
 can cause contraction
 or deflaggelation

Needs neutralization
 w/sodium tetraborate

Can cause contractual
 reaction
Can cause contractual
 reaction
Does not produce a
 sterile sample
Stains other material;
 also toxic
 Stains
 9.  Lugols's solution
Stains algae; aids
 settling by releasing
 gases
Samples stable only
 one year
                                      98

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             TABLE 3.1.4  RECOMMENDED PRESERVATION AND HANDLING METHODS (U.S. EPA, 1976)
Item
Preservation Method
Holding Time
Container
BENTHIC MACROINVERTEBRATES
 Count/identification

 Wet and dry weight

 Ash-free weight


 Calorimetry



 Radio-tracer studies

 Flesh tainting

 Tissue analysis

FISH

 Count/ident if ica t ion


 Weight/length

 Flesh tainting



 Acetylcholinesterase
70% ethyl alcohol

Refrigerate at 4°C or ice

Filter and refrigerate at
 4°C

Refrigerate at 4°C or ice.
 Once filtered, store in
 desiccator

Freeze

Freeze

Freeze
1 year

Immediate to 24 hours

6 months
Glass or plastic

Glass or plastic

Glass or plastic
Immediate to 24 hours    Glass or plastic
1 year

Indefinite

Indefinite
10% Formalin, add 3 g borax     Indefinite (1 year;
 and 50 ml glycerin per liter*   sooner is better)
None - analyze immediately

Clean, then freeze



Freeze
None

Indefinite



Indefinite
Glass or plastic

Glass or plastic

Glass or Plastic
Borosilicate glass
 or polyethylene

None

Borosilicate glass
 or polyethylene
Aluminum foil

Aluminum foil
                                                                                           (continued)

-------
                                       TABLE 3.1.4  (Continued)
      Item
                         Preservation Method
Holding Time
 Container
o
o
       Tissue analysis
       Stomach contents
                         Freeze
                         Remove stomach from fish
                          and preserve in 10%
                          Formalin  (as for count/
                          identification)
MACROPHYTES AND MACROALGAE

 Count/identification    5% Formalin

 Wet and dry. weight      Refrigerate at 4°C or ice

 Ash-free weight         Freeze

 Chlorophyll a           Freeze at -20°C


PERIPHYTON

 Count/identification    5% neutral Formalin
      Diatom species  pro-
       portional  count

      Wet and dry weight
      AslHEree weight
                         5% neutral Formalin
                         Refrigerate at 4°C or ice
                          (do not freeze)

                         Freeze at -20°C
Indefinite
Indefinite (1 year,
 prefer sooner)
1 year

Immediate to 24 hours

6 months

1 month (keep out of
 light; acid)



6 months
 Borosilicate  glass
 or  polyethylene
 Aluminum  foil

 Glass or  plastic
Glass or plastic

Glass or plastic

Glass or plastic

Glass or plastic
                                                                                       Opaque glass or
                                                                                        plastic
6 months to indefinite   Glass or plastic
Immediate to 24 hours    Glass or.plastic
6 months
Glass or plastic

         (continued)

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                                   TABLE 3.1.4  (Continued)
Item
Preservation Method
Holding Time
Container
 Chlorophyll deter-
  mination
 ATP determination



PHYTOPLANKTON

 Count/identification


 Wet and dry weight


 Ash-free weight


 Chlorophyll a
 Diatom species
  proportional count

 Calorimetry
 ATP determination
Immediate extraction in
 90% aqueous acetone;
 store at -20°C

Extract by boiling with
 Tris Buffer; store
 extract at -20°C
a. 5% neutral Formalin
b. Merthiolate

Refrigerate at 4°C or ice
(do not freeze)

Filter and freeze at
 -20°C

Extract immediately or
 filter and freeze in desic-
 cator at -20°C

5% Formalin
Refrigerate at 4°C or ice;
 once filtered, store in
 desiccator

Extract by boiling with-Tris
 Buffer, freeze extract at
 -20°C
1 month (keep out
 of light and acid)
6 months
Glass or plastic
Glass or plastic
a. Indefinite
b. 1 year

Immediate to 24
 hours

6 months
Opaque, glass or
 plastic

Glass or plastic
Glass or plastic
1 month (keep out of     Glass or plastic
 light and acid)
6 months to indefinite   Opaque  glass or
                          plastic

Immediate to 24 hours    Glass or plastic
6 months
Glass or plastic
                                                                                          (continued)

-------
                                  TABLE 3.1.4  (Continued)
Item
Preservation Method
Holding Time
Container
ZOOPLANKTON
 Count/identification
 Wet and dry weight
 Calorimetry
 ATP determination
5% Formalin or Lugol's solu-
 tion plus 50% glycerin, or
 70% ethanol plus 50% glycerin

Refrigerate at 4°C or ice
 (do not freeze)

Refrigerate at 4°C or ice
 (do not freeze); once fil-
 tered, store in desiccator
1 year
Immediate to 24
 hours

Immediate to 24
 hours
Immediately extract by boiling  6 months
 with Tris Buffer; store
 extract at -20°C
Glass or plastic



Glass or plastic


Glass or plastic



Glass or plastic
*  Replace solution with alcohol after 1 week.

-------
3.1.4.2  Design of Experiment—

     This section should contain:

          •  Identification of the biological subject
          •  Identification of the test material
          •  Route of administration of test material
          •  A table giving groups, group sizes and dose levels
          •  Information on how dose levels were selected
          •  Exposure schedules and duration of test
          •  Special instructions for administration of doses
          •  Description of controls
          •  Description of special test equipment
          •  Special instructions necessary to complete the plan
             of work

     The test subject should have been selected with all the criteria for
appropriate test species in mind.  The test material will have been selected
for some particular purpose or with accepted rules for prioritization in
mind.

     The animal species, the nature of the test material, the milieu of the
experiment and the purposes of the experiment all have a bearing on the
route of administration of the test material.  A feeding study, for example,
usually implies incorporation of the test material in the diet.  A problem
arises if the material is highly volatile or is a gas.  It is unlikely that
large quantities of these materials would remain incorporated in the feed
and be ingested.  It is possible to administer gases orally through use of a
carrier, such as water or corn oil, in which the material is soluble, and to
incorporate that into the diet.  Alternatively, microencapsulation could be
used, if a nontoxic material through which the gas is not diffusable could
be found.  Intubation, or gavage, of the gas and the carrier is also a possi-
bility.  As a quality control procedure, fresh solutions would have to be
prepared frequently in intubation studies.

     Soluble materials can be administered in the drinking water.  If vola-
tile, a closed system of glass and stainless steel is required and rubber
washers, etc., must be avoided.  Analytical chemical analysis will be re-
quired to verify that the stock solutions are fresh.  To ensure integrity of
the closed system, water should be supplied from glass bottles with plastic
screw caps fitted with stainless steel siphon tubes containing stainless
steel balls.

     Inhalation routes, skin or eye applications, aquatic experiments, in
the laboratory or in the field, plant experiments, and so on, all require
careful description of the route of administration in the Design of Experi-
ment Section.  Procedures for quality control of administration of the test
substance should be included in the Conduct of Experiment section of every
protocol.  More detail will be found in Sections dealing with different
kinds of bioassays.
                                    103

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     The table of groups, group sizes and dosage levels may take the form
shown below for a chronic mouse feeding study:
                     No. of Animals
Group No.

    1

    2

    3

    4

    5

    6
Male
50
50
50
50
50
50
Female
50
50
50
50
50
50
               Dose Levels*

               Control

               Low (1/8 MTD)

               Medium low (1/4 MTD)

               Medium (1/2 MTD)

               Medium high (3/4 MTD)

               High (MTD)
* Active material

•  Control - no-treatment diet
•  Maximum tolerated dose (MTD) calculated from available subacute
   or subchronic data
•  Test material will be incorporated in the diet over a 24-month period
•  Feed and water will be offered ad libitum

     The number of groups, the number of animals per group, the scale sel-
ected for dose levels and the proposed method of data analysis are all
proper subjects for review by a statistician at the Experiment Design stage.
As a matter of good practice, it is desirable to have the statistician's
signature on the protocol to indicate that the design is adequate.

     The statistical design of experiments has been described in Section 2.2
of the Quality Assurance Guidelines.  As an illustration of the criticality
of number of animals per group, the following tabulation gives an example of
how number is influenced by the expected frequency of finding of an effect
in the control group.  The number of animals per group required to show a
15% difference between the control and a treated group with 95% probability
is:
              Percent Animals Affected

          Control          Treatment Group
             0
             10
             20
15
25
35
Group Size
 Required

    36
    66
    85
      Note that  the  statistician's interest is in the magnitude of the diff-
 erence it is  desired  to  detect,  the acceptable level of significance of
 that difference,  and  sufficient  history of the use of the assay procedure
                                     104

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(including the particular test subject) to determine the expected frequency
of the occurrence of an effect in the control.

     Quality control over the design of experiment processes is exercised
through a review which may be by a peer committee from the study director's
lab or it may involve outside consultation.  The approval system should in-
clude signing of the written protocol before start of work by the study
director and the head of the laboratory,  and by a statistician.

3.1.4.3  Conduct of Experiment—

     In this section of the protocol, each procedure for conduct of experi-
ment should be spelled out in sufficient detail that there can be no mistake
regarding the details of day-to-day operations of the laboratory.  In these
Quality Assurance Guidelines, we recommend that the operational steps and
the quality control activities be laid out in parallel columns or otherwise
highlighted in association with each other.  The first example given here
illustrates the parallel column arrangement.  The other examples illustrate
a different, and more space-saving format.


                  EXAMPLE 1:  CHRONIC MOUSE FEEDING STUDY

Quarantine

•  Quarantine all animals upon receipt.
   QUALITY CONTROL ~ Hold in quarantine for 1 week.

Animal Identification, Randomization, and Housing

•  Assign to study group following quarantine.
   QUALITY CONTROL — Use randomization procedure (see Section 2.7)

•  Prior to study initiation, all animals will be weighed and appropriate
   adjustments made to achieve an equivalent mean body weight value between
   the groups.

•  Identify by cage, group, and individually.
   QUALITY CONTROL — Use ear tags and durable cage markers.

•  All animals will be housed by sex and dosage, five per cage.
   QUALITY CONTROL — Follow NCI Guidelines for cage space per animal.

Test and Control Materials

   QUALITY CONTROL — Conduct stability tests; return samples to sponsor for
   analysis, if requested.

Feed

•  The basic diet will consist of a commercial rodent ration.
                                    105

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   The diet will be analyzed for the parameters listed below:
     Polychlorinated biphenyls and chlorinated hydrocarbons
     Antibiotics
     Lead, arsenic, mercury
     Estrogen
     Aflatoxins
     Nutritional content
   QUALITY CONTROL — The frequency of these analyses is to be determined by
   the sponsor.

   The test material will be incorporated into the basal diet on a weight/
   weight basis and thoroughly mixed in a twin-shell blender to provide the
   appropriate diet level for each group.
   QUALITY CONTROL — The uniformity and concentration of the test material
   In the feed will be demonstrated prior to administration.

   Fresh batches of the diet will be prepared weekly.
   QUALITY CONTROL -- Samples will be taken and tested from each batch of
   feed.
Water
   Offer ad libitum.
   QUALITT~CONTROL -- Sample on a quarterly basis and analyze for heavy
   metals and coliforms.
            EXAMPLE 2.  PRIMAL DERMAL IRRITATION STUDY IN RATS
                (Illustration of special procedures only)

Preparation of Treatment

•  The hair will be clipped from the backs, and on one side a 1-inch square
   will be abraded by making minor incisions through the stratum corneum,
   but not deep enough to disturb the derma (that is, not sufficiently deep
   to produce bleeding).
   QUALITY CONTROL — Observe for bleeding.

•  Treatment will be applied with animals immobilized in an animal holder.
   The entire trunk will be wrapped with a rubber dam or Saran wrap for 24
   hours.
   QUALITY CONTROL — Follow plan in design of experiment.


               EXAMPLE 3.  CHRONIC INHALATION STUDY IN RATS              ,f
                (Illustration of special procedures only)
                                                                        '~,  ^
Generation of Atmospheres

•  Generate atmospheres by method appropriate to the test material.  For
   volatile liquids, generate high concentrations by passing compressed air
   through the liquids at constant rates.  Reduce to dilution with filtered

                                    106

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   warm air drawn through the chambers which operate under negative pressure.
   QUALITY CONTROL -- Monitor continuously prior to exposure of animals.
   Calibrate the monitoring equipment (such as hydrocarbon analyzer) with
   the substance being tested.  The range of calibration points will encom-
   pass the selected dosage levels.

   QUALITY CONTROL — Aliquots of the test substance will be introduced into
   large gas sampling bottles of known volume.  After vapor concentration
   reaches equilibrium the aliquot will  be introduced into the analyzer.

•  Analyzer should be equipped with 10-point automatically timed solenoid
   system:  1-8, Level of substance in eight chambers; 9, Room atmosphere;
   10, Combined stack effluent.
   QUALITY CONTROL — Sample four times  each day for 10 minutes per sampling
   point.Adjust flow as required.


           EXAMPLE 4.  IN VITRO TRANSFORMATION OF BALB/3T3 CELLS

Seeding

   QUALITY CONTROL — The vehicle for the test chemical is used in the nega-
   tive control plates.

•  Approximately 10,000 cells are seeded into a 60-mm plastic plate and incu-
   bated 24 hours to firmly attach the cells.  This plate will  be used to
   assess transformation.  Simultaneously with seeding, separate plates will
   be seeded at 200 cells per plate to obtain toxicity determinations.

Dosing

•  The positive control  and four doses of test chemicals are added to the
   transformation and toxicity plates.  Treatment with the test chemicals
   will consist of exposing the cells in an airtight enclosed chamber to
   either vapors or a gaseous state of the test materials.  Various dose
   levels will be achieved by varying the length of exposure to a fixed
   level of the vapors or gas.  Treatment will be terminated by removing the
   plates from,the chamber and replacing the media with fresh growth media.

Incubation

.  Following treatment,  the cells will be incubated for 3 to 4 weeks before
   they are scored for transformed foci.  The toxicity plates will be scored
   after only 1 week.  During the incubation periods, growth media will be
   changed twice weekly.
3.1.4.4  Observations and Tests—

     All observations and tests required should be described fully in this
section of the protocol.  Quality control procedures should be identified.
Detail will vary sharply from one type of bioassay to another.  Typical
examples are given below.

                                    107

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                EXAMPLE 1.  TERATOGENICITY STUDY IN RABBITS

Observations and Tests

•  Weekly records will be made on Individual female parents with respect to
   body weight (day 0. 6, 12. 18, 29), appearance, behavior, and survival.

•  At termination, brain, liver, and kidney weights, and the calculation of
   liver/brain weight ratios will be done on all adult females In each
   group.  The following observations will be recorded on does killed at
   termination and on their progeny:
      Number and placement of uterine sites
      Number and placement of live, dead, and resorbed fetuses
      Number of corpora lutea
      Fetal weight and length (crown to rump)
      External fetal anatomy
      Any gross abnormalities
      Gross necropsy evaluation on all fetuses, pups, and does.

•  All fetuses and pups will be cleared and stained with Alizarin Red S for
   evaluation of skeletal effect.  The reproductive organs of the female
   parents will be preserved In 10% neutral formalin and held for possible
   future hlstologlc evaluation.
   QUALITY CONTROL — All data will be evaluated statistically.


          EXAMPLE 2.  PRIMARY DERMAL IRRITATION STUDY IN RABBITS

Observations and Tests

•  After 24 hours of exposure, the patches will be removed and the resulting
   reactions will be evaluated on the basis of scores Indicated In the
   following table:

                       EVALUATION OF SKIN REACTIONS

      I.  Erythema and Eschar Formation

          0  No erythema
          1  Very slight erythema (barely perceptible)
          2  Well-defined erythema
          3  Moderate to severe erythema
          4  Severe erythema (beet redness) to slight eschar forma-
              tion  (Injuries In depth)
             Total possible erythema score
                                     108

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      II.  Edema Formation

           0  No edema
           1  Very slight edema (barely perceptible)
           2  Slight edema (edges of area well defined by definite
               raising)
           3  Moderate edema (raised approximately 1 mm)
           4  Severe edema (raised more than 1 mm and extending
               beyond area of exposure)
           4  Total possible edema score

   Readings will be made again at the end of 72 hours.  The reading on each
   rabbit will be recorded.
                    EXAMPLE 3.  PATHOLOGICAL PROCEDURES

     In chronic studies, whole-animal test pathological procedures are re-
quired and these can be standard.  A proposed standard for pathological pro-
cedures 1s as follows:

Personnel
•  A board-certified veterinary pathologist with experience 1n laboratory
   animal pathology will be responsible for all pathology procedures,
   evaluations, and reporting.  Histology technldan(s) will be supervised
   by an HT/ASCP certified technician.  Personnel trained and experienced
   1n laboratory animal dissection to recognize gross abnormalities will be
   prosectors.  Qualified personnel will be available for weekend coverage
   to necropsy dead or moribund animals, or to refrigerate them for necropsy
   at the earliest possible time.

Facilities

•  Refrigeration 1s available for holding dead animals until necropsy.
   Animals will not be frozen.  The histology laboratory 1s separated from
   the necropsy area and 1s equipped with automatic tissue processors,
   microtomes, embedding and stirring equipment, and supplies.

•  Adequate storage facilities are available to store and file hlstologlc
   slides, tissue blocks, and wet tissues for the duration of the contract.
   This facility 1s vermin proof and temperature controlled.  The area pro-
   vided for trimming of fixed tissues has adequate ventilation and exhaust
   hoods for removal of formaldehyde fumes.

Gross Necropsy

•  A blood smear will be taken from all animals at the time of necropsy.
   Whether this smear ultimately will be read or not will depend on the
   observations made during gross necropsy or hlstopathologlc review.  There-
   fore, all smears will be fixed and retained for possible future use.
                                    109

-------
A complete gross necropsy is defined as an examination and harvest of all
of the following tissues:

   Gross lesions and tissue masses..    ..      Ileum
    (and regional lymph nodes, if             Colon
    possible)                                 Cecum
   Skin                                       Rectum
   Mandibular lymph node                      Mesenteric lymph node
   Mammary gland                              Liver
   Salivary gland                             Pancreas
   Thigh muscle                               Spleen
   Sciatic nerve                              Kidneys
   Sternebrae, vertebrae or femur,            Adrenal glands
    including marrow                          Bladder
   Costochondrial junction, rib               Seminal vesicles
   Thymus                                     Prostate
   Larynx                                     Testes
   Trachea                                    Ovaries
   Lungs and bronchi                          Uterus
   Heart                                      Nasal cavity *
   Thyroid                                    Brain
   Parathyroids                               Pituitary
   Esophagus                                  Spinal cord
   Duodenum                                   Eyes *
   Jejunum
   *  Always examined; harvested only when lesions are present.

All animals that die or are killed will receive a complete gross necropsy
(unless cannibalism or autolysis preclude all or part of the examination).

The gross dissection and evaluation will be performed by or under the
direct supervision of the pathologist(s).

Peripheral blood smears from the heart, tail, or toe will be prepared for
animals in those cases where neoplasia of the lymphoid system or of the
bone marrow is suspected (as evidenced by an enlarged spleen, liver or
lymph node, or by a watery appearance to the blood, indicating an anemic
condition).  Smears will be air-dried, then fixed in absolute methanol
within 24 hours.  Touch preparations will be prepared from any enlarged
spleen.  If lymphoid organs other than the spleen are enlarged, then a
touch preparation will be made from those affected organs.  Smears will
not be stained unless requested by the pathologist.

All tissues and/or organs will be examined in situ, then dissected from
the carcass, re-examined and fixed in 10$ neutral buffered Formalin.
Lungs of mice and rats will be fixed in their entirety after opening and
examining the trachea and main-stem bronchi.  The calvarium will be re-
moved and the dorsal nasal bone removed for examination of nasal turbinates.
The entire skull will be fixed with the brain in situ.  Other tissues will
be fixed at a thickness not exceeding 0.5 cm.

                                110

-------
         I
 •  Urinary bladders will be opened and examined before fixation.  Contracted,
   empty bladders may be partially distended with Formalin before opening to
   reveal small lesions that may be obscured by epithelial folds.  One
   kidney will be longtJtM
-------
   Complete histopathologic examination is defined as examination of the
   following:

      Gross lesions and tissue         Seminal vesicles
       masses (and regional            Testes
       lymph nodes, if possible)       Ovaries
      Blood smear (as required         Uterus
       by the pathologist)             Brain (three sections including
      Mandibular lymph node             frontal cortex and basal ganglia
      Mammary gland                     cortex and thalamus; and cere-
      Salivary gland                    bell urn and pons)
      Sternebrae, femur or             Thymus
       vertebrae, including            Trachea
       marrow                          Lungs and main-stem bronchi
      Thyroid                          Heart
      Parathyroid                      Pancreas
      Esophagus                        Spleen
      Stomach                          Kidneys
      Small intestine (one             Adrenal glands
       section)                        Urinary bladder
      Colon                            Pituitary
      Liver                            Spinal cord (if neurological signs
      Gallbladder                       are present)
      Prostate                         Eyes (if grossly abnormal)

•  Tissues will be blocked in a systematic manner to enhance efficiency in
   histopathologic examinations.  All pathologic diagnoses will be made or
   confirmed by the pathologist(s).

Submission of Pathology Results (Individual Animal Data Record Form)

•  Histopathologic diagnoses of all lesions will  be entered under Organ and
   Diagnosis.  Primary versus metastatic tumors,  e.g., liver hepatocellular
   carcinoma; and lung, hepatocellular carcinoma  (metastatic) will be indi-
   cated.

•  Descriptive narratives of gross necropsy findings will be provided for
   all animals.  The number as well as description of tissue masses will be
   Included.  If they are confluent or too numerous to count (TNTC), this
   will be noted.

Residual  Material
   All blocks, wet tissues, and slides of chronic animals (test, vehicle
   control, and untreated control) will be retained in a vermin-proof, tem-
   perature-controlled area until  termination of the bioassay investigation.
   At completion of the program, these residual  materials will  be organized,
   packed, marked, and shipped to the sponsor.   Clearance to ship will be
   requested before any action is taken to ship.

   Wet tissues (residue from harvested tissues,  not carcasses)  will  be
   stored in two sealed plastic bags one inside  the other and organized by

                                    112

-------
   histology numbers.   A permanent Ink label  will  be placed between the two
   bags showing the name of the contractor and the histology number.  Once
   the bags are organized, they will  be packed In  350 Ib-test double-wall
   cardboard boxes and marked on one  end to show:

      Name of contractor

      Contract number

      Chemical number

      Animal group number(s)

      Histology numbers 1n that box.

•  These boxes will be sealed shut with shipping tape, bound with filament
   tape, and shipped to the sponsor upon receipt of clearance to do so.

•  Blocks will be resealed with paraffin, organized by histology number, and
   labeled or permanently marked with the name of  the contractor and the
   histology number.  When Mstopathology 1s  complete and the residual
   material 1s to be prepared for shipment to the  sponsor, blocks will  be
   placed Into single-wall cardboard  boxes the size of approximately 80
   blocks and then these smaller boxes placed Into 350 Ib-test double-wall
   cardboard containers approximately 16" x 18" x  7-1/2".   Boxes will be
   marked on one end to show the Information  Indicated In the above.  Ship-
   ping cartons will be sealed with pressure  tape  and bound with filament
   tape for shipment.

•  Slides will be organized by histology number.  They will be placed in
   metal slide cabinets.  Each metal  slide cabinet will be marked to show
   the range of histology numbers and the name of  the contractor.  These
   cabinets will contain a 11st Identifying the name of the contractor, the
   number of slides, and the cross-reference  Information,I.e., animal
   numbers, histology numbers, and chemical numbers, which will allow com-
   plete identification of the contents.

•  A master log of histology number assignments will be provided to the
   sponsor along with the first shipment of slides.  Since this log may not
   be complete when the first shipment of slides 1s made, updated versions
   of the log will be provided.

Pathologic Material to be Retained by the Contractor Until Termination and

Final Reporting of Study

•  All wet tissues will be stored In  plastic bags, sealed, clearly and per-
   manently labeled and retained in a vermin-proof area.  All histologic
   slides and paraffin blocks will be sealed with  paraffin.

•  The grouping of mouse tissues on the micros!ides will be as follows:
                                    113

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Slide 1:   Brain (2);  Pituitary:  Thyroid/Parathyroid/Trachea/Esophagus
Slide 2:   Heart;  Kidney (2);  Adrenal glands (2);  Liver with gallbladder
          (2);  Thymus;  Spleen;  Pancreas
Slide 3:   Lung and main-stem bronchi
Slide 4:   Stomach;  Small intestine (2);  Large Intestine (2);  Urinary
          bladder
Slide 5:   Testes/Epididym1s/Sem1nal vesicles (2)/Ovary (2);  Prostate/
          Uterus;  Salivary gland with mandlbular lymph node;  Mammary
          gland;  Skin
Slide 6:   Bone;  Bone marrow;  Spinal cord (1f neurological signs are
          present)
Slide 7:   Tissue masses  (suspect tumors)
Slide 9:   Multiple sections of skin
Slide 10: Blood smear (or eye, if abnormal)

-------
3.1.5  References
American National Standards Institute.  1971.  Laboratory Qualification
     Guidelines for ANSI Certification Programs.  ANSI, 1430 Broadway,
     New York, N.Y.

American Society for Testing and Materials (ASTM).   1977.  Standard
     Practice for Generic Criteria for Use in the Evaluation of Testing
     and/or Inspection Agencies, ASTM Designation E548 A.S.T.M., 1916
     Race St., Philadelphia, Pa.

Bicking, C. A., and W. E. Deming.  1971.  A Reappraisal of the Contribution
     of Statistical Methods to Quality Assurance Proceedings.  International
     Statistical Institute, Washington, D.C.

Center for Disease Control.  1975.  Guide to Proficiency Testing.  Atlanta,
     Ga.

FDA.  1976a.  Compliance Program Manual.  Transmlttal No. 76-209, Food and
     Drug Administration.

FDA.  1976b.  Proposed Regulations for Good Laboratory Practice.  Food and
     Drug Administration. Fed. Reglst. 41(225):  51206-51230.

Huibregste, K. R., and V. H. Moses.  1976.  Handbook for Sampling and Sample
     Preservation of Water and Waste Water.  EPA 600/4-76-049.

National Bureau of Standards.  1973.  Clinical Laboratory Performance
     Analysis Using Proficiency Test Statistics.  NBSIR 73-197 (Authors:
     P. W. Finkel and J. W. Rowen).

National Cancer Institute.  1976.  Guidelines for Carcinogen Bioassay In
     Small Rodents.  NCI-CG-TR-1.  Bethesda,  Md.

National Research Council.  1972.  Guide for the Care and Use of Laboratory
     Animals.  DHEW No.  (NIH) 74-023.  U.S. Government Printing Office,
     Washington, D.C.

Office of the Secretary of Commerce.  1976.  Procedures for a National Vol-
     untary Laboratory Accreditation Program.  Fed. Regist. 41(38):  8163-
     8168.

OSHA.  1974.  Proposed Rules for Accreditation of Testing Laboratories.
     Occupational Safety'and Hazard Administration.  Fed. Reglst. 39(191).

Slack, K. V., R. C. Averett, P. E. Grewson, and R.  G. Lipscomb.  1973.
     Methods for Collection and Analysis of Aquatic Biological and Micro-
     biological Samples.  Book 5, U.S. Department of the Interior, Washing-
     ton, D.C.
                                    115

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U.S. EPA. 1975.  Handbook for Evaluating Water Bacteriological Laboratories.
     Second Edition.  U.S. Environmental Protection Agency, EPA-670/9-75-
     006, Cincinnati, Ohio.

U.S. EPA. 1976.  Handbook for Sampling and Sample Preservation of Water and
     Wastewater.  U.S. Environmental Protection Agency, EPA-600/4-76-049,
     Cincinnati, Ohio.
                                      116

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3.2  FIELD RESEARCH

3.2.1  Field Sampling

     A general requirement for satisfactory sampling is valid and representa-
tive samples (McFarren, 1974).

     A formal sampling plan includes:

          •  the selection of sampling site (Section 3.2.1.1)
          •  the frequency of sampling (Section 3.2.1.2)
          •  the calibration and maintenance of sampling equipment
             (Section 3.2.1.3)
          •  sample preservation (Section 3.2.1.4)
          •  the selection of sampling methods (Section 3.2.3)

3.2.1.1  Selection of Sampling Site—

     The selection of the sampling site is the beginning, inevitable task of
any field biologist.  There is limited, scattered information on selection
of sampling sites in field biology literature.  The following criteria should
be taken into consideration when selecting the sampling site:

          •  familiarity with historical data including biological,  chemical,
             and physical nature of the site
          •  good definition of the study objective
          •  degree of accessibility
          •  whether or not the stands (or stations) appear to be
             representative
          •  availability of satisfactory adjacent stands, since it is con-
             venient to establish more that one station at each field locality
             (Davis and Gray, 1966)
          •  organism-specific:  For fish, sample in the obscure and unlikely
             areas as well as at obvious locations; sample all depths, not
             just surface and bottom.  For other organisms, sample to suit
             the special requirements
          •  habitat-specific:  In rivers, one sample upstream and another
             downstream from the pollution source.  In lakes, reservoirs, and
             other standing-water bodies where the zones of pollution may be
             arranged concentrically, locate stations in an area adjacent to
             the waste outfall and in an unaffected area
          •  for aquatic vegetation:  Three criteria are applied in the deci-
             sion to include or reject a particular side (Auclair et. al. ,
             1976).  They are:

             o  Following an initial survey prescribe samples in proportion
                to the area covered in each existing emergent vegetation  type
             o  Sample at different water depths
             o  At Least one emergent species has to be present

          •  In benthic studies, station positions must be stratified to
                                      117

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            reflect both natural abiotic environmental gradients and pollu-
            tlonal gradients.

    "Criteria for locating stations must receive more attention.  Preliminary
cruises should be designed simply to determine the position of future stations.
Justifications for sampling grids should be included in all research reports"
(Swartz, 1976).

    Weber (1973) gave some suggestions on selecting sampling sites for plank-
ton studies with regard to pollution.  First of all, it was suggested that
sampling be widespread to define the quantity and nature of all plankton in
the body of water in long-term studies.  In short-term studies, sampling
sites might be more restricted because of limitations in time and manpower.
Secondly, it was recommended to locate the sites upstream and downstream from
a suspected pollution source in a small stream or river and to locate sampling
sites in lakes, reservoirs, estuaries, and the oceans in grid networks or
along longitudinal transects.  Thirdly, if pollutants are discharged from
various sources, locate sampling sites in such a manner as to separate their
effects, i.e., antagonism, synergism or additivity.  Finally, on the basis
of historical data, choose sampling sites including areas from which plankton
have been collected in the past.

    For studying pesticide residues in the water environment, Lauer (1974) em-
phasized that the location of the sampling station must make it possible to
obtain samples representative of the water body being sampled.  The greater
the variability of the water mass, the more sampling stations must be selected.

    If the objective of a study is   qualitative in nature (to describe the
flora and fauna of an area with a high degree of accuracy), a relatively
large number of samples must be collected from a large number of habitat types
(Slack et al., 1973).

3.2.1.2  Frequency of Sampling—
    Frequency of sampling is of critical importance.  In a sampling program,
it evidently influences the validity of data, particularly  the precision and
accuracy of data.  In general, the more frequent the sampling, the more pre-
cise and accurate the data.

    There are many elements that determine the frequency of sampling.  Among
these elements some important ones are:

            •  the objective of study
            •  the organisms being studied
            •  the availability of manpower      •  -   *
            •  the availability of historical information
            •  the limitation of time
            •  the limitation of money
            •  the adequacy of sampling equipment
            •  environmental factors
                                      118

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    Prior to the beginning of sampling, study objectives must be defined
clearly and carefully.  For example, the frequency of sampling may vary from
hourly, for a detailed study of diel variability, to quarterly (every third
month), for a general estimation of seasonal variations, depending on the ob-
jectives (Rand et al., 1975).  If the objective of the study is quantitative
in nature, the increase in the frequency of sampling may increase the preci-
sion of the data., e.g., of the estimation of fish population in a body of
water.  Frequent samplings are also necessary in a pollutional study if the
characteristics of effluents change or if spills occur.  This will help
biologists more precisely to locate the effluents or spills in a given study
area.

    Available manpower, time and money always determine the scope of study
and the frequency of  sampling, too.  The sampling frequency must be adjusted
to limitations in personnel, time and money.

    If sampling can be automated, more frequent samplings can be made than
are otherwise possible.  For example, automatic monitors can sample air,
water or other media  continually, e.g., hourly through day, month, and year.

    Biological factors such as organisms being studied determine the fre-
quency of sampling.   That is, the frequency of sampling for plankton may
differ from that for  fish or other organisms because each studied organism
has its unique biology, e.g., habitat types, and natural variability.

    The frequency of  sampling is sometimes determined by the available histor-
ical information attainable by searching literature (or work) by previous
investigators.

    Environmental factors may also influence the determination of sampling
frequency.  For instance, sudden meteorological changes such as a hurricane
storm may force biologists to sample more frequently in its aftermath.

3.2.1.3  Calibration  and Maintenance of Sampling Equipment—

    Table 3.2.1 summarizes the equipment used currently in biological field
sampling.  It is generally agreed that no type of sampling equipment 13
applicable to all biological communities.  Instead, there is sampling equip-
ment available for each biological community, such as the special nets, pumps
and water bottles applicable to a plankton community.

    Traditionally, little importance is attached to the calibration of field
sampling equipment.   This is probably due to two things:

            •  most field sampling equipment is designed for qualitative studies
            •  for quantitative studies, sampling frequency and site selection
               affect the precision of data much more than calibration errors
                                      119

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           TABLE 3.2.1  A LIST OF BIOLOGICAL  SAMPLING  EQUIPMENT
                   (U.S.EPA,  REGION II, 1975;  U.S.DI, 1972)
Organisms
Field Sampling Equipment
Mammals
Birds
Reptiles

Plants
Fish
Macroinvertebrates
Zooplankton
 Periphyton
Mouse traps (mouse, rat, etc.)
Conibear traps (bear, etc.)
Snares (deer, etc.)
Box traps (chipmunk, muskrat, etc.)
Beaver traps (beaver)
Herd traps (deer, etc .)
Nets (monkeys, etc.)
Guns (rabbits, deer, etc.)

Box or enclosure traps  (gregarious seed-eaters)
Net or rocket trap  (wild turkey, etc.)
Drive and drift traps (water fowl)
Mist nets (commercial birds)
Nest traps (water fowl)

Drift traps (snakes)

Transportation
Survey gear
Base maps
Specimen containers

Electric shocker
Gill nets
Trammel nets
Seines
Trawls
Others (hook and line, chemicals, etc.)

Grab samplers (Ponar, Peterson, Ekman, Tall Exman,
 Orange Peel, Shipek, Smith-Mclntyre, etc.)
Surber sampler
Corers

Nets
Clark-Bumpus
Pumps <
Integrated (tubular) samplers
Kemmerer  or Van Dorn  water bottles
Juday trap
Artificial substratum
Natural substratum
                                                                   (continued)
                                      120

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                          TABLE 3.2.1  (Continued)
Organisms                    Field Sampling Equipment

Phytoplankton                Nets
                             Clark-Bumpus
                             Pumps
                             Integrated (tubular) sampler
                             Kemmerer or Van Dorn water bottles
                             Others

Macroalgae (e.g. chara)      Same as aquatic plants

Macrophytes
 (Aquatic vascular           Transportation gear of survey
     plants and              Base maps
     aquatic plants)         Specimen containers

Microorganisms               Water sampling bottle, e.g.,
                             Kemmerer type


     The following information on equipment used ought to be included on the
field data sheet:

          •  date of use
          •  user's name
          •  operating conditions
          •  special remarks on maintenance and repair

     For sampling equipment, maintenance and repair are more important than
anything else.  Regular maintenance work consists of:

          •  good, thorough cleaning after use
          •  drying before storage
          •  proper storage

For example, nets for plankton or fish collection need attention.  In particu-
lar, sampling equipment employed in brackish or marine water requires a fresh-
water rinse to prevent rusting or rotting.  The repair of equipment should be
scheduled and done on time and by the right personnel.  Replacement must be
considered if repaired equipment does not do an adequate sampling job.

     The maintenance of mobile laboratory facilities should be also considered
as an Important task.

3.2.1.4  Sample Preservation—

     Upon obtaining a valid and representative sample in the field, sample
preservation is an Important consideration.  Biological sample preservation
                                      121

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normally emphasizes:

          •  sample holding container
          •  type of preservative used
          •  sampling labelling information
          •  holding time between sampling and analysis

The containers for biological sample material can be divided into two princi-
pal categories:  glass and plastic.  There are many types in each category.
All containers have their disadvantages.  In general, the major disadvantage
of glass is that it is breakable and heavy-weight.  This can be a strict handi-
cap in field use.  Polyethylene, on the other hand, is durable, light-weight
and easy to handle.  So plastic containers are more widely accepted.  Never-
theless, both kinds of containers require the use of proper chemicals to pre-
serve field biological materials.

     The chemical preservatives most often used for general field preservation
include formaldehyde, ethyl alcohol, borax, and arsenic trioxide.  The use
of these preservatives for organisms varies from microscopic protozoa to large
mammals.  Table 3.2.2 presents recommended techniques for using these preserva-
tives   with a number of biological materials.  Each recommended technique is
briefly described.

     As shown in Table 3.2.2, in addition to chemical preservation, physical
preservation of biological material is also recommended.  Two means are em-
ployed in physical preservation:  refrigeration and freezing.  Refrigeration
(approximately 1-2°C) is an excellent way to preserve most biological materials
for a short period of time.  For longer periods of preservation deep freezing
(approximately -20°C) is considered as an excellent method to preserve many
specimens.  Either way, it must be kept in mind that the specimen should be
placed in a watertight container, e.g. a plastic bag, and packed in a second
container with either dry or natural ice surrounding the inner-most container.
The sample (or specimen) must be shipped immediately to a central laboratory
for analysis.


 TABLE 3.2.2  TECHNIQUES RECOMMENDED FOR PRESERVATION OF BIOLOGICAL MATERIAL
                           (MOSBY AND COWAN,  1971)


  Biological Material      Recommended techniques, listed in order of
                              preference


Mammals
     whole, small          (1) Ethyl alcohol (70%);  (2)  5% Formalin

     whole, large          Formalin (7-10%);  also injection  of preservative
                           into internal organs by hypodermic-perfusion via
                           circulatory route
                                                                  (continued)
                                     122

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                           TABLE 3.2.2  (Continued)
   Biological Material
 Recommended  techniques,  listed in order of
    preference
 Skins,  pelts



 Skins,  study*



 Food material stomachs


 Droppings

 Reproductive tracts


 Birds
      whole


 Skins,  pelts)
 Skins,  study)

 Stomachs

 Droppings and pellets

 Reptiles  & Amphibians
      whole


 Snake skins

 Salamanders
Amphibian skins
      (to preserve color)

Fish

Jnsects
     Hard bodies
     Soft bodies
 (1)  Clean  thoroughly  and  air dry;  (2)  clean and
 salt thoroughly  (NaCl);  (3)  use  alum on pelts which
 appear  to  be  "slipping"

 (1)  Borax  (not to be  used on. skins  having red
 pelage); (2)  arsenic  trioxide-borax mixture in
 equal proportions;  (3) arsenical soap

 Small stomachs-5% Formalin;  large stomachs-5 to 10%
 Formalin (wrap stomachs in cheesecloth)

 Dry  quickly,  fumigate with carbon disulfide

 (1)  AFA (preferably)  or Bouin's  fluid;  (2)  10%
 Formalin
 (1) 70% alcohol;  (2) 5% Formalin, both with inter-
 nal injection
 (1) Borax;  (2) arsenic-borax mixture

 5% Formalin

 Dry quickly and fumigate with carbon disulfide
 (1) 35-40% isopropyl alcohol or 70% ethyl  alcohol;
 (2) Formalin-specimens should be slit or injected

Rolled flat, placed in 70% alcohol

Kill with chloretone or 20% alcohol; harden with
5% Formalin and store in 70% alcohol

Kill with ether; skin and place skin in water;
float onto cardboard; dry quickly

(1) 70% alcohol; (2) 10% Formalin
Kill with KCN bottle; store dry

Kill and store in 5% Formalin or 10% alcohol
                                         (continued)
                                      123

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                          TABLE 3.2.2   (Continued)
  Biological Material
Recommended techniques, listed in order of
   preference
Miscellaneous
     Skeletons-field
     Skeletons-in
     laboratory
     Fumigants-for all
     specimens in pelt,
     study or standing
     mount form

Pathological Material
     General
Hematological
Bacteriological
Virological
      Rabies
      Other Viruses
 Parasitological
      Ectoparasites
(1) Clean thoroughly and dry quickly; treat with
arsenical soapt  for shipment; (2) place in alcohol
(Formalin, unless neutralized, dissolves calcium of
bones)

(1) Boll gently in 3% hydrogen peroxide to remove
meat and to bleach bones, degrease in carbon tetra-
chloride; (2) clean by use of dermestid beetles

Carbon disulfide as gas insecticide to kill insects;
paradlchlorobenzene as insect deterrent and DDT
as insect contact killer
(1) Refrigerate  (30°-40eF); (2) deep freeze and
transport to laboratory as quickly as possible

(1) Make several blood or tissue smears;  (2) blood
serum;  (3) cell counts:  either sodium oxalate 2-4
mg/ml or sodium citrate 2-4 mg/ml; refrigerate;
(4) whole blood or serum dried on paper discs

(1) Refrigerate entire specimens; (2) take blood,
pus or  fluids in sterile containers; refrigerate;
(3) saturate cotton swabs with blood, pus, or
tissue  juices; transport in special medium; (4)
make smears from blood, serous fluids, tissue juices
If possible confine the animal and wait until death
occurs.  Refrigerate and rush the head (if possible
the entire carcass) to Public Health Laboratory
 (1) refrigerate;  (2)  freeze;  (3) put  1 cm
 of tissue in glycerol
cubes
Remove by hand or with aid of ether, chloroform,
or  sorptlve silica powder  (Dri Die).   (1) ship
live In non-airtight container with moist cotton,
refrigerate if possible; (2) kill with ether, chlor-
oform or HCN and ship dry between layers of cotton;
(3) freeze and ship frozen
                                         (continued)
                                        124

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                          TABLE 3.2.2   (Continued)
  Biological Material
Recommended techniques, listed in order of
   preference
Helminths



Protozoa



Histological


lexicological
Plants
     Terrestrial
     Aquatics, or other
     plants with a mass
     of tissue
 (1) relax in cold water.  Fix nematodes in hot
 70% alcohol.  Fix cestodes, trematodes in warm
AFA; (2) 70% alcohol-95 parts, glycerol-5 parts

 (1) refrigerate tissues, feces, citrated blood
 (2) make smears of blood, feces, tissue impressin
 (3) fix tissues in 10% Formalin

Fix small pieces of tissue in 10% Formalin (10 'o
20 x volume of tissue).  Do not freeze

Refrigerate or freeze blood, liver, kidneys, br •• -;n.
stomach with contents, small intestine
Place between folded paper, dry quickly between
corrugated cardboards and with slight pressure !•
plant press

(1) alcohol-acetic acid-Formalin solution; (2) 2
to 4% Formalin
* Injection with embalming fluid (equal parts of Formalin, glycerine, and
  phenol plus 85 parts water) will keep birds and mammals fresh enough to
  skin for study mounts for about a week without refrigeration.
t Poisons should not be used on skeletons which are to be cleaned by derme -
^ Clean large skulls and skeletons by boiling in 4 oz. sodium sulfate and
  8 oz. ammonia to 6 gallons of water.

     Samples are useless unless adequately labelled.  The samples or sarapl;
containers must have attached the following information, written with a'.wat
proof marker on durable paper:

          •  date
          •  name of study area
          •  site of sampling station
          •  type of sample (qualitative or quantitative)
          •  volume of water represented or weight where applicable
          •  number of subsamples of sample
          •  type of analyses desired for sample
          •  name of collector
          •  method of sample collection
                                      125

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     It is always a good practice to duplicate full data on a second label
and to pack it with the sample container so that at least one set of sample
information is preserved.  This labelled sample should go to the project
manager or laboratory supervisor with a completed field data sheet (Figure
3.2.1) and a completed chain of custody form (Figure 3.2.2).

     Holding time has been defined in the following ways:

          • the entire period of time from the point of the
            initial sample collection to the beginning of the
            analysis
          • the period of time between the point of receipt of the
            sample at the laboratory and analysis
          • the period of time between the point of the formation
            of composite sample and analysis

     Neither of the latter two show the real length of time a sample has been
moved away from its environment.  Therefore, the results of analysis may not
be valid due to the inaccurate reflection of possible changes.  This may be
critical when analyzing for the population of mlcrobiologic bacteria which
change fast in water, but it may not be important for fish scale samples
that are commonly preserved for age and growth study.  Consequently, the
holding times between the beginning of sampling in the field and analysis
in the laboratory must be specified.

     Under no circumstances should the laboratory supervisor or project
manager delay the analyses on any field biological samples.  When a sample
enters the laboratory, the material with the shortest holding time should
be analyzed first.  In the meantime, a composite sample must be formed if
needed and further preservation in the laboratory must be accomplished If
required.  Then the relatively stable samples can be analyzed.  Thus, the
problem of delayed analyses is reduced to a minimum.
                                     126

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    Location _
    Collector
                  Sea. Depth (Ft.)
                                    Air Temp. (°F)
    SAMPLING METHOD (Circle)

     Kemmerer Petersen Surber Manual
     Plankton Net Seine Trawl. Bucket
     Other   	
                              COMPOSITE DATA (Circle)
                                  Flow   Space
                              Observed Flow 	
                              Avg. Daily Flow _
                                         Time
    OBSERVATIONS (Circle)
     weather
Wind
Ft. Wave
Surface
Bottom Z
TIDE CONDITION
      Clear
North
            Clean
             Ooze
    P. Cloudy
East
              Oil
             Sand
     Overcast
South
            Garbage
             Gravel
       Fog
West
             Trash
             Clay
     Drizzle
      Rain
0-5
MPH
          Gas Bubbles
             Rubble
          Dead Fish
             Rock
      Snow
5-15
MPH
           Sewage
             Shell
                                 Ind. Waste
                         Organic
               Over 15
               MPH
                  Float Solids
           LW      HW    LW
          Slack  Slack  Slack

               FloodEbb

           Tide Stage (Height
                  Low
                 Normal
          	High	
    WATER-Color
           Odor
                 From Plankton, Waste, Sediment, Other
                          Fresh/Brackish/Salt
    STREAM-Width (Ft.)
           Rapids 	

    ANIMALS-
                  Depth (Ft.)
            I Pools
                   Z Riffles
                 Low/Normal/Flood
                "	 Z
    PLANTS-Floating
         Fish:  Adults, Fry
        	 Z Emergent
                     Perlphyton
                          Insects:  Adults, Larvae
                                Z Sumberged 	
                                    Algae
Samples to;
 Bact  Bio  Chem  Other
              Collection (Ending) Date
7
Mo
1
Day
1
                                    Sample Temp. (°C)
Station No.
              Ending Time (24 Hr)
                                    DO (mg/1)
Sample Depth (Ft.)
              Beginning Date
Yr
1
Mo
1
Day
1
                                    Cond. (uMHOS/CM)
Lab Number
              Beginning Time (24 Hr)
                                    Salinity (Z.)
Type of Sample
  Grab  Composite  Sediment
              PH
                                    Other
  Remarks
                                                             (EPA, Region II)
           Figure 3.2.1   Field data sheet (U.S. EPA,  Region II)
                                      127

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                             of Unit And JuUress:
                             r     Bait
                        of
                                              tllicy for S««Bl**
K>
OS
»il lM"U*»d »y;   rimt   tot*
                                        tUted fcy:
                                             By:
                 TIM
tec*
               »j;
Tlw
                                                                                    tot*
                                                   tote
                                                                                 tote
             •HMO*,  foe niangr of Custody
for fliain* of
                                                               for f>>i>gf of Cusrotfjr
                                                                                             (IT*.  IGBJOM II)
                                 Figure 3.2.2   Chain of  custody for* (U.S. EPA,  Region II).

-------
3.2.2  Field Analyaia

     Blologlbta and analytical chemlats have become mort and more  Interested
In having analyaea done In Che field because Che holding and preaervaCion of
aamplea have been ahown Co affect Che quallCy of reaulta, i.e. Che accuracy of
data.  For example, Che addition of the common, preeervative, HgCl2, that la
applicable Co Che meaaurement of nutrlenta In Che aample, InCerferea with Che
meaaurement of BOD (Biological Oxygen Demand).  The bacterial Inhibition by
auch chemicalB reducea Che BOD reading.  For biological material,  Che pre-
aervatlon of moaC aamplee changea Che original natural colors of organlama.
Thla change aomeClmea ma Icea lc more tlme-conaumlng and more tedloua to Iden-
tify organlama.  The prolonged holding of the preaerved samplea  (eapeclally
in ethanol) often cauaea an undereaclmace of peatlcide realdueu.  Lauer (1974)
recommended the rouClne check of peatlcldea In Che preaervatlvfc with Che
reaulC to be added to the total obtained from the organlama before final
computation of concentratlone.  Becauae of conalderatlona like thla. the Task
Group on Biologic Quality and Organlca of the Federal intraagency  Work Group
on Dealgnatlon of Standarda for Water Data Acquialtlon auggeated that some
analyaea that are usually done In the laboratory be practiced In the field
(U.S. Department of Interior, 1972).

3.2.2.1  EPA Field Methode—

     The U.S. Environmental Protection Agency (1973) haa publlahed a manual,
entitled "Biological Field and Laboratory Methoda for Meaaurlng the Quality of
Surface Watera and Effluente" (C.I.  Weber, editor),   it contains  field and
laboratory  methodology for aampllng, Identifying and quantifying plankton,
parlphyton, macrophyton, macrolnvertebratea, flah and bloaaaay, and haa a
chapter on "Blometrlca".  The manual la periodically reviewed,and  revlaed exiat-
ing methoda and new methoda are added aa Che need arlaee.  The aecond edition
of the EPA Methoda Manual will be algnlficantly expanded Co Include the
following addleional materials:

        Non-parametric atatiatlcal analyela
        Adenoalne triphoaphate analyaia
        Nitrogen fixation (acetylene reduction) methoda
        Liquid acintlllatlon technlquea for primary productivity
        Periphyton primary productivity methoda
        Sediment oxygen demand
        Scuba technlquea
        Hlatopathology and hlecochemietry
        AceCychollneateraae analyaia
        EffluenC bloaaaay
        Field and laboratory biological quality aaaurance guldellnea

3.2.2.2  Inatfuraent Calibration—

     Table 3.2.3 Hate inatrumenta and equipment commonly used in  the
biological field analyaia.
                                     120

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               TABLE 3.2.3 INSTRUMENTS AND EQUIPMENT FOR LABOR-
               ATORY AND FIELD ANALYSIS IN BIOLOGICAL RESEARCH
ATP photometers               Fluorometers           pH meters
Audial instruments            Freezers               Refrigerators
  (recorders, etc.)           Gas chromatographs     Salinometers
Balances                      Incubators             Spectrophotometers
Current meters                Light meters           Thermometers
DO probes                     Microscopes:           Visual instruments
Drying ovens                   Compound                (binoculars, etc.)
                               Electron              Volumetric glassware


     Calibration procedures for spectrophotometers are described in "Spectro-
photometer Calibration and Performance", ASTM E225-67.  Rand et al. (1975)
detail procedures for the calibration of microscopes, fluorometers, analytical
balances and other instruments.  According to Rand et al.  (1975), balances
shall provide a sensitivity of at least 0.1 g at a load of  150 g, with
appropriate weights.  An analytical balance having a sensitivity of 1 rag
under a load of 10 g shall be used for weighing small quantities (less than
2 g) of materials.  Single-pan rapid-weight balances are most convenient.  See
Chapter 3, Instrumental Quality Control, of the U.S. EPA's Handbook for
Analytical Quality Control in Water and Wastewater Laboratories (U.S. EPA,
1972).

     A good calibration system for any instrument for field and laboratory
analysis should-be based on the following requirements:

     •  Develop a calibration plan and follow it
     •  Use calibration standards.  For example, solutions  containing
        chlorophyll a, h and c and the degradation product  pheophytin a
        are available for spectrophotometric analysis by writing to:
        U.S. Environmental Protection Agency, EMSL - Laboratory
        Evaluation and Quality Assurance Branch, 26 West St. Clair Avenue,
        Cincinnati, Ohio   45268.  A quality control sample is also available
        from the above address for fluorometric analysis for chlorophyll
     •  Adequate environmental conditions should be provided during
        calibration
     •  A calibration interval for recalibration should be  assigned to all
        instruments and equipment listed in Table 3.2.3 and calibration
        standards should be specified
     •  A record of calibration should be maintained for each instrument
        or piece of equipment.  This record consists of:

        o  Date
        o  True value of standards and calibration value
        o  Factor, if any required to correct reading from  meter
        o  Amount of drift
        o  Initials of person performing calibration

      •  Written calibration procedures should be provided  for all  listed
        instruments and equipment.  These are usually collected in a quality

                                      130

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        control manual
     •  The calibration record should include the traceability of the
        standard used in the calibration
     •  A calibration checklist should be provided and instruments
        and equipment should be checked and adjusted periodically by a
        laboratory service man or consultant if service is not avail-
        able locally, following manufacturer's instructions as closely
        as possible

3.2.2.3  Field Sampling with Laboratory Analysis—

     Assuming that all samples are collected properly in the field and
handled adequately, and field analysis is not dictated by preservation
problems, laboratory analysis can then be initiated by the project manager.

     The analysis of samples is basically in two groups:  qualitative and
quantitative analysis.  Table 3.2.4 lists the major analyses for field-
collected samples of common organisms.  Qualitative analysis is primarily for
organisms and species identification.  Quantitative analysis includes other
functional tests such as:

     •  Number
     •  Productivity
     •  Growth
     •  Bioassays
     •  Chemical analyses (tissue analyses)

Recently, the taste test (flesh tainting) of commercial macroinvertebrates
and fish has come into the territory of laboratory analysis.

     In a broad sense, bioassay can be divided into field bioassay and
laboratory bioassay.  Most biologists are familiar with laboratory bioassay
which in general comprises aquatic and mammalian bioassay.  Aquatic and mam-
malian laboratory bioasnays are discussed in Section 3.3 and 3.5, resoectivelv.
Field bioassays will come under further discussion later in this section.


              TABLE 3.2.4  MAJOR ANALYSES OF COMMON ORGANISMS
              IN FIELD SAMPLING WITH LABORATORY ANALYSES


         Organisms                     Major Analysis

         Viruses                       Identification

         Bacteria                      Identification
                                       Colony count

         Phytoplankton                 Identification and counts
                                       Diatom species proportional count
                                       Ash-free weight
                                       Chlorophyll analyses
                                                                (continued)

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                  TABLE 3.2.4 (Continued)
Organisms
Major Analysis
Zooplankton
Periphyton
Macrophyton
Macroinvertebrates
Fish and other
   vertebrates
Plants
ATP determinations
Primary productivity
Bioassay

Identification and counts
Dry weight
Ash-free weight
Bioassay

Identification and counts
Diatom species proportional counts
Ash-free weight
Chlorophyll analyses
ATP determinations
Primary productivity
Bioassay

Identification
Dry weight
Ash-free weight
Chlorophyll analyses
Bioassay

Identification and counts
Dry weight
Ash-free weight
Age and growth
Bioassay

Identification and counts
Age determinations
Growth measurement
(in length and/or weight)
Bioassay

Identification
Dry weight
Ash-free weight
Bioassay
                             132

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     Chemical analysis is usually done to determine the amount of three
groups of major environmental contaminants, pesticides, metals, and radio-
isotopes, in each trophic level of organisms.  Tissues are often employed for*
histopathological analysis and histochemical (or enzyme) analysis.

3.2.3 Sampling Method

     This section covers sample collection, sample preparation, preservation
and storage, and sample analysis for the following test subjects (for viruses
and bacteria, see Section 3.4.1):

     •  Plankton
     •  Periphyton
     •  Macrophyton
     •  Macroinvertebrates
     •  Fish
     •  Birds
     •  Mammals
     •  Plants

3.2.3.1  Plankton—

     In "Biological Field and Laboratory Methods" (U.S. EPA, 1973), plankton
is defined as organisms suspended in a body of water which, because of their
physical characteristics or size, are incapable of sustained motility  in
directions counter to the water currents.  In fresh water they are generally
microscopic; in sea water, they are more frequently larger.  All of them
drift with currents.

     Plankton consists of both plants and animals.  The planktonic plants are
referred to as "phytoplankton" and animals are "zooplankton".  Reports have
shown that complex and intimate relationships exist among the various com-
ponents of plankton.  Phytoplankton such as algae occur as unicellular,
colonial, or filamentous forms, and usually constitute the greatest portion
of the biomass of plankton.  These chlorophyll-bearing plants carry on
photosynthesis and serve as primary producers.  The zooplankton in fresh water
comprise primarily protozoans, rotifers, cladocerans, and copepods; in marine
waters, a much greater variety of organisms is encountered.  Zooplankton and
other herbivores graze upon the phytoplankton and, in turn, are preyed upon by
other organisms, thus passing the stored  energy along to larger and usually more
complex organisms.  In this manner nutrients become available to large
consumers such as macroinvertebrates and fish.

     For the following reasons, plankton have been used extensively by
pollution engineers and biologists as indicator organisms for environmental
assessment studies (Rand et al., 1975):

     •  Because of their short life cycles, plankton responded
        sensitively to environmental changes, and hence the species
        composition and standing crop indicate  the quality of the
        water mass in which they reside


                                      133

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     •  Because of their small size and great numbers* they not only
        strongly influence certain non-biological aspects of water
        quality (e.g., pH, color, taste, and odor), but in a practical
        sense, they are a part of water quality.  However, because of
        their transient nature, plankton communities may be of limited
        value in assessing water quality

     The decision on selecting the sites and stations for plankton samples
should be made according to the following:

     •  Formulate a study design which includes study objectives,
        the limitations of manpower, time and money
     •  Select the same sampling sites selected by previous
        investigators if consistent with study aims, for a better
        understanding of current results
     •  Select the sampling stations as near as possible to those
        selected for chemical and bacteriological sampling to
        insure maximum correlation of findings
     •  Select a sufficient number of stations in as many sites
        as necessary to define adequately the kinds and quantities
        of plankton in the waters studied
     •  Understand the physical nature of water (such as currents,
        depths, and volume of flow) that influences greatly the
        selection of the sampling stations

     Keeping of field notes and inserting of sampling labels must be taken
into consideration in plankton collection.  Both labels and marker should
be waterproof.  Record the following information on all labels:

     •  Sample identification number
     •  Location, including name of water body, distance and
        direction to nearest city, county and state, latitude
        and longitude, or other description
     •  Date and time
     •  Name of collector
     •  Type of sample, including equipment used, sample
        volume, tow length if net is used, vertical or
        horizontal tow
     •  Preservatives used and concentrations
     •  Special preparation of samples desired
     •  Types of analyses to be performed, as a reminder and a
        cross-check

Keep a field notebook containing all information written on the label, plus
pertinent additional notes.  These notes should include, but not be limited
to:

     •  Weather conditions:  wind direction and intensity, and
        cloud cover
     •  Physical nature of water:  smooth water surface or rippled,
        water color and turbidity, and depth at station
                                     134

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     •  A list of all types of samples taken at station
     •  Information on direction, distance, and description
        of effluents in the vicinity
     •  Other general descriptive information

     Sample size depends on the type and number of determinations to be made;
the number of replicates depends on the statistical design of the study and
the statistical analyses selected to assist in data interpretation (Rand et
al., 1975).

                 TABLE 3.2.5  PRESERVATION OF PHYTOPLANKTON
Preservatives
Preparation
Usage
Formalin plus sodium
tetraborate (neutra-
lized Formalin)
Neutralized Formalin
plus cupric sulfate
Neutralized Formalin
plus detergent
solution
Merthiolate
Neutralize Formalin with   Preserve the samples for
tetraborate to pH«7.0-7.3. more than 1 year, but this
Five milliliters of the    preservative will cause
neutralized formalin are   many flagellated phyto-
added for each 100 ml of   plankton to lose flagella
sample
Add saturated cupric sul-
fate solution to the pre-
served samples.  One
milliliter of the satu-
rated solution per liter
of sample is adequate

One part of surgical
detergent to five parts
of water makes a stock
solution.  Add 5 ml of
stock per liter of sample

Dissolve 1.0 gram of
merthiolate, 1.0 gram of
aqueous saturated iodine-
potassium iodide solution
(prepared by dissolving
40 grams of iodine and
60 grams of potassium
iodide in 1 liter of
distilled water), and 1.5
gram of borax in 1 liter
of distilled water.  Add
37.3 ml of this stock
solution to 1 liter of
sample
Maintains the green color
of phytoplankton samples
and aids in distinguishing
photoplankton from detritus
Prevents clumping of settled
organisms
Stain cell parts to simplify
identification.  But this
preservative will cause
blue-green algae to lose gas
from their vacuole and so
enhances settling
                                      135

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                        EXAMPLE:  PHYTOPLANKTON

|He Collection

Sample equipment:  Nets, pumps, tubular equipment and cylindrical type of
samplers are generally used for phytoplankton sampling (see Table 3.2.1).
However, the U.S. Environmental Protection Agency has recommended the use
of the cylindrical type of sampler with stoppers (U.S. EPA, 1973).  Net
collection of phytoplankton is recommended for quantitative analysis.
Pumping may harm delicate algae when tubing is flushed between stratified
samplings.
QUALITY CONTROL -- Use only nonmetallic samplers when metal analysis,
algae assays, or primary productivity measurements are being performed.

Sample volume:  When phytoplankton densities are less than 500 units per
milliliter   collect a 6-liter sample.  In richer waters, a sample of 1
to 2 liters is sufficient.
QUALITY CONTROL — For quantitative analysis, caution must be taken to be
exact on sample volume.

Sample preservation:  See Table 3.2.5 for preservatives used, their prepara-
tion and usage.  Each preservative has its advantages.
QUALITY CONTROL — When diatom slides are to be made, DO NOT use detergent
solution which prevents clumping of settled organisms.
QUALITY CONTROL — If merthiolate is used as preservative, the preserved
samples are not sterile, and SHOULD NOT be stored for more than 1 year.
After that period of time, Formalin should be used.

;\fter collection and preservation, phytoplankton samples sometimes must be
concentrated in the laboratory before analysis.  Three common techniques
used for concentrating are:  sedimentation, centrifugation, and filtration.
Sedimentation is preferred (U.S. EPA, 1973).  Because of the different sedi-
mentation rates of the various sizes and shapes of phytoplankton, caution
must be exercised during sedimentation.

From the sample concentrates, a subsample is always withdrawn for phyto-
plankton semipermanent wet mounts, phytoplankton membrane filter mounts,
or diatom mounts.  See Standard Methods, 14th edition (Rand et al., 1975)
for the detailed preparation of mounting slides.  The mounted slides will
be ready for microscopic examination for species composition and count.

Sample Analysis

Qualitative analysis—Phytoplankton identification:  Identify the phytoplank-
ton to species level whenever possible.  When identifying phytoplankton, it
is useful to examine fresh, unpreserved samples.  An initial examination is
needed because most phytoplankton samples contain a diverse gathering of
organisms.
QUALITY CONTROL — Use a good quality compound binocular microscope with a
mechanical stage.  Require a substage condenser for high magnification.
QUALITY CONTROL — For exact magnification, the microscope must be adequately
calibrated.

                                  136

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 jUALITY CONTROL — Utilize all  available references for exact
 dentlflcatlon and consult the  authority for questionable Identification.

Quantitlve analysis-Phytoplankton count:  Identify and count the phyto-
plankton directly.  In samples  with very low populations, concentrate
organisms and then count.   In those samples where algae concentrations
are extreme, or where silt or detritus may Interfere,  carefully dilute
a small portion of the sample 5 to 10 times with distilled water, and
then count.  The apparatus (five types) used in counting phytoplankton
are listed below.  For procedures of using each apparatus, see Standard
Methods, 14th edition (Rand et  al., 1975), or Biological Field and
Laboratory Methods (U.S. EPA, 1973).
QUALITY CONTROL — Use an adequately calibrated microscope.
QUALTIY CONTROL — The analyst  should carefully manipulate the dilution
and concentration of the samples that may Introduce error.

(1) Sedwlck-Rafter (S-R) cell is 50 mm long by 20 mm wide by 1 mm deep
and the total volume is 1000 mm3 or one ml.
QUALITY CONTROL —  The diluted or concentrated samples must be well
mixed before transfer Into counting chamber.
QUALITY CONTROL —  Be exact on the volume of the well-mixed sample to
be transferred Into the chamber, e.g., 1.0 miliniter.
QUALITY CONTROL — Examine the  underside of the cover  slip and add these
organisms to the total count.          0
QUALITY CONTROL — Always randomly select the strips or fields for count.
QUALITY CONTROL — Be consistent on counting phytoplankton that lie only
partially within the grid or that touch one of the edges.

(2) Palmer-Maloney (P-M) Nannoplankton cell:  The cell has a circular
chamber 17.9 mm In diameter and 0.4 mm deep, with a volume of 0.1  ml.
QUALITY CONTROL — Use P-M cell only for nannoplankton count.

(3) Bacterial Counting cells and Hemocytometers:  The  cell (Petroff-
Hausser cell) 1s 1 mm x 1  mm x  1/50 mm which gives a volume of 1/50 mm3.
The depth in the hemocytometer  1s 1/10 mm (compared to 1/50 mm in a P-H
cell), and thus the total  stabilization volume 1s 1/10 mm3.
QUALITY CONTROL— Do not attempt routine counts until  experienced 1n use
of the bacterial counter and the statistical validity  of the results
is satisfactory.
QUALITY CONTROL — Employ these cells for counting high-density
populations (50,000 cells/ml) that may be found in sewage ponds or in
laboratory cultures.

(4) Membrane Filter:  A special filtration apparatus using a vacuum of
0.5 atmospheres and 1-inch, 0.45 u membrane filters.
QUALITY CONTROL — Be exact on  the amount of water to  be filtered.
QUALITY CONTROL — The filtered samples from estuarlne and sea waters
must be rinsed with distilled water to remove salts.
 UALITY CONTROL — Record the occurrence of each species 1n 30 random
 ields.
                                137

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(5) Inverted microscope with cylindrical counting chambers:
Precision-made, all-glass counting chambers in a wide variety of
dimensions are available.  The chambers can also be easily and inexpen-
sively made in the laboratory.
QUALITY CONTROL — Allow complete sedimentation before making a count.
On the average, allow 4 hours per mm of height.
QUALITY CONTROL — Make random counts.  For field counts, as a general
rule, count a minimum of 100 of the most abundant species.  At higher
magnification, count more fields than under lower power.

Diatom Analysis:  Identification and Count.  Prepare diatom slides as
directed in Standard Methods, 14th edition (Rand et al., 1975) or
Biological Field and Laboratory Methods (U.S. EPA, 1973).  Identify and
count the diatoms at high magnification under oil.  Randomly examine
lateral strips the width of the Whipple grid, and identify and count all
diatoms until 250 cells are counted.
QUALITY CONTROL — The slides must be labelled with all relevant
information.
QUALITY CONTROL — Use "A  Guide to the Common Diatoms at Water Pollution
Surveillance System Stations", as a basic reference (Weber, 1971).
Utilize all other available references and experts for identifying
purposes.
QUALITY CONTROL -- Adopt a consistent system on counting.  Count all
diatoms within the borders of the grid.  Ignore small cell fragments.

There are two other counting methods for quantifying phytoplankton:
Lackey Drop Microtransect Counting Method; and Particle Counters (Rand
et al., 1975; Lackey, 1938; Maddux and Kanwisher, 1965).  The former
method is a simple method of obtaining counts of considerable accuracy
with samples containing a dense plankton population.  It is similar to
the S-R strip count.  The particle counters are used effectively for
counting pure culture but are not suited for enumerating natural
plankton populations in surface water grab samples because they do not
discriminate between the plankton and other particles such as silt or
organic detritus.
Biomass determination:  Chlorophyll can be measured in vivo fluoro-
metrically or in acetone extracts (in vitro) by fluorometry or spectro-
photometry.  The measurements can be categorized into four types:  (1)
spectrophotometric determination of chlorophyll a, b, and c (Trichromatic
Method), (2) fluorometric method for chlorophyll a, (3) spectrophoto-
metric determination of pheophytin a (a common degradation product of
chlorophyll a), and (4) fluorometric determination of pheophytin a.
QUALITY CONTROL — Keep the stored samples in the dark to avoid photo-
chemical breakdown of the chlorophyll.
QUALITY CONTROL -- Mix the phytoplankton sample thoroughly to ensure a
homogenous suspension of algal cells (in vivo measurement).
QUALITY CONTROL — Calibrate the spectrophotometer or fluorometer with
calibration standards.  See Section 3.2.2.2.
QUALITY CONTROL — Stopper the cuvettes to minimize evaporation of acetone
during the time the spectrophotometric or spectrofluorometric readings
are being made (in vitro measurement).
                                138

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   See Standard Methods,  14th Edition (Rand et al.,  1975) for details
   concerning equipment and reagents used,  procedures,  and calculations.

   Phytoplankton productivity measurements  indicate  the rate of conversion
   from inorganic carbon  to an organic form by phytoplankton during photo-
   synthesis.  These measurements are useful  in determining the effects of
   pollutants and nutrients on the aquatic  community (U.S. EPA, 1973).   Two
   widely used methods of measuring phytoplankton productivity in situ  are:
   the oxygen method of Gaarder and Gran, and the carbon-14 method of
   Steeman-Nielsen.
   QUALITY CONTROL — DO  NOT use phosphorus-containing  detergents to clean
   BOD bottles.   Acid-clean them, JUST BEFORE use, rinse with the water being
   tested.
   QUALITY CONTROL — Double precaution must be taken to insure light
   exclusion of the  dark  bottles used.
   QUALITY CONTROL — Build supporting line or rack  that DOES NOT shade the
   suspended bottles.

   (1)  Productivity, Oxygen Method:  See Rand et al. (1975), pp. 1037-1039
   and 440-454.
   QUALITY CONTROL — Water used to fill duplicate clear, darkened, and
   initial-analysis  bottles SHOULD come from the same grab sample.
    'UALITY CONTROL — Incubate the BOD bottles for at least 2 hours, but
    EVER longer than it takes for oxygen-gas bubbles  to form in the clear
   bottles.

   (2)  Productivity, Carbon-14 Method:   General  directions for this method
   are found in Rand et al.  (1975),  pp.  1039-1041,  pp.  278-282, pp. 293-302
   and 633-682.
   QUALITY CONTROL — Water used to  fill  BOD bottles  SHOULD come from the
   same grab sample.
   QUALITY CONTROL — Incubate the samples for up to  4  hours.
    lUALITY CONTROL — There should be at least 1,000  cpm (counts per minute)
    n the filtered sample for statistical  significance  (Strickland and
   Parsons,  1968).
I
•  Cell  Volume of Phytoplankton:   Determine the shape of a cell  and then the
   volume of a cell  by using the  simplest geometric configuration.
   Calculate the total  volume of  any phytoplankton species by multiplying
   the average cell  volume in cubic micrometers by the number per liter.
   QUALITY CONTROL — For better  representation of cell  volume,  measure
   20 individuals of each species to get average cell  volume for each sam-
   pling period.
   QUALITY CONTROL — Be exact on the subsample volume from the  well-mixed
   sample.
   QUALITY CONTROL — Keep a consistent counting system.

•  Cell  Surface Area of Phytoplankton:   Same as above, but measure the cell
   surface area instead.
   QUALITY CONTROL — Same as described for cell volume of phytoplankton.
                                    139

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      TABLE 3.2.6  THE PRESERVATION OF ZOOPLANKTON (U.S. EPA, 1973)
Preservatives
Preparation
Usage
Formalin
Add sodium tetraborate to
obtain pH of 7.0 to 7.3.
Obtain a final concentration
of 5% neutral Formalin.
Preserve grab
samples.
Formalin plus glycerin
Ethanol plus glycerin
Rose Bengal stain
Add 5% glycerin to 5% neutral  Preserve the con-
Formalin,                      centrated net
                               samples.
Freezing
Add 5% glycerin to 70%
ethanol.
Add 0.04% Rose Bengal stain
to 5% neutral Formalin.
The concentrated sample is
placed in a fine-meshed bag,
drained of excess water,
placed in a plastic bag,
and frozen for laboratory
processing.
Preserve the con-
centrated net
samples.

Differentiate
animal and vege-
tative material
in turbid samples

For chemical
analysis of zoo-
plankton samples.
                          EXAMPLE:  ZOOPLANKTON
Sample Collection
    Sampling equipment:  a messenger-operated water bottle, or metered
    plankton net is often used for collecting quantitative samples.   Filter
    surface-water samples through nylon netting or tow an unmetered
    plankton net through the water to obtain semi-quantitative samples.
    Towing from an outboard motor boat and casting of nets are two common
    techniques in  sampling.  Tows can be vertical, horizontal or oblique
    tow for different purpose of study.  Net casting is used to obtain a
    qualitative estimate of relative abundance and species present.  To
    sample most sizes of zooplankton, two nets of different mesh size can
    be attached a short distance apart on the same line.
    QUALITY CONTROL --  When towing with a boat is employed, maintain speed
    to ensure a wide angle (near 60°) for easy calculation of the actual
    sampling depth of the net.
    QUALITY CONTROL — Clean nylon nets thoroughly,  rinse with clean water
    and dry before storing.
                                    140

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    QUALITY CONTROL -- Rinse messenger-operated samplers with clean water,
    dry and lubricate all  moving parts with light machine oil.

    Sample Volume:  In lakes, large rivers, estuaries  and coastal  waters,
    filter 1.5m3 (horizontal tow) to 5m3 (oblique tow) of water through nets
    for adequate representation of species present.   For samples in flowing
    streams and ponds, filter 20 liter surface water through  a  No.  20 net  to
    obtain an estimate of  zooplankton present.
    QUALITY CONTROL — Be  sure to obtain the exact volume of  sample for
    quantitative analysis.

•   Sample Preservation:   Preserve zooplankton samples with 70% ethanol, 5%
    neutral Formalin (pH of 7.0 to 7.3), or LugoVs  solution  (Rand  et al,
    1975).  Freeze the concentrated samples for chemical  analysis
    (U.S. EPA, 1973).   See Table 3.2.6 for the detailed description of
    zooplankton preservation.
    QUALITY CONTROL — Usually, use Formalin to preserve samples obtained
    from coastal waters.
    QUALITY CONTROL — If  the sample is taken from estuarine  or sea water,
    the nylon bag (used to hold concentrated net samples for  chemical
    analysis)must be dipped several times in distilled water  to remove the
    chloride from interstitial seawater, as chloride can interfere  with
    carbon analysis.

Sample Preparation

.   Concentrate zooplanktbn samples by sedimentation and then mount them on
    slides as directed in  Standard Methods, 14th Edition (page  1020) if
    desired.
    QUALITY CONTROL -- Must recover organisms (especially cladocera)  that
    cling to the surface of the water in the settling  tube.

Sample Analysis

    Qualitative Analysis:   Make an initial  examination.  Identify  the small
    (nanno) zooplankton during the routine phytoplankton qualitative analysis.
    Identify Copepoda. Cladocera and other larger forms with  the use of a
    binocular dissecting microscope at a magnification of 20  to 40.  Identify
    rotifers at 100.  All  animals should be identified to species  if possible,
    QUALITY CONTROL -- Use all available, appropriate  taxonomic reference
    at the bench.  See a list of recommended references (U.S.  EPA,  1973).
    QUALITY CONTROL -- Use   taxonomic expertise in  identification  of
    questionable specimens.

    Quantitative Analysis  — Pi pet Method:   Dilute the concentrated sample.
    Withdraw 1 ml of subsample from the center of well-agitated water-
    plankton mixture with  a 1-ml  Stempel  pipet.   Transfer the subsample
    to a gridded culture dish (110 x 15 mm) with 5-mm  squares.   Enumerate
    (about 200 zooplankters) and identify under a dissecting  microscope
    (U.S. EPA, 1973).
    QUALITY CONTROL -- Randomly select 10 strips for  rotifer count.
                                    141

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    QUALITY CONTROL -- Accurately determine the volume of the counting
    chamber from  its  inside dimensions because this volume changes the
    outcome of  the calculated count.

    Biomass Determination  — Dry and Ash-free Weight:  Determine dry weight
    by  placing  the aliquot of concentrated sample  in a tared porcelain
    crucible and  drying at 105°C for 24 hours.  Subtract the weight of the
    crucible to obtain the dry weight.  After the  dry weight is determined,
    place  the crucible in  a muffle  furnace at 500°C for 1 hour.  Cool,
    wet the ash with  distilled water, and bring to a constant weight at
    105°C.  Subtract  the weight of  crucible and ash from the dry weight to
    obtain ash-free weight.  This method is sometimes used for phytoplankton
    biomass determination.
    QUALITY CONTROL — Wash the sample well with distilled water by
    settling to reduce the amount of contamination.
    QUALITY CONTROL -- Must collect sufficient sample to provide several
    aliquots each having  100 mg wet weight or 10 mg dry weight because at
    least  two replicate aliquots must be processed for each sample.  Must
    keep the temperature  in the oven or furnace  constant for all drying
    processes.

    In  addition to the aforementioned techniques for biomass determination,
    there  is a  recently developed method of measuring adenosine triphosphate
    (ATP)  in plankton that provides a means of determining the total viable
    plankton biomass.  According to Weber  (1973),  the ratio of ATP to bio-
    mass varies somewhat  from species to species,  but appears to be constant
    enough to permit  reliable estimates of biomass from ATP measurements.
    The method  is simple  and relatively inexpensive.  The instrumentation
    is  stable and reliable.  The method also has many potential applications
    in  entrainment and bioassay research, especially plankton mortality
    studies.  See equipment and reagents used, procedure, and calculation of
    ATP in Standard Methods, 14th Edition  (Rand et a!., 1975).

    Moreover, the "nitrogen fixation" idea  is introduced by aquatic
    physiologists to  measure metabolic rates of plankton communities in
    the water.  The two methods for estimating nitrogen fixation rates in
    the laboratory are the 15N  isotope tracer method and the acetylene
    reduction method.  It  is found  that the great  variation in the rate of
    nitrogen fixation with different types of organisms and with the con-
    centration  of combined nitrogen in the water makes it impossible to
    use nitrogen  fixation  rates to  estimate biomass of nitrogen-fixing
    organisms in  surface waters.  But the acetylene reduction method is
    useful in measuring nitrogen budgets and in algal assay work (Stewart
    et  al.,  1967  and  1970; Weber, 1973).

3.2.3.2  Periphyton—

    Periphyton  is also known as "Aufwuchs" in German, which can be seen in
some literature.  It  is defined  as "a community of  microscopic plants and
animals  associated with the surface  of submersed objects.  Many of the
protozoa and  other minute  invertebrates and algae that are found in the


                                    142

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plankton also occur In the periphyton" (Rand et al.,  1975).

     Two types of sampling are generally used for periphyton sample
collection:  qualitative and quantitative sampling.   Qualitative studies
concerned only with systematics of periphyton require no  elaborate or
complicated apparatus for the collection of samples.   Knives,  scrapers,  and
similar implements have sometimes been modified for specific habitats,  e.g.,  a
curved knife for scraping epiphytic periphyton from bulrushes  (Wetzel and
Westlake, 1974).  For the measurement of biomass,  artificial substrate is a
most widely accepted sampling method compared to those devices that have been
developed for the collection of quantitative samples  from irregular
surfaces.

     Since the periphyton community is an excellent indicator  of water
quality, the selection of a minimum of two sampling stations will be
required to provide data on the community in both the pollution-free zone
and the polluted zone in a body of water.  However, a more intensive
sampling program is recommended if possible.

                            EXAMPLE:  PERIPHYTON

Sample Collection

     Natural substrate method:  qualitative samples may be taken by scraping
     submerged rocks, sticks, and other substrates available at the station.
     QUALITY CONTROL -- This method is not recommended for the collection of
     quantitative samples because of inaccurate measurements of sampling
     areas.

 •   Artificial substrate method:  The standard (plain, 25 x 75 mm) glass
     microscope slide is a most suitable artificial substrate for quantita-
     tive sampling.  Plexiglas slides may be used in  place of glass slides.
     In large rivers or lakes, a floating sampler (Rand et al., 1975, p.
     1046) is advantageous when turbidities are high  and the substrates must
     be exposed near the surface.  In small, shallow  streams or littoral
     areas of lakes where turbidity is not a critical factor,  substrates may
     be exposed in two possible ways:  (a) attach the substrates with PLASTIC
     TAK adhesive to bricks or flat rocks in the stream bed, or (b) anchor
     Plexiglas racks to the bottom to hold the substrates.  In areas where
     the siltation is a problem, hold the substrate  in a vertical position to
     avoid a covering of silt (U.S. EPA, 1973).
     QUALITY CONTROL — The depth of exposure must be consistent for all
     sampling sites.
     QUALITY CONTROL — Because of unexpected fluctuations in water levels,
     currents, wave action, and the threat of vandalism,  duplicate samplers
     should be used (U.S.  EPA, 1973).
     QUALITY CONTROL — A minimum of four replicate  substrates should be
     taken for each type of analysis (U.S. EPA 1973).

     After taking samples, further separations may be needed to obtain the dif-
     ferent components of periphytes (e.g., algae, diatom) relatively free  from
     detritus and mineral matter.  Sample preparation varies according to  the
     method of analysis; see the 14th edition of Standard Methods, Section
                                      143

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     1003C (Rand et al . , 1975).  Generally, preserve samples that are taken
     for counting and identification in 5% Formalin or other suitable material.
     Wetzel and Westlake (1974) suggest that Lugol's iodine (made up of 10 g
     of pure iodine, 20 g of KI, 200 ml of distilled water and 20 g of glacial
     acetic acid combined a few days prior to using; store the solution in dark
     glass bottles; added to the samples in a 1:100 ratio) and 5% mercuric •
     chloride are particularly suitable.  If the material is for chlorophyll
     analysis, store it at 4°C in the dark in 100 ml of 90% aqueous acetone.
     Use bottle caps with a cone-shaped polyethylene seal to prevent evaporation.
     Sladeckova (1962) gives detailed suggestions for the collection, preserva-
     tion, and transport of periphyton on artificial substrates.
Sample Analysis
     Identification
     QUALITY CONTROL -- Use all available taxonomic references for each pos-
     sible composition of periphyton community: algae, fungi, protozoae,
     rotifer, microcrustacea.
     QUALITY CONTROL -- Consult the taxonomic authority whenever necessary.
     Counting:  Sedwick- Rafter count is a universal method.  The quantitative
     determination of organisms on a substrate can be expressed as:
                                 -  C x 1QQQ mm 3 x V x pF
                  M«
                  No.
                                      L x u x D x $ x A
     where  C  =  number of cells counted (tally)
            V  -  sample volume, ml
           DF  =  dilution factor
            L  =  length of strip, mm
            W  =  width of strip (Whipple grid image width), mm
            D  =  depth of a strip (S-R cell depth), mm
            S  =  number of strips counted
            A  =  area of substract scraped, mm2

     QUALITY CONTROL -- Thorough mixing must be done by vigorous shaking prior
     to counting.
     QUALITY CONTROL -- If a material is too concentrated for a direct count,
     a proper dilution must be made.
     QUALITY CONTROL — Avoid clumps of cells in the counting cell  because
     these clumps could result in inaccuracy of the count.

     Diatom proportional count:  Mount diatom slides as described in Standard
     Methods (see Plankton, 1002 D.3) or "Biological Field and Laboratory Meth-
     ods" (U.S. EPA, 1973, page 11 in Plankton Section).  Identify and count
     all diatoms within the borders of the grid until 250 cells (500 halves)
     are tallied.
     QUALITY CONTROL — The slides must be labelled with all  relevant information.
     QUALITY CONTROL — Use "A Guide to the Common Diatoms at Water Pollution
     Surveillance System  Stations" (Weber, 1971) as a basic  reference.

3.2.3.2  Macrophy ton —
     Macrophytes are all aquatic plants possessing a multi-cellular structure
with cells differentiated into specialized tissues.  Their communities range
from completely submerged stands of large algae (e.g.,  Chara, Cladophora) ,
mosses  (e.g., Fontinalis) , pteridophytes (e.g., Isoetes) and angiosperms

                                     144

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(e.g., Elodea, Ranunculus ssp.),  through stands of rooted plants with float-
ing leaves (e.g.,  Nymphaea)  and mats of floating plants with emergent leaves
(e.g., Eichhornia. Lemna)~to wetlands with plants with little except their
underground parts  submerged  (e.g.,  Equisetum,  Phragmites, Rhizophora).

     As usual, there are two types  of studies  in relation to macrophyton:
qualitative and quantitative sampling.   Before beginning a quantitative
investigation it is desirable to  have a statistical design which will assist
in determining the best sampling  procedure, sampling area size,  and number
of samples.  It is recommended that the appropriate TP (Terrestrial Produc-
tivity) techniques should be adopted (Milner and Hughes, 1968; Blackburn
et al., 1968; Edwards and Owens,  1960;  Forsberg, 1959; Jervis, 1969; West-
lake, 1966; Westlake, 1968).

     Due to natural phenomena, there are frequent shifts in plant population
of a particular site or location.  Quality control is generally obtained
by standardizing the time of the  year and accumulating data over a long
period of time.  The specific quality control  in sample collection, sample
preparation and sample analysis will be summarized and briefly discussed
below.

              TABLE 3.2.7 SAMPLING  EQUIPMENT FOR MACROPHYTES
                              (Westlake, 1974)
Type of Equipment
Suggested Application
Scoop, diver operated
Ekraan dredge

Petersen dredge
Petersen dredge, modified

Cylindrical sampler



Quadrate frame sampler



Pronged grab
Important root systems

Mud; small root system

Hard bottom; poor sampling
Hard bottom; better sampling

Soft bottom; upright plants, small

root system

Soft bottom; tall plants, small
root systems

Luxuriant vegetation; roots from
soft bottom
                           EXAMPLE:  MACROPHYTON

Sample Collection

    Selection of sampling site and frequency:  The general aim will be to

                                     145

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   remove and weigh the vegetation from enough known areas to obtain
   a mean biomass sufficiently accurate to show significant differences
   between sampling periods and sites (Westlake, 1974).
   QUALITY CONTROL — Individual plants should be collected at each
   sampling site sufficient to establish the frequency and diversity
   of the population.  At least four sites should be selected for each
   location   (U.S. Department of the Interior, 1972).
   QUALITY CONTROL — Normal statistical methods must be applied with
   caution because the spatial variation is often nonrandom (Westlake, 1974).
   QUALITY CONTROL — Select the size and shape of the sampling area to
   reduce the variability, e.g., large quadrates, rectangular quadrates in
   contagious (clumped) communities, summed quadrates along transects
   parallel to gradients (Westlake, 1974).
   QUALITY CONTROL -- In stands of limited area care must be taken to
   avoid damaging the community excessively and affecting subsequent
   samples (Westlake, 1974).
   QUALITY CONTROL -- Avoid sampling or experiments in previously
   disturbed areas (Westlake, 1974).

•  Sampling equipment:  See Table 3.2.7.
   QUALITY CONTROL -- Select appropriate gear for personnel and nature of
   the survey, types of plants.

•  Sampling techniques:  Approach the sampling areas by wading, in boats or
   by diving; remove plants by hand or by sampler.
   QUALITY CONTROL -- Collect base maps and detail information related to
   terrain concerning the safety of personnel.
   QUALITY CONTROL -- Use appropriate mode of transportation related to the
   area.
   QUALITY CONTROL -- Mark off areas for hand sampling with stakes and
   strings if large, or quadrate frames if smaller, to avoid overlap.
   QUALITY CONTROL -- Use a net set downstream of the sampling area to
   collect the cut submerged plants.

Sample Preparation for Macrophytes (including washing, sorting, sub-sampling,
and drying for future analysis)

•  Wash in a shallow sloping trough with a jet of water (approx. 2.5 atm.)
   to remove soil,  epiphytes, and animals.
   QUALITY CONTROL — Be sure to wash well because the total weight of
   unwanted material may exceed the weight of the plants.
   QUALITY CONTROL -- Recover plant fragments by flotation by passing the
   water through a 1/2-inch (approx. 12.7-mm) mesh net.

•  Sort into different species for productivity studies.
   QUALITY CONTROL -- Requires trained and experienced personnel but no
   special equipment is needed.

•  Preservation.
   QUALITY CONTROL -- Small, delicate samples should be preserved in
   buffered 4% Formalin solution.
                                    146

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     QUALITY CONTROL -- All  other samples  may be dried in a plant press and
     mounted for further identification.

  •  Record and label.
     QUALITY CONTROL —  All  necessary information must be recorded on the
     label and field notebook (or data sheet).

  •  Dry:  Use a domestic spin-drier and/or general  purpose laboratory oven.
     QUALITY CONTROL — Dried samples must be cooled in a desiccator and
     sealed in polyethylene  bags  before weighing,  as many samples can take up
     to 10% moisture from air.

  •  Subsample.
     QUALITY CONTROL — Random sampling process  should be used.
     QUALITY CONTROL — The  weed  should be chopped and well  mixed before
     taking subsamples.

  •  Individual specimens should  be properly prepared (mounted or preserved)
     and annotated with recorded  data before the sample analysis  begins.
     Sample analysis of macrophytes includes, in general, identification,
     biomass (or standing crop) and productivity.   Dryweight biomass measure-
     ment may be summarized  as  follows:  A sample  is taken from a small
     defined area with  conspicuous borders.   The wet weight of material  is
     obtained after the plants  have drained for  a  standard period of time.
     The sample is then dried for 24 hours at 10°C and reweighed.   The dry
     weight of vegetation per unit area is then  calculated.

Sample Analysis

  •  Identification:  Identify  samples according to family,  genus,  and
     species.
     QUALITY CONTROL — Use  appropriate taxonomic  texts for identification.
     See reference list relevant  to aquatic plants in Section 3.2.4.2.

  •  Biomass or standing crop:  See description  of method just above.
     QUALITY CONTROL —Balances capable of holding bulky samples, weighing up
     to 5 to 10 kg of fresh  weights, will  be needed for samples from 1
     square meter.
     QUALITY CONTROL — Balances  capable of weighing up to 1 kg are most
     convenient for dry weight  determination.
     QUALITY CONTROL — For  consistent results,  the oven must be  calibrated
     to 105°C.

  •  Productivity:  Use of isolated shoots for emergent macrophytes.
     QUALITY CONTROL ~ Never use this method for  productivity of benthic
     plants.
     QUALITY CONTROL — The water used for incubation needs  to be taken from
     the same location  as the plants because of  the stratification of
     nutrients, temperature,  etc., in many habitats.
                                     147

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     Oxygen exchanges in light and dark enclosures in situ for submerged
     macrophytes .
     QUALITY CONTROL -- Possible sources of error in the application of the
     oxygen techniques are lacunal storage of oxygen, and irregular utili-
     zation of oxygen for respiration due to intermittent current stirring.
     QUALITY CONTROL — The results must be interpreted with extreme caution.
         technique in situ for submerged macrophytes.
     QUALITY CONTROL — The incubation chambers are recommended to be
     cylinders made of clear Plexiglass in various sizes to permit placement
     in situ around different species of plants.
     QUALITY CONTROL — The volume of the chamber must be calibrated.
     QUALITY CONTROL — The rooted organs of macrophytes must be included in
     the chambers.
     QUALITY CONTROL — Keep the incubation to a short mid-day period (e.g.,
     from 10:00 to 14:00 hr) of four hours because evidence suggests  that the
     production rates of this mid-day increment are good mean values  under a
     majority of light and other environmental conditions.
     QUALITY CONTROL — The excretion of organic matter, i.e., carbohydrates,
     during the photosynthesis by macrophytes presents a possible source of
     error in the employment of the 14C techniques.

  •  Chlorophyll determinations:  Analyze for chlorophyll a, b, c, and d.
     QUALITY CONTROL — Ensure thorough acetone extraction by grinding or
     homogenizing material.
     QUALITY CONTROL — Spectrophotometer must be adjusted and calibrated
     according to manufacturer's manual at regular time intervals.


3.2.3.4 Macro invertebrates —

     The macroinvertebrates, as discussed in this section,  are animals that
are large enough to be seen by the unaided eye and can be retained by a U.S.
Standard Number 30 sieve (28 meshes per inch, 0.595 mm opening).  Many small
or slender individuals and early life stages of these invertebrates will pass
through the sieve and not be included.  The sieve, however, is a practical
and rapid method of sorting most macroinvertebrates from their substrate.
They may be collected by various methods using equipment such as grabs (or
dredges), Surber samplers, corers, nets, seines,  artificial substrates,
trawls, or other specialized samplers.  A few basic requirements for  field
invertebrate sampling are:

     •  The selection of the best sampler requires evaluation of the
        physical conditions in which the sampler will be used.  These
        conditions include substrate type, and depth
     •  The kind of sampler selected is used consistently for a
        particular area so that population characteristics may be compared
     •  Use more than one sampler type to obtain good representation  of
        the fauna which reside in natural substrates
                                      148

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                       EXAMPLE:  MACROINVERTEBRATES

Sampling Equipment/Methods

 •  Grab devices.
    QUALITY CONTROL — Understand the patchy distribution of some organisms
    in nature.
    QUALITY CONTROL — Use grab sampling only for qualitative studies, i.e.,
    estimate of numbers of taxa.  Due to the problems  in depth of pene-
    tration, angle of closure, completeness of closure of the jaws and loss
    of sample material during retrieval, creation of a "shock" wave and
    consequent "wash-out" of near-surface organisms, and stability of the
    sampler at the high-flow velocities often encountered in rivers, grab-
    collected samples provide an imprecise estimate of aquatic macro-
    invertebrate populations (U.S. EPA, 1973).
    QUALITY CONTROL -- Collect additional samples to increase precision in
    the selected method.

 •  Sieving devices.
    QUALITY CONTROL -- Collect the samples from downstream to upstream.
    QUALITY CONTROL — Stand on the downstream side of a sieving device and
    take replicates in an upstream or lateral direction.

 •  Coring devices.
    QUALITY CONTROL — Best suitable for sampling the  relatively homogeneous
    soft sediments of the deeper portions of lakes.

 •  Nets.
    QUALITY CONTROL — In the aquatic environment, place the top of the drift
    nets just below the surface to lessen the chance for collection of float-
    ing terrestrial insects.
    QUALITY CONTROL — For field insects study, use sweep-net method to com-
    pare populations from one area at different times, or from different
    areas.  Bear in mind that three major difficulties encountered in
    sampling are:  daily changes in the environment, differences in the
    growth habits and structure of the vegetation, and differences in the
    agility and tenacity of the insects (Davis and Gray, 1966).

 •  Artificial substrates.
    QUALITY CONTROL — Use EPA-recommended samplers (multiple-plate sampler
    and rock basket sampler) for studying a macroinvertebrate community.
    QUALITY CONTROL — Caution should be exercised in  the reuse of samplers
    that may have been subjected to contamination by toxicants, oils, etc.
    QUALITY CONTROL ~ Adoption of a 6-week exposure period is provisionally
    recommended as standard (Rand et al., 1975).
    )UALITY CONTROL — Unless the water is exceptionally turbid, a 1.2-meter
    [4-foot) depth is recommended as standard.
    QUALITY CONTROL — Never use artificial substrates to measure the
    productivity of a particular environment.

Sample Preparation
                                    149

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  Sieving.
  QUALITY CONTROL — Use  a U.S. Standard No. 30 sieve to separate samples
  collected with.conventional  sampling devices.
  QUALITY CONTROL — Sieving should be  done  in the field Immediately after
  sample collection.

  Preservation.
  QUALITY CONTROL — Preserve  the  samples  in 70 percent ethanol.  Do not
  use  Formalin.  When  necessary, specimens could be  transferred from
  alcohol to  pins.
  QUALITY CONTROL — Samples are preserved immediately in plastic or glass
  containers.
  QUALITY CONTROL — Rose Bengal stain  at  a  concentration of  approximately
  200  mg/1  in the preservative may be  used to stain  the animals to aid in
  sorting  (Rand  et  al.,  1975;  Slack et  al.,  1973).

  Records and labelling.
  QUALITY CONTROL — WEite all information  (see Section 4.2.1.6) on water-
  resistant labels  with  a waterproof marker.
  QUALITY CONTROL — This information  must be recorded in a permanent
  record.

  Sorting and subsampling.
  QUALITY CONTROL — Subsampling may be used for samples containing
  excessively large numbers  of organisms before sorting.  But be sure that
  sample is thoroughly mixed and distributed evenly  over the  bottom of a
  shallow  tray before  delineation.
    lUALITY  CONTROL — All  organisms should  be sorted  into major categories
    i.e.,  insect orders,  molluscs,  worms) and placed  in vials  containing
   70 percent ethanol.
  QUALITY  CONTROL — All  vials from a  sample should  be labelled internally
  with the  sorter's name and the sample identification (log)  number and
   kept as  a unit in a  suitable container until organisms are  identified,
  counted and the data are recorded on the bench sheets.  See a typical
   laboratory bench  sheet in  Table  3.2.8.
  QUALITY  CONTROL -- A check on the sorting  procedure can be  done by re-
  examination of the sample  or by  aliquot  analysis.

Sample Analysis

•  Identification.
   QUALITY CONTROL  — The accuracy  of  identification  will depend greatly  on
   the available taxonomic literature.   See Section 3.2.4.2.
   QUALITY CONTROL  —  Store identified  specimens  in a reference collection
   for quality control  checks.
   QUALITY CONTROL  — Mount the whole  organisms  or  parts  thereof on  glass
   slides for examination at high magnification  to  make species identifica-
   tion whenever necessary.  Make  proper labelling  on the prepared slides.
   QUALITY CONTROL — Rear the collected insect  larvae in  the  laboratory  to
   aid in identifying the difficult-to-identify  species.
                                    150

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   tUtM of water body
   Collected by _
   Sorted by ^^_^_^
          Lot Ho. 	
          Station No.
          Date collected
*
DIPTERA















TR1CHOPTERA




PLECOPTERA '




EPHEMEROPTERA




ODOHATA




NEUROPTERA

HEHIPTERA


COLEOPTERA


T /U\i.
^\"f











































pi











































TOTAL











































DRY WGT











































*



CRUSTACEA















HIRUDINEA




NEMATODA




BIVALVIA




GASTROPODA




OTHER







TOTAL
*











































DRY WCT











































   Total * of organisms
   Total t of taxa
Total dry weight
Ash-free weight
   * Initials of  taxonomists in this column       L»larvne, N • nymph, H - pupae
                                                             (Weber, 1973a)
Figure 3.2.3  Laboratory bench sheet for aquatic macroinvertebrates
                               (Weber,  1973a).
                                      151

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    QUALITY CONTROL —  Identification can be checked by re-examination or by
    multiple analysis.

 •  Biomass.
    QUALITY CONTROL —  Use "ash-free dry weight" method.
    QUALITY CONTROL —  Hard parts, e.g., shells, etc., can introduce errors.
    QUALITY CONTROL —  Determine the wet weight with a good, calibrated
    analytical balance  to the nearest 0.1 mg.  Do the same for ash-free
    weight.
    QUALITY CONTROL —  Use of weight is not recommended unless it can be
    equated to dry weight by determination of suitable conversion factor.
    QUALITY CONTROL --  Use appropriate manuals for biomass determination,
    e.g., a Manual on Methods for the Assessment of Secondary Productivity in
    Fresh Waters  (Edmondson and Winberg, 1971).

 •  Bioassay.
    QULAITY CONTROL —  See Section 3.2.4.4 and 3.2.4.5.

 •  Counting.
    QUALITY CONTROL —  See Section 3.2.4.3, and Table 3.2.9.
    QUALITY CONTROL —  Refer to Edmondson and Winberg's manual.
3.2.3.5 Fish-

    Many sampling methods have been available to assess the fish populations.
The methods vary greatly in their precision and the cost-effectiveness
required to obtain information.  A creel census or other catch record from
commercial and sport fisheries is useful for showing the harvestable nature
of the fish population.  Other methods in which all species and sizes of
fishes in a body of water may be sampled include draining the body of water,
seining, use of chemicals, netting, trapping, or electroshocking.

                               EXAMPLE:  FISH

Sampling Equipment/Methods

 •  Catch records/recording.
    QUALITY CONTROL — Standard forms should be designed to record the
    desired information.

 •  Seines/seining.
    QUALITY CONTROL — Cotton seines should be treated with a fungicide to
    prevent decay.   Nylon seines are recommended.
    QUALITY CONTROL — Seining is only effective in shallow water and is more
    useful in qualitative study.

 •  Nets/netting (gill nets, trammel nets, etc.).
    QUALITY CONTROL -- Gill  nets made of multifilament or monofilament nylon
    are recommended.
    QUALITY CONTROL — Replace the individual floats (usually supplied with
    nets) with a float line  made with a core of expanded foam and use a lead

                                     152

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   core leadline instead of individual  lead weights  to  reduce  net
   entanglement problems.
   QUALITY CONTROL  —  Gill  and  trammel  netting  are in extensive  use  to
   sample fish populations  in estuaries,  lakes,  reservoirs  and large
   rivers.  When drifting gill  or  trammel  nets  are set,  they require
   constant surveillance.

•  Traps/Trapping (Trap  nets, hoop nets,  fyke net, etc.).
   QUALITY. CONTROL  —  Trap  and  hoop nets  made of nylon  have a  longer life.
   Protect cotton nets from decay  by treatment.

•  Trawls/trawl ing  (fry  trawl,  otter trawl, etc.).
   QUALITY CONTROL—  The use of trawls requires  experienced personnel.
   QUALITY CONTROL  —  Trawls are best used to gain information on a
   particular species  of fish rather than to estimate the overall  fish
   population.

•  Chemicals/chemical  fishing  (rotenone,  antimycin,  etc.).
   QUALITY CONTROL  —  The most  widely used toxicant  is  rotenone.
   Recommended concentrations of the 5% preparation:  0.1 ppm  for sensitive
   species, 0.5 ppm for  most species, and 1 to  2 ppm for resistant species.
   QUALITY CONTROL  —  Chemical  sampling is usually employed on a spot basis,
   e.g., on embayment  of a  reservoir or a short reach of a  river.
   QUALITY CONTROL  —  An appropriate efficient  spraying  equipment must be
   selected to apply rotenone emulsion.

•  Electroshocker (AC, DC,  etc.).
   QUALITY CONTROL  —  Before deciding which design to use,  the biologist
   should carefully review  the  literature. See more than 30 listed
   references in "Biological Field and Laboratory Methods for  Measuring the
   Quality of Surface  Waters and Effluents"  (U.S. EPA,  1973).
   QUALITY CONTROL  —  The crew  should wear rubber boots  and electrician's
   gloves and adhere strictly to safety precautions.
   QUALITY CONTROL  —  Electrofishing is more effective  and  efficient for
   sampling fish population at  night.
   QUALITY CONTROL  —  Electrofishing devices are effective  in  collecting
   most sizes and species of fish  from many different environments.

•  Fish studies are usually dependent on  data collected in  the field and
   include fish identification, weight, length  and other observations.  The
   collected samples should be  prepared as described in the following for
   further studies, e.g., age,  growth and condition  of  fish, and fish kill.

Sample Preparation

•  Preservation and storage.
   QUALITY CONTROL  —  A  10% Formalin is usually used as a fish preservative.
   Preserve fish in the  field.  Add 3 to  5 g borax and  50 ml glycerin per
   liter of Formalin.
   QUALITY CONTROL  ~  Fish  longer  than 75 mm should  be  slit on the right side
   of body to allow penetration of the preservative.


                                    153

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    QUALITY CONTROL -- For permanent preservation, specimens must be washed
    in running water for at least 24 hours and placed in 40 percent isopropyl
    alcohol.
    QUALITY CONTROL — Only plastic or glass containers should be used.
    QUALITY CONTROL — Samples may also be iced or placed in dry ice for
    preservation.

 •  Data Recording.
    QUALITY CONTROL -- See Section 3.2.1.4.
    QUALITY CONTROL — Use the metric system for length and weight
    measurements.

 •  Sample analysis is usually done in the laboratory and after preservation
    and includes identification, age, and growth determination, condition
    factor, histopathology, and flesh-tainting.

Sample Analysis

 •  Identification.
    QUALITY CONTROL — Use appropriate manuals for fish identification.
    See reference list in (U.S. EPA, 1973).
    QUALITY CONTROL — Confirm questionable identification with Federal,
    state and university fish taxonomists.

 •  Age and growth.
    QUALITY CONTROL — Use appropriate personnel for age determination.
    QUALITY CONTROL — Use adequate handbook for the age and growth study.
    For example, Carlander's Handbook is good for freshwater fishes
    (Carlander, 1969).
    QUALITY CONTROL — Use available written computer package for the back
    calculation of fishes' growth history.

 •  Condition (including natural and man-induced mortalities).
    QUALITY CONTROL — Use trained and experienced personnel.
    QUALITY CONTROL — The speed of response to fish kill is a key to success

 •  Counti ng.
    QUALITY CONTROL — See Section 3.2.4.3, and Table 3.2.8.
    QUALITY CONTROL — Use adequate handbook for fish population study,  e.g.,
    Ricker's handbook is good for fish in freshwater (Ricker, 1971).

 •  Flesh tainting.
    QUALITY CONTROL — Uniform taste quality should be assured before
    exposure of test fish.
    QUALITY CONTROL — A test panel should be trained in flesh tainting  and
    should be given acceptable samples for comparison.

 •  Bioassays.
    QUALITY CONTROL — See Sections 3.2.4.4 and 3.2.4.5.

 •  Biomass.
    QUALITY CONTROL -- See Section 3.2.4.6.

                                     154

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    QUALITY CONTROL — Use Ricker's Handbook (Ricker, 1971).


 3.2.3.6 Birds—

                              EXAMPLE:  BIRDS

 Sample Collection

 •  Qualitative study:  Both shooting and trapping techniques are used by
    bird collectors for collecting qualitative specimens.   A shotgun armed
    with different-sized shots (e.g., Nos. 10, 6, 4, 2 and BB) is necessary
    for general collecting of birds.  Various traps are indicated in Table
    3.2.1.
    QUALITY CONTROL — Use proper shooting equipment.  Never use a rifle to
    collect birds as the rifle bullet tears them all up.

 •  Qualitative study:  Trapping is only the means for a catch-mark-recapture
    (CMR) study for estimating avian population.  Sampling plans which are
    very critical in the quantitative study of birds should include site
    selection of sampling, the frequency of sampling, number of sampling units
    and size of sampling plots.   The size of the sampling  unit (or plot)
    depends on the size, mobility and abundance of the species.  For
    partridge, 100 hectares may  be recommended (Petrusewicz and Macfadyen
    1970).  The number of sample units depends on the homogeneity of the
    habitat as well as on the numbers and characters of the distribution of
    birds in it.  In a normal heterogeneous habitat, an  average of 5 to 10
    sampling units is usually adequately representative.  In an unknown
    habitat a larger number is recommended (Petrusewicz and Macfadyen, 1970).
    QUALITY CONTROL — The project supervisor should consult a statistician
    for a final decision on a formal sampling plan.  The complete review
    of historical information on areas and species studies would be greatly
    helpful.
    QUALITY CONTROL —  Use appropriate means for catch-mark-recapture (CMR)
    study.
    QUALITY CONTROL —  Trap sites, marked birds and other pertinent
    information should be recorded permanently.   All entries should be in
    carbon ink.

Sample Preparation

 •   Skinning and Mounting:   Anderson (1964)  has  discussed  these techniques in
    Chapter IV, Collecting and Skinning Birds of his book  entitled, "Methods
    of Collecting and Preserving Vertebrate  Animals."
    QUALITY CONTROL — No samples or specimens will  be analyzed without
    proper identification labels.
    QUALITY CONTROL — There must be a capture sheet for every bird.
    QUALITY CONTROL — Avoid the use of abbreviations  and  laboratory jargon-
    in ten years or less  they may be difficult to  be understood.           '
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 •  Preservation:   Alcohol  and formaldehyde are two commonly used liquid
    preservatives  for preserving soft parts of birds,  stomach contents,  and
    bird droppings.  A mixture of powdered  arsenic and powdered arsenic plus
    borax (in about equal  proportions by volume) is the most satisfactory
    preservative for the birds'  skins.
    QUALITY CONTROL --If laparotomy is carried out, laparotomy sheets
    including sex, band number,  the date and time of laparotomy, name of
    operation, etc., must be completed and filed permanently.

 •  Labelling:  Label all  prepared specimens with the  pertinent information,
    e.g., identification number, location and date of  collection, etc.,  in
    accordance with the pertinent record.

Sample Storage

 •  Deepfreeze or refrigerate the samples which are delayed for preparation
    or analyses.
    QUALITY CONTROL — No samples should be delayed for further analyses,
    e.g.,chemical  residue analysis in the laboratory.

 •  Fumigate the skinned and stuffed birds for long-term storage.  DDT  or
    moth balls can be used as fumigants.
    QUALITY CONTROL -- All  skinned, or preserved specimens should be stored
    with labels for permanent records.

Sample Analysis

 •  Identification:  Identify all specimens to species level whenever
    possible.
    QUALITY CONTROL — Be exact in identification with available taxonomic
    references.  Refer to an authority for identification of questionable
    birds.

 •  Number of Birds:  The methods of studying bird populations are greatly
    varied depending on the species studied, the habitat, technical means,
    time and money available.  Table 3.2.8 shows the various methods that
    have been used by wildlife biologists.  Two of the most familiar
    methods are direct count  and mark-and-recapture study.
    QUALITY CONTROL — Good eyes of the individual making count, and a  good
    pair of  binoculars are essential in estimating bird population.
    QUALITY CONTROL — If a sample census is used, a census datum should be
    accompanied by a clear statement of constraints and definitions under
    which it was collected and by a critical evaluation of its accuracy.

  •  Weight and Biomass:  Obtain individual bird weight by weighing a repre-
    sentative number of birds and calculating average (X).  Measure biomass
    by  adding up the weights of all birds or calculate by multiplying the
    average  (X) by numbers estimated (N).
    QUALITY CONTROL - Choose the individuals that represent either classes
    or  a succession of known time intervals in the history of their
    population.


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    QUALITY CONTROL -- The accuracy of biomass measurement is dependent
    completely on the accuracy of determinations of numbers and of weighing.

  •  Bioassay.  See Section 3.2.4.4 and 3.2.4.5.


3.2.3.7 Mammals—


                              EXAMPLE:  MAMMALS

Sample Collection

  •  Qualitative study:  The larger mammals are almost invariably taken by
    shooting, and some of the smaller species, such as rabbits and squirrels,
    are more often shot than trapped.  A shotgun is indispensable for
    general collecting of mammals, too.  A double-barrelled gun is pre-
    ferable, and shells loaded with different size shots,  Nos. 10, 6, 4,
    2, and BB.
    QUALITY CONTROL — Use appropriate shooting equipment  for collecting
    mammal specimens.  For example, the rifle is not ideal for collecting
    the smaller mammals as the rifle bullet tears them up  too much.

  •  Quantitative study:  Trapping is more often used for mark-and-recapture
    studies to estimate animal population.  Traps used by  collectors vary
    with animals to be trapped and collecting individuals.  See Table 3.2.1.
    Sampling plans are a must in the quantitative study of mammals.   The
    plans, as usual, include sampling frequency, sampling  site selection,
    number of sampling units and size of sampling plots.  The first two
    elements depend heavily on the objective of the study.  The size of the
    sample unit depends on the size, mobility, and abundance of the species.
    For small mammals, 2 to 6 hectares may be recommended, and for hares
    and deer, 100 hectares (Petrusewicz and Macfadyen, 1970).  The number
    of sample units depends mainly on the homogeneity of the habitat as well
    as on the numbers and character of the distribution of animals in it.
    In a habitat of normal heterogeneity, an average of 5  to 10 sampling
    units is usually adequately representative.  In an unknown habitat a
    larger number is recommended  (Petrusewicz and MacFayden, 1970).
    QUALITY CONTROL -- Trap sites and other records must be noted in
    permanent notebook.
    QUALITY CONTROL — Use appropriate traps for various sizes of animals
    and their habitats.
    QUALITY CONTROL — Consult statisticians to adopt a formal sampling
    plan.

Sample Preparation

 •  Sample labelling.   Label  all  prepared samples with necessary information
    on waterproof paper and in waterproof ink.
    QUALITY CONTROL — Specimens  should always be fully labelled at the time
    they are prepared, as  a specimen without an authentic  record has no
    scientific value.
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       TABLE 3.2.8  THE METHODS FREQUENTLY USED BY WILDLIFE BIOLOGISTS
                FOR ESTIMATING NUMBER OF ANIMALS IN THE FIELD

Methods involving direct counts of animals:

   Territory-Mapping methods
   Drive counts
   Temporal censuses
   Extermination or total capture
   Sample censuses
   Pseudo sample censuses (e.g., The Kind Method, Frye's strip census,
    time-area counts, etc.)

Methods involving animal signs and related objects:
    Auditory index
    Pellet counts
    Miscellaneous indices (e.g., counts of leaf nests for squirrels)

Methods involving marked animals:
    Petersen or Lincoln Index
    The Schnabel Method
    Jolly's Method for multiple recapture experiments
    The Frequency of Capture Method
    Miscellaneous methods (e.g., Schumacher-Eschmeyer Method)

Methods involving "reduction" of population size and rate of "capture":
    The Graphical Solution
    The Leslie Method
    DeLury's Method

Method of selective reduction or Increase (Dichotomy method or the change in
 composition method) (Overton, 1971):
    Age and sex determinations, birth and death rates, etc.  See "Criteria of
     Sex and Age" by Taber  (1971), and "Population Analysis" by Eberhardt
     (1971).  The former article describes explicitly the techniques of de-
     termining sex and age  of birds and mammals vhile the latter article
     directs the wildlife biologists how to estimate the survival and re-
     cruitment rate, to analyze population structure, and to predict popula-
     tion size and trends.  For these determinations, the following QUALITY
     CONTROL measures must  be used:
       All bird specimens should have the sex verified by dissection.

       Use trained and experienced personnel.
       Use available computer packages for analyzing complex, dynamic bird
     populations.  See "Using Computers in Wildlife Management" by Adams, in
     Giles (1971).
       Follow standard procedures for weighing and preserving avian gonads
     used by Avian Physiology Laboratory, Fish and Wildlife Service.
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 •  Skinning and Preservation.   See  Chapter  III,  Skinning  Mammals  of  "Methods
    of Collecting and Preserving Vertebrate  Animals"  (Anderson,  1964)  for
    full  details.  The prepared  skins, whole animals  and any  parts of animals
    can be preserved by recommended  techniques  described in Table  3.2.2.  A
    mixture, in about equal  proportions  by volume,  of powdered arsenic and
    powdered borax is the most satisfactory  preservative for  the skins of
    small mammals.  Alcohol  and  Formalin (formaldehyde) are most commonly
    used for preserving entire specimens or  any soft  parts of animals,
    stomach contents, droppings, etc.
    QUALITY CONTROL — Use proper preservatives in  right concentrations  for
    various animals or animal parts.

Sample Storage

 •  Fumigate the skinned and stuffed animals when storing with naphthaline
    flakes, moth-balls or insecticides  (e.g.  DDT).
    QUALITY CONTROL — Store all specimens with labels for permanent
    records.

 •  Deepfreezing or refrigerating of some samples is  recommended.

Sample Analysis

 •  Identification:  Identify all specimens  to  species level  whenever
    possible.
    QUALITY CONTROL — Use all available references for exact identification
    and consult the proper authority, e.g., museum curators for unidentifiable
    animals.

 •  Number of mammals:  The methods  of  determining  population size are many
    and greatly varied, depending on the qualities  of the  species  studied,
    the habitat, and technical means and time available.   Main categories of
    methodology are total count, sample  counts, catch-mark-recapture  (CMR)
    methods, and many other specialized  methods.  See Table 3.2.8  for a
    list of methods that are described  in details by  Overton  (1971).
    QUALITY CONTROL -  If a "total census" is used, there  is  no  question of
    variance or confidence limits in the sampling sense.   If  not,  a census
    datum should be accompanied  by an explicit  statement of constraints and
    definitions under which it was collected and  by a critical evaluation
    of its accuracy.
    QUALITY CONTROL — Use the "census"  methods in  consistent ways through-
    out the study period.
    QUALITY CONTROL -- Personnel who count animals  should  have good eyes and
    be equipped with a good pair of  binoculars.

 •  Weight and Biomass:  Individual  weights  are obtained by collectively
    weighing a representative number of  animals and calculating  the average
    (X).   Biomass can either be  measured by  summing up the weights of all
    animals or calculated by multiplying the average  individual  weights (X)
    obtained at a census by numbers  (total estimated  population, N).
    QUALITY CONTROL — Individuals should be chosen to represent either age
    classes or a succession of known time intervals in the history of their

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    population.
    QUALITY CONTROL -- The accuracy of biomass  estimates  depends
    principally  on the accuracy of determinations  of numbers  and  of
    weighing.

    Population analysis including age and sex composition,  birth  and  death
    rates,  and numerical  abundance.  Taber (1971)  has described clearly  and
    in detail  the criteria of age and sex for birds and mammals and the  ways
    of determining their sex and age.  For other elements of  population
    analysis,  see Eberhardt's (1971) article, "Population Analysis" that
    instructs  wildlife managers how to estimate the rate  of survival  and the
    rate of recruitment,  to analyze population  structure  and  finally  to
    predict population size and trends.
    QUALITY CONTROL — Use trained and experienced personnel.
    QUALITY CONTROL — Use available computer packages for  studying complex,
    dynamic wildlife populations.  See Adams on "Using Computers  in Wild-
    life Management" in Giles (1971).

    Bioassays.  See Sections 3.2.4.4., 3.2.4.5., and 3.3.
3.2.3.8 Plants—


    The following example which is in part derived from "Taxonomy of
    Vascular Plants"  (Lawrence, 1951), includes sample collection,  sample
    preparation, sample preservation and sample analysis,  with respect to
    quality control.
                              EXAMPLE:   PLANTS
Sample Collection
    Certain items of equipment are indispensable to plant collecting,  particu-
    larly a collecting pick (for digging up rhizomes, deep-seated bulbs or
    conns, and the roots of most herbaceous plants), a strong knife or a
    machete, and a pair of pruning shears (for cutting woody material  to
    pressing size).  Besides, a garden rake or potato digger is useful in
    collecting submerged aquatic plants.
    QUALITY CONTROL -- Use standard collecting equipment.  Most required
    collecting equipment is available from biological supply houses.
    QUALITY CONTROL -- Use one method consistently through the study period.

    Basically, there are three major ways to handle freshly collected plant
    material.  The first, and most satisfactory method, is to press each
    plant as it is collected.  Secondly, the plant materials are accumulated
    in a metal collecting can or vasculum.  The third method, used more in the
    tropical rain forests than in temperate regions, is to carry collected
    specimens in a rucksack.
    QUALITY CONTROL — Plants should be pressed or processed as soon as
    possible.


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Preparation of Specimens

    Pressing:   Conventionally, most  presses  comprise a  pair of wood or metal
    frames, blotters,  pressing papers, and straps or strong cord.  The
    specimen to be pressed  is arranged within  the folded sheet of  pressing
    paper that has been placed on  a  blotter, and another sheet is  placed over
    it.   If the plants are  to be dried with  aid of artificial heat, a sheet of
    corrugated material (ventilator)  is  used between each pressing paper and
    its  specimen,  otherwise no corrugates are  used and  the press is built up
    by an alternation  of  blotter-pressing paper-blotter, and so on.  The
    press frames are on the top and  bottom of  the press, and it is then
    "locked up" by means  of straps or stout  cord.
    QUALITY CONTROL -- Select specimens  that are free from evidence of Insect
    feeding, rust  infections, and  other  obvious pathological symptoms.
    QUALITY CONTROL — Avoid depauperate individuals.
    QUALITY CONTROL -- Ensure that the specimen is either in flowering or
    fruiting condition.
    QUALITY CONTROL -- When an herbaceous specimen is collected, always in-
    clude enough of the underground  parts to show their character.

    Keeping wet material  without its  spoiling  is a problem faced by
    collectors working in tropical regions,  or under emergency situations
    when adequate  drying  facilities  are  lacking.  Two techniques have been
    demonstrated as useful  in these  cases, but the results are inferior to
    those from the usual  method of processing.  In either case, the objective
    is to keep the material  from decomposing after it has been collected and
    arranged in pressing  papers, until such  time as it  can be dried by normal
    procedures. These two  techniques are use  of a solution of two parts of
    concentrated formaldehyde (40%)  and  three  parts water, or use of a
    solution of one part  of formaldehyde and two parts  of 70 percent alcohol
    for  temporary  preservation of  plant  specimens before drying.

    Drying:   There are two  types of  drying techniques:  those accomplished
    without heat and those  with the  aid  of artificial heat.
    QUALITY CONTROL — No corrugate  should be  employed  when using the drying
    technique  without  heat.
    QUALITY CONTROL — Either technique  can  produce specimens of poor quality
    and  because the drying  process is much accelerated  when heat is used
    greater care must  be  exercised during all  its stages to produce quality
    specimens.

•   Mounting:   Usually specimens are  mounted on sheets  of standard size
    herbarium  paper (11%  by 16% inches).  After mounting, they are stored
    in   special cases built to fit  sheets of  this size.  Herbarium papers in
    a selection of qualities are available from biological supply sources.
    Mounting is accomplished by the  use  of glue or paste, the use of
    adhesive linens, or the combination  of both.  There are three techniques
    most commonly  used in mounting specimens with paste or glue.  The first
    technique, the glass  plate method, requires the use of a piece of plate
    glass at least 14  by  20 inches.   The paste is spread thinly over most of
    the  surface with a brush.  The specimen  is removed  from the pressing sheet
    and  placed face upward  on the  prepared plate, with  all parts of the lower

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   side in contact with paste.   It is  then  transferred  carefully  to  the
   sheet of mounting paper.   A  pressing  sheet  of  newsprint  is  placed over
   the specimen,  pressed firmly,  and taken  off and  discarded.   Reapply fresh
   paste on the plate for each  mounting.  The  second  technique requires no
   glass plate.  The paste is  brushed directly on  major  protions  of the
   specimen.  The third technique is designed  for mounting  specimens with
   very light weight and thin texture.   The specimen  is laid,  lower  side
   uppermost, on a piece of cheesecloth,  sprayed  with a diluted solution of
   paste by means of an atomizer, and  then  flipped  over onto the  sheet of
   herbarium paper.
   QUALITY CONTROL —  Use the  longest-lasting and  most durable paper for
   permanent museum collections.
   QUALITY CONTROL —  Use special  "A" Tin  Paste  and  Improved  Process Glue.
   Both products can be kept indefinitely when covered, and require  no
   thinning or heating before use.
   QUALITY CONTROL -- The glass plate  should be kept  clean  for each  mounting,
   and washed and set to dry after each  mounting  period.

•  Labelling:  For all specimens, whether pressed and mounted,  preserved, or
   stored, herbarium labels  are an essential part of  its  permanent preser-
   vation.  The purpose of the  label is  to  provide  the  user with  pertinent
   information in relation to specimen.
   QUALITY CONTROL -- The label should be large enough  to accomodate the
   data to be placed on it.
   QUALITY CONTROL — Under no  circumstances should a label be  so  large as  to
   require folding.
   QUALITY CONTROL — Data on labels should be typed.   Data written  on labels
   in longhand are always acceptable,  but must be legible.

•  Storing of fresh plant material  for residual analysis  of pesticides or
   other chemical substances.
   QUALITY CONTROL — Use proper  refrigeration equipment  for storage.

Sample Preservation

•  The preservation of herbarium  collections from insect  damages  is
   accomplished most effectively  by insecticides  used in  herbarium manage-
   ment including cyanide gas,  paradichlorobenzene, carbon  tetrachloride, or
   DDT.  The two principal  repellents  used  are naphthalene  compounds and
   paradi ch1orobenzene.
   QUALITY CONTROL — Use preservatives  properly  and cautiously.
   QUALITY CONTROL — If liquid preservation is used, the plant material
   should be photographed in sufficient  detail  to show the  form and  such
   other significant details as may otherwise  be  lost.

•  The preservation of juicy materials include the  use of formaldehyde (5$),
   alcohol (70%), or aqueous hydroxyquinoline  sulfate (1-2%).

•  Quick-freezing techniques are  also  used  for quantitative samples.
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Sample Analysis

  • Qualitative analysis:  Identify the prepared plant specimens to the
    species level.
    QUALITY CONTROL — Use available taxonomic references in relation to
    geographical flora.
    QUALITY CONTROL -- Use type specimens and consult experienced taxonomists
    for accurate identification.

  • Productivity
    QUALITY CONTROL — See Section 3.2.4.4 and 3.2.3.3.
3.2.4 Functional Tests

3.2.4.1 CulCuring—

    The objective of the culturing of organisms is to provide healthy
organisms, i.e., disease-free and toxicant-free, for bioassays.

    Assuming that organisms are transported under favorable conditions, stress-
free, uncrowded and at favorable temperatures, from the field to the labora-
tory,  these field-collected organisms must still be held in quarantine for at
least seven days for observation for parasites and disease in order to avoid
the transfer of such infections  to the laboratory culturing tanks or living
quarters.  During this period, the organisms can recover from the stresses
arising from treatment for disease or parasites during transit or upon arrival
in the laboratory.  Moreover, a sample of individuals can be used to determine
if they have accumulated potential toxicants in their body tissue.  This
check becomes extremely necessary and crucial because toxicant-contaminated
organisms, e.g., fish, are always more resistant if such toxicant is also
used as a test substance.

    During the quarantine period, the following quality assurance procedures
must be carried out to ensure healthy organisms for bioassays:

    •  Organisms should be fed daily
    •  Crowding should be avoided
    •  Dead and abnormal organisms must be discarded.  If the mortality
       is more than 10%, due to stress, parasites or diseases, destroy
       the lot.  Clean and sterilize all affected containers and equip-
       ment, and collect another supply of organisms from a new area, if
       possible (Rand et al., 1975)
    •  Organisms should be observed carefully for unhealthy signs and
       closely attended by experienced personnel

    Other important items that must be taken into consideration are:

    •  Laboratory animal management must be adequate.  Adequate manage-
       ment, e.g., housing and care, permits animals to grow, mature,
       reproduce, or behave normally, and to be maintained in physical
       comfort and good health

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    •  Personnel should be well trained and experienced and must care
       about the welfare of animals
    •  Animal facilities should be well designed and properly maintained.
       For example, the water supplies, freshwater or marine, are essential
       to assuring the success of rearing aquatic organisms

    Culturing procedures and attendant quality control procedures for a number
of organisms frequently used in bioassay follow.

    •  Phytoplankton, including freshwater and estuarine or marine algae

       References:  Rand et al. (1975), pp. 697-703; U.S. EPA (1976b)
                    pp. 19-25.

    Quality Control:

       o  Proper adjus  lent of nutrient concentrations, pH, light intensity,
and temperatures are essential prerequisites for the successful cultivation
of algae.
       o  Sterilization must be done on the culturing utensils when pre-
paring culturing media and whenever the algae are transferred.
       o  Use proper references that illustrate the instructions for the
cultivation of the respective algae.
       o  Use available pure cultures from culture collections all over the
nation or world.  See Table 3.2.10.

    •  Protozoa, e.g., Tetrahymena pyriformis

       Reference:  Rand et al. (1975), pp. 759-760.

    Quality Control:

       o  Use standard bacteriological techniques to prepare and autoclave
culture media and to t  nsfer axenic cultures of T\ pyriformis.
       o  Use available standard cultures.  See Table 3.2.10.
       o  Maintain Btock cultures at 26*0.5°C in a suitable incubator, i.e.,
Revco Model IB-1650 from Revco, Inc., Scientific Industrial Division, 1100
Memorial Drive, V/est Columbia, S.C. 29169.

    •  Freshwater cladocerans, Daphnia

       References:  Rand et al. (1975), pp. 763-764; Needham et al. (1937);
                    Parker and Dewey (1969).

    Quality Control:

       o  Use an appropriate culture medium for Daphnia culturing, e.g.,
manure-soil, a medium developed by Banta and modified by Anderson (1964).
       o  Once the cultures are initiated, the culture medium need not be
changed.
       o  When the stock Daphnia reach old age and the reproductive rate
drops, replace them with young females in fresh media.

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    •  Marine copepod, Acartia tonsa

       References:   Rand et al. (1975),  pp.  768-772;  U.S.  EPA (1976b);
                    Heinle (1969);  Mullin and Brooks  (1967);  Zillioux  and
                    Wilson (1966).

    Quality Control:

       o  Use an appropriate diet and proper concentrations of diet for
various stages of copepod.
       o  Use a generation cage that allows  the eggs  to pass through the net
and hatch, eliminating the possibility of cannibalism by adults.

    •  Crustaceans, including grass shrimp,  blue crabs, etc.

       References:   Rand et al. (1975),  pp.  795-806;  Hughes et al. (1974);
                    Spotte (1970);  Smith et  al. (1974); Cook (1967); Mock
                    (1974);  U.S.  EPA (1976b).

    Quality Control:

       o  Use a favorable water supply and accomplish the control of
competitors, predators and disease through filtration and sterilization by
ultraviolet light treatment.
       o  Handle the test subjects carefully and as little as possible.
       o  Avoid cannibalism by holding young stages of crayfish in separate
compartments.
       o  Routinely clean the sides and bottoms of compartments to remove
organic material, growth, and wastes.
       o  Feed the newly hatched nauplii of brine shrimp, Artemia salina to
the lobster larva to avoid cannibalism and to decrease the possibility of
developmental variability.
       o  Control the essential environmental factors such as DO, temperature,
and salinity as precisely as possible.
       o  The chelipeds of grass shrimps must be removed with fine surgical
scissors to prevent removal of eggs by the females.
       o  The crustacean larvae should be removed from containers containing
ovigerous females each morning and mixed together to insure uniformity of
test animals.

    •  Larvae of aquatic insects, including those of stoneflies, mayflies,
caddlsflies, and Diptera.

       References:   Rand et al. (1975),  pp.  829-830;  Fremling (1967);
                    Prater and Anderson (1976).

    Quality Control:

       o  All insects collected must be examined for injuries before rearing
in the laboratory.
       o  Avoid overfeeding which will cause DO difficulties.
       o  Supply suitable substrates for various insects,  e.g., highly

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organic ooze for chironomids.
       o  Keep the water temperature under control for nymphal growth, e.g.,
the maintenance of temperature at 14  to 17 C for limited nymphal growth of
mayflies.
       o  If dechlorinated, deionized tapwater is used, the water need never
be drained and changed.
       o  To reduce  the amount of turbidity, the charcoal filters should be
flushed clean on a monthly basis and the charcoal replaced on a semi annual
basis.

     •  Mollusks, such as oysters, clams, scallops and mussels

       References(   Rand  et al. (1975), pp. 836-839; Loosanoff and Davis
       (1963); Castagna and Duggan  (1971).

     Quality Control:

       o  Provide an abundant water supply rich in planktonic food organisms.
       o  Clean regularly the intake pipe and the water system to insure
that growth of fouling organisms in the pipes does not remove plankton
organisms before the water reaches the holding tank.
       o  Clean accumulated feces and silt from the holding tray at least
once a week, preferably twice a week.
       o  Thermal conditioning, e.g., induced spawning for scallops by
raising the water temperature to 27  to 30 C, should be well controlled.
Discard females once they have spawned.

     •  Fish

       References:   Rand et al. (1975), pp. 846-847, 849-853, 869-870;
                     National Academy of Sciences (1973); Stalnaker and
                     Gresswell (1974); Carlson and Hale (1972); Hokanson
                     et al. (1973); McCormick et al. (1972); Siefert (1972);
                     May (1970); Hirano and Oshima (1963); Hansen and Parrish
                     (1977); Middaugh and Dean (1974); Middaugh and Lempesis
                     (1976).

     Quality Control:

       o  Limit the  possibility of injuring fish during collection in the
field.  For example, the loss of some fish scales may cause disease problems
raising fish mortality.
       o  Always avoid rearing fish in unusually high densities in the
laboratory because disease becomes a very important factor that can alter
bioassay results or  even nullify bibassays by killing the test subjects after
they are weakened by the stress of the test substance or condition under study.
       o  Parasites  and diseases must be controlled in order to get reliable
bioassay results.  Prevention of disease is preferred.

     •  Animals, including birds and mammals

       References:  "u.s. DHEW, 1974"

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     Quality Control:
        o  Provide adequate veterinary care.
        o  Insure proper quarantine and isolation of animals.
        o  Be sure of absolute separation by  species.
        o  Appropriate diagnosis, treatment,  and control of diseases.
TABLE 3.2.9
Organisms
 MAJOR SOURCES  OF  STANDARD, PURE OR TYPE CULTURE COLLECTIONS  FOR
	ALGAE AND PROTOZOA	
  Source  of  Culture  Collection                            	
Algae
Protozoa
  (1)   Graduate  School  of Oceanography, University of  Rhode
       Island, Narragansett, Rhode Island, U.S.A.

  (2)   Department  of  Botany, University of Indiana, Bloomington,
       Indiana,  U.S.A.

  (3)   Eutrophication Research Program, Pacific Northwest
       Environmental  Research Laboratory, 200  S.W. 35th  Street,
       Corvallis,  Oregon 97330, U.S.A.

  (4)   Virginia  Institute of Marine  Science, Gloucester  Point,
       Virginia  23062,  U.S.A.

  (5)   Chesapeake  Biological Laboratory, Box 38, Solomons,
       Maryland  20688,  U.S.A.

  (6)   Dr.  Robert  Gviillard, Woods Hole Oceanographic Institution,
       Woods  Hole, Massachusetts, U.S.A.

  (7)   The  Institute  of Applied Microbiology,  University of
       Tokyo, Bunkyo-ku, Tokyo, Japan

  (8)   The  Botany  School of  the University of  Cambridge, Downing
       Street, Cambridge, Great Britian

  (9)   Department  of  Botany, The Hebrew University of  Jerusalem,
       Algal  Laboratory, Jerusalem,  Israel

 (10)   Sammlung  von Algenkulturen des Pflanzenphysiologischen
       Instituts,  Universitat GBttingen, Nikolansberger  Weg 18,
       34 GBttingen,  Germany
 (11)   Czechoslovak Academy of Sciences, Vinicha 5, Praha  2,
       Czechoslovakia

  (1)   The  American Type Culture Collection  (ATCC), 12301  Park-
       lawn Drive, Rockville, Maryland 20852,  U.S.A.

  (2)   The  Botany  School of the University of  Cambridge,
       Downing Street,  Cambridge, Great Britian

  (3)   Czechoslovak Academy of Sciences, Vinicha 5, Praha  2,
       Czechoslovakia
                                     167

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3.2.4.2 Identification—

    The published taxonomic works on organisms are comprehensive in scope and
to list them here, even in condensed form for one specific organism, is not
feasible.  A reference list for the identification of the following aquatic
organisms is given in "Biological Field and Laboratory Methods for Measuring
the Quality of Surface Waters and Effluents" (U.S. EPA, 1973):

      Organisms                          Page No. of Section

   Phytoplankton                         7, 8  PLANKTON
   Zooplankton                           12    PLANKTON
   Periphyton                             3    PERIPHYTON
   Macrophyton                            3    MACROPHYTON
   Macroinvertebrates                          MACROINVERTEBRATES
       Coleoptera                        33
       Diptera                           34
       Crustacea                         34
       Ephemeroptera                     35
       Hemiptera                         36
       Hirudinea                         36
       Hydracarina                       36
       Lepidoptera                       36
       Megaloptera                       36
       Mollusca                          36
       Odonata                           37
       Oligochaeta                       37
       Plecoptera                        37
       Trichoptera                       37
       Marine macroin-
          vertebrates
   Fish                                  16-18 FISH

    In Section 3.2.7 is a bibliography which includes other organisms than
those just mentioned and lists books,  manuals or reports most frequently
used in laboratories in the scientific community in which studies on
organisms are in progress.  This bibliography is organized in general
taxonomic orders,  i.e.:

                Virus                          Amphibia
                Fungi                          Reptilia
                Bacteria and Actinomycetes      Birds
                Protozoa                       Mammals
                Other Microinvertebrates        Plants
                Fish                           Aquatic Plants

    The U.S.  Environmental Protection  Agency has prepared many identification
manuals for selected organisms.   For example,  11 identification manuals for
aquatic macroinvertebrates have been prepared in the Agency's series, "Biota
of Freshwater Ecology Systems" since the  Agency's establishment (U.S. EPA


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 1976c).  These are:

    No.  1.  Freshwater Planarians  (Turbellaria) of North America
    No.  2.  The Genus Argulus  (Crustacea:  Branchiura) of the United States
    No.  3.  Freshwater Spaericean  clams  (Mollusca:  Pelecypoda) of North
            America
    No.  4.  Freshwater Polychetes  (Annelida) of North America
    No.  5.  Freshwater Amphipod Crustaceans (Gammaridea) of North America
    No.  6.  Aquatic Dryopoid Beetles (Coleoptera) of the United States
    No.  7.  Freshwater Isopoda (Asellidae) of North America
    No.  8.  Leeches (Annelida:  Hirudinae) of North America
    No.  9.  Crayfishes (Astacidae) of North and Middle America, 1972
    No.  10. Genera of Freshwater Nematodes (Nematode) of Eastern North
            America
    No.  11. Freshwater Unionacean  Clams  (Mollusca - Pelecypoda) of North
            America.

    They may be obtained without cost from the Aquatic Biology Section,
 Biological Methods Branch, Environmental Monitoring and Support Laboratory,
 U.S. Environmental Protection  Agency, Cincinnati, Ohio  45268.

    According to EPA's Newsletter  of Analytical Quality Control (April 1977,
 No. 33)  the following identification manuals are being prepared by various
taxonomic authorities:

    •  A key for the identification of 300  taxa of freshwater gastropods
       found in the North America  (John  B. Burch, Mollusk Division, Museum of
       Zoology, The University of  Michigan, Ann Arbor, Michigan)

    •  A key to the identification of the common species of rotifers and
       a summary of their environmental  requirements and pollution tolerance
       (John Gannon, University of Michigan Biological Station, Douglas Lake,
       Michigan)

    •  The classification, geographical  distribution and ecology of the
       mussels of the United States (U.S. Environmental Protection Agency
       and Tennessee Valley Authority)

    The  availability of taxonomic  references at the bench, and the skill and
 the systematic knowledge of the biologist, will determine the data quality
 resulting from identification  efforts.   No single biologist masters readily
 the sophisticated classification of living organisms, even an order of Insecta,
 e.g.,  Diptera.  No single reference is completely appropriate for the Order
 Diptera.  However, to ensure the validity and integrity of data in
 identifying organisms the biologist must be sure to do the following:

    •  Consult with appropriate experts  for good, adequate bench references
    •  Use the available EPA identification manuals
    •  Develop and use a reference specimen collection  (Weber, 1975)
    •  Utilize "outside" experts to solve difficult problems in specimen
       identification (Weber,  1975)
    •  Access the EPA "BIO-STORET" to verify the identification  (Weber,  1976;

                                      169

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       Nacht and Weber,  1976)
    •   Adopt computer  data storage  and  retrieval  system  similar  to
       "BIO-STORET"  for  geographical  regions,  e.g., Master  Species
       for New England States

3.2.4.3   Counting—

    The  many and diverse schemes  for  counting  numbers of organisms,  that  is,
estimation of  population size  (numbers  and density) fill a  voluminous
literature.  THese methods have been  briefly discussed and  referenced by
organism in Section  3.2.3.

    The  goal of population estimation appears  to  be  twofold.   First of all,
one wishes  to  obtain the best  possible  estimates  commensurate with  the
objectives of  the  study  and the time, money, and  personnel  available.  It is
also  desired to be able  to make a statement about the precision  of  the
estimate, i.e., how  well the assumptions  are met  and the influence  of sampling
error.   Overton (1971) gives considerable attention to the  problems  of
collecting concomitant information  to be  used  in  validating assumptions,
modifying the  estimator  if assumptions  are ill-founded,  and evaluating
variances and  confidence limits.

    The  quality control  of counting includes the  following  activities:

     • Apply a formal  sampling plan.  Count at least two samples.   Use
randomization  in   sample selection.   Samples must be labelled with  identifi-
cation number and other related information when they arrive at the  laboratory

     • Train and organize personnel for quality.   The same  individual should
be assigned to count the number of  organisms throughout  the study to
optimize consistency of  results

     • Use the "total  census"  method whenever  possible,  to  eliminate
sampling errors.   When other census methods are used, use them consistently
throughout study period  and compare the results from different methods.
Each  set of data should  be accompanied  by an explicit statement  of
constraints and definitions under which it is  collected  and by a critical
evaluation of  its  "precision and accuracy"

     • Utilize available automated  counting equipment (counters) for
counting of microorganisms

     • Establish regular audits of performance  in the field and  laboratory

     • Sign and witness  all the data collected  and all calculations

3.2.4.4   Biomass/Productivity—

    Productivity and biomass should not be confused.  Biomass is the
summation of the weights of all individual organisms measured at a  given
time, while productivity is "rate of production".  To avoid confusion, the
time  interval, e.g., year,  month, etc., should  be always  stated  when speaking

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of productivity.

    The definition of productivity has been elaborated upon by Odum (1971)
as follows:

    •  Primary productivity.  The rate at which radiant energy is stored
by photosynthethic and chemosynthetic activity of producer organisms (chiefly
green plants) in the form of organic substances which can be used as food
materials.

    •  Gross primary productivity.  The total rate of photosynthesis,  in-
cluding the organic matter used up in respiration during the measurement
period.  This is also known as "total photosynthesis" or "total assimilation".

    •  Net primary productivity.  The rate of storage of organic matter in
plant tissues in excess of the respiratory utilization by the plants during
the period of measurement.  This is also called "apparent photosynthesis"
or "net assimilation".

    •  Net community productivity.  The rate of storage of organic matter
not used by heterotrophs (net primary production - heterotrophic consumption)
during the growing season.

    •  Secondary productivities.  The rate of energy storage at consumption
levels.

    Methods for measuring productivity are summarized in Table 3.2.1Q.

3.2.4.5.  Physical Characteristics of the Environment—

    The principal physical characteristics of the environment that are of
interest are temperature, color, turbidity (or suspended solids), oil and
grease and airborne  particulates.  Water temperature is among the more
important of these characteristics because:

    •  The water covers a major part of the earth.  The life associated
with the water environment has its species composition and activity regulated
by water temperature.  Essentially all of the organisms are "cold-blooded"
or poikilotherms.  The temperature of the water regulates their metabolism
and their ability to survive and reproduce effectively.

    •  Industrial uses by man for process water and for cooling are likewise
regulated by the temperature of the water.  According to a report by the
Federal Water Pollution Control Administration (1967), "Temperature, a
catalyst, a depressant, an  activator, a restrictor, a stimulator, a killer,
is one of the most important and most influential water quality characteris-
tics to life in water."

    Standard experimental protocols for testing physical characteristics in
the field do not appear to have been developed:  Nakatani (1969) believes
that "the best practical method to investigate the effects of elevated
temperatures on salmon or other desirable species in the Columbia River is


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Methods
                           TABLE 3.2.10  METHODS FOR MEASURING PRODUCTIVITY
Uses
Quality Control
Harvest method          Terrestrial plants such as:
                         Cultivated crops, e.g.,
                          wheat, corn, rice.
                         Noncultivated ragweed
                          field, or where plants are
                          little consumed until
                          growth has been completed.
                         Young forests or crop-like
                          forest plantations.
                         Cattle range (expressed in
                          terms of the number of
                          cattle that can be sup-
                          ported by so many acres).

Oxygen measurement      Phytoplankton, macrophyton
 e.g., "dark-and-light"  in freshwater ecosystems
 bottle method, di-      and in marine ecosystems.
 urnal curve
 method
Carbon dioxide method
 e.g., enclosure
 method
Terrestrial plant com-
 munities, such as
 crops, grasslands, etc,
                             This method can only be used in situations in which
                              herbivore animals are not important and in which
                              a steady-state condition is never reached.
                             Since food used by the plants themselves and asso-
                              ciated microorganisms and animals is not included,
                              this method always measures net community pro-
                              duction.
"Dark-and-light" bottle method must be limited to
 a short duration, e.g., one 24-hour cycle or less.
 The combination dark-and-light bottles measure
 primary production, and the light bottle measures
 net community production.
The use of large plastic spheres instead of bottles
 reduces the inner surface^to-volume ratio and is
 presumed to reduce the effect of surface bacterial
 growth.
The "diurnal curve" method is particularly appli-
 cable to streams or estuaries and is especially
 useful in dealing with polluted waters.  It meas-
 ures gross primary production.  Reasonable cor-
 rections should be made for a source of errors in-
 troduced by diffusion, if any.

Equivalent to the aquatic Hdark*«and<«light" bottle
 method, enclosure method measures gross and net
 primary production.  Refrigerating or air

                                        (continued)

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                                            TABLE 3.1.10  (Continued)
    Methods
Uses
Quality Control
    Radioactive methods
     (14C, 32P are used)
Aquatic plants, phyto-
 plankton.
u>
    pH method
Aquatic ecosystems,
 laboratory
 micro-ecosystems,
 conditioning the chamber often becomes necessary
 if measurements are to extend over an appreciable
 period of time.
As in the diurnal curve method, the accuracy of
 the aerodynamic method depends on the accuracy of
 the corrections that must be made for mass move-
 ments of air and for gas evolution from soil that
 may contain CC-2, which is not a product of metabo-
 lism during the period of measurement.  Use of
 remote sensing and continuous monitoring tech-
 niques should increase the validity and integrity
 of data.

The 14C method is one of the most sensitive and
 widely used methods for measuring aquatic plant
 production (radioactive carbon [^CJ added as
 carbonate).
Use precisely and adequately calibrated radioactive
 counting device.
Trained, experienced personnel should be assigned
 on the control and handling of radioactive mate-
 rial

The investigator must first prepare a calibration
 curve for the water in the particular system to
 be studied because (1) pH and CC-2 content are not
 linearly related, and (2) the degree of pH change
 per unit of C(>2 change depends on the buffering
 capacity of the water.  See Beyers, et al., 1963,
 Publ. Inst. Mar. Sci. Univ. Texas 9/. 454-489, and
 Beyers, 1964, Amer. Bio. Teacher 26;491-498 for
 the details of a pH calibration curve.
Use precise instrumentation of remote sensing and
 continuous monitoring techniques.
                                        (continued)

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                                        TABLE 3.2.10  (Continued)
Methods
Uses
Quality Control
Disappearance of
 raw materials such
 as phosphorus and
 nitrogen
Chlorophyll method
Marine phytoplankton.
Aquatic communities,
 such as phytoplankton,
 macrophyton, and terres-
 trial communities.
This method measures the net productivity of the
 whole community during the period of spring
 growth of phytoplankton.
The method must be used with caution since non-
 living forces may also cause the disappearance of
 these raw materials.

This method measures primary productivity.
Follow standard extracting (pigment) procedures.
Spectrophotometer must be regularly and adequately
 calibrated for precision.

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              TABLE 3.2.11  PHYSICAL CRITERIA FOR WATER QUALITY
                        (NAS, 1974;  U.S.  EPA, 1976d)
SOLIDS (SUSPENDED. SETTLEABLE)  AND TURBIDITY

  Freshwater fish and other aquatic life:   Settleable and suspended solids
   should not reduce the depth  of the compensation point for photosynthetic
   activity by more than 10 percent from the seasonally established norm for
   aquatic life.

COLOR

  Waters shall be virtually free from substances producing objectionable color
   for aesthetic purposes.

  The source of supply should not exceed 75 color units on the platinum-cobalt
   scale for domestic water supplies.

  Increased color (in combination with turbidity) should not reduce the depth
   of the compensation point for photosynthetic activity by more than 10 per-
   cent from the seasonally established norm for aquatic life.

OIL AND GREASE

  For domestic water supply: Virtually free from oil and grease, particularly
   from the tastes and odors that emanate from petroleum products.

  For aquatic life:
   (1)  0.10 of the lowest  continuous flow 96-hour LC50 to several important
    freshwater and marine species, each having a demonstrated high suscepti-
    bility to oils and petrochemicals.
   (2)  Levels of oils or petrochemicals in the sediment which cause deleteri-
    ous effects to the biota should not be allowed.

   (3)  Surface waters shall be virtually free from floating nonpetroleum oils
    of vegetable or animal  origin, as well as petroleum-derived oils.

TEMPERATURE

  Freshwater aquatic life:   For any time of year, there are two upper limiting
   temperatures for a location  (based on the important sensitive species found
   there at that time).
   (1)  One limit consists  of a maximum temperature for short exposures that
    is time dependent and is given by the species-specific equation:

                         T   =  (1/b)  loglo (t-a) - 2°C

    where    T  =  temperature  (°C)
             b  =  slope of the line fitted to experimental data and
                   available from Appendix II-C, NAS, 1974, for some species
         logic  =  logarithm to base 10 (common logarithm)
                                                                     (continued)
                                      175

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                          TABLE 3.2.11  (Continued)
           t  =  time (minutes)
           a  =  intercept on the "y" or logarithmic axis of this line
                 fitted to experimental data and which is available from
                 Appendix II-C, NAS, 1974, for some species

 (2)   The second value is a limit on the weekly average temperature that:
  a.   in the cooler months (mid-October to mid-April in the north, and Decem-
  ber to February in the south) will protect against mortality of important
  species if the elevated plume temperature is suddenly dropped to the
  ambient temperature, with the limit being the acclimation temperature
  minus 2°C when the lower lethal threshold temperature equals the ambient
  water temperature (in some regions this limitation may also be applicable
  in summer);
  b.   in the warmer months (April through October in the north, and March
  through November in the south) is determined by adding to the physiologi-
  cal optimum temperature (usually for growth) a factor calculated as one
  third of the difference between the ultimate upper incipient lethal
  temperature and the optimum temperature for the most sensitive important
  species (and appropriate life state)  that normally is found at that
  location and time; or
  c.   during reproductive seasons (generally April through June and September
  through October in the north, and March through May and October through
  November in the south) the limit is that temperature that meets site-
  specific requirements for successful migration, spawning, egg incubation,
  fry rearing, and other reproductive functions of important species.  These
  local requirements should supersede all other requirements when they are
  applicable.
  d.   There is a site-specific limit that is found necessary to preserve
  normal species diversity or prevent appearance of nuisance organisms.

Marine aquatic life:  In order to assure protection of the characteristic
  indigenous marine community of a water body segment from adverse thermal
  effects, the following must be observed:

  a.   the maximum acceptable increase in the weekly average temperature due
  to artificial sources is 1°C (1.8°F)  during all seasons of the year, pro-
  viding the summer maxima are not exceeded;  and
  b.   daily temperature cycles characteristic of the water body segment
  should not be altered in either amplitude or frequency.

  Summer thermal maxima,  which define the upper thermal limits for the
  communities of the discharge area,  should be established on a site-specific
  basis.  Existing studies suggest the  following regional limits:
                                           Short-term       Maximum
                                            Maximum      True Daily Mean*
  Sub-tropical regions (south of Cape      32.2°C (90°F)    29.4°C(85°F)
   Canaveral and Tampa Bay,  Florida,
   and Hawaii)
                                                                (continued)
                                     176

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                          TABLE 3.2.11  (Continued)
                                             Short-term        Maximum
                                              Maximum      True Daily Mean*
   Cape Hatteras, N.C., to Cape              32.2°C (90°F)    29.4°C (85°F)
    Canaveral, Florida

   Long Island (south shore)  to Cape         30.6°C (87°F)    27.8°C (82°F)
    Hatteras, N.C.
   * True Daily Mean  =  average of 24 hourly temperature readings

  Baseline thermal conditions should be measured at a site where there is no
   unnatural thermal addition from any source,  which is in reasonable proxim-
   ity to the thermal discharge (within 5 miles) and which has similar hydrog-
   raphy to that of the receiving waters at the discharge.

the direct approach of working on-site, using local fish and Columbia River
water".  So he drifted juvenile chinook salmon (0-age) in live-box through
the plumes produced by the Hanford Reactor in the Columbia River and warmed
shoreline areas*used an inclined plant scoop-trap in the river downstream
from a reactor outfall to sample the natural run of seaward migrants, and
scored mortalities.  In both the live-box drifts and the trap collections,
no mortalities attributable to heat were observed.  The water temperature
observed at the fish trap anchored about 400 meters downstream in a center of
a reactor discharge plume showed a range of 10.5  to 15.5 C.

    In addition to cage or trap studies, biologists have suggested other
means to study the effect of heated effluents on anadromous fish by counting
natural fish populations or observing fish swimming behavior  (or runs).
Observations are made using aerial surveys by planes or using sonic  tags
on fish (Nakatani, 1969), and direct observations on fish in a runway or
channel (Alabaster, 1969).

    Table 3.2.11 lists the physical criteria for water quality  (U.S. EPA,
1976d;  National Academy of Sciences, 1974).

3.2.5 Field Bioassay

3.2.5.1 Aquatic Field Tests—

    Three terms are often used to describe field tests.  These are "field
survey", "monitoring program" and "field test"  (Livingston et al., 1974;
U.S. EPA, 1975a).

    •   "In a field survey, a sampling method is devised  to include:   a
broad range of the animal and plant life, both perturbed  and  unperturbed
areas, seasonal changes; and where possible, before and after effects  of
some event, such as the application of a potentially registrable  pesticide."
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     •   Monitoring implies continuous measurement of some variable.

     •   "In field tests, organisms are maintained in cages or confined areas
in the field environment.  Such systems can continually assess the effect of
the application on a series of representative species."

     A portion of the real world can be partitioned off and purposely contam--
inated.  The advantages of this type of research are that the spill or contam-
ination is under control of the investigator so that pre-stress data can be
assembled and the actual stress manipulated and measured, while the complexity
of the real world is retained to a greater degree than in a laboratory study.
The problems are:
     •   Deciding whether deliberate damage to even a small portion of environ-
ment is justified by the information that will be obtained;

     •   Confining the damage to the area under study;
     •   Deciding whether the portion of the environment under study is repre-
sentative; and
     •   Achieving sufficient control over the test area (U.S. EPA, 1975a).

     However, it appears that the approach with the greatest possibility for
standardization is cage-type or confined-area exposure.

                       EXAMPLE:  AQUATIC FIELD TESTING

Design of Experiment

•  The design of the experiment is one of the first tasks in aquatic field
   testing and a crucial factor to achieving the ultimate goal — "Quality
   Assurance" in the field test.

•  Use statistical consultation in the design of the experiment.  At the very
   least, suitable replication and control areas are a must, and the value of
   pre-application field data becomes obvious.

•  Apply a formal sampling plan.  Notice the great difficulties of sampling
   with mobile species and species with nonrandom distribution.  Different
   communities and localities may require different sampling procedures.  The
   frequency of sampling depends greatly on the objective of the study.  For
   example, the "reproductive success" study of an individual species requires
   less frequent sampling than the mortality study of the same species.

•  Choose an appropriate test area.  The area to be used should be as homo-
   geneous as possible with respect to the biotic, physical and chemical en-
   vironment.  Every effort should be made to choose an area which allows the
   investigator to prevent, control, or minimize the spread of the applied
   toxicant.

•  A high degree of knowledge of the biology of the various species is required.

Personnel

•  The team which conducts the field test must be adequately organized.  It
   should consist of at least one aquatic toxicologist or biologist as team


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   supervisor,  one or two technicians,  and  have  access  to an analytical  chemist
   and a biostatistician.

•  Train all  personnel  for quality.

Facilities and  Equipment

•  The maintenance of field equipment and/or  instruments and cage  construction
   are major concerns.   Among  these  the cage  construction is of  paramount
   importance for the field toxicity test.

•  Cage construction:  The construction of  cages for  specific taxa depends  on
   the species, its predators, the habitat, and  the properties of  the  chemical
   being tested.   Heitmuller and Nimmo (1972)  constructed a holding cage for
   exposing penaeid shrimp to  bottom sediment  and suggested that the cage is
   also suitable  to hold mollusks, crabs, or  fish for field tests.

•  Bioassay trailer:   A bioassay trailer (Zillich, 1969) has been  proven useful
   for testing  the biological  effects of many  industrial wastes  in the field.
   Federal, State and local agencies have just begun  utilizing mobile  bioassay
   units in applied research areas as aids  for engineering design, in  investi-
   gations to determine water  quality criteria,  in enforcement of  water quality
   standards, and in aquatic pest control studies.  Ideally, the design of
   these units  should be guided first by the  mission  of the sponsor and then
   by considerations of economy and  flexibility  (Gerhold, 1973).

•  Portable apparatus for acute toxicity bioassays:   The apparatus is  simply
   designed for conducting acute toxicity bioassays in  the field,  particularly
   effluent tests.  Falk (1973) designed an apparatus which proved to  be very
   satisfactory under field conditions, being  inexpensive, light,  and  portable
   as well as giving satisfactory results..  Fish in the control  tests  survived
   with no mortality.  Through mixing by aeration, the  temperature did not  vary
   more than 2°C  over a 96-hour period.  Results obtained from experiments  were
   comparable with those obtained from bioassays conducted under controlled
   laboratory conditions.  Additional advantages of this portable  apparatus
   were:  it is much cheaper to set  up than the  controlled  laboratory  for  the
   effluent.   Burress (1975) employed large plastic bags to  contain 284 liters
   of water and used more and  larger fish in  96-hour  tests of antimycin without
   employing either aeration devices or bulky supports  for  rigid vessels as
   indicated in Falk (1973).  Burress highly  recommended his method for con-
   ducting on-site toxicity tests.

Test Methods

•  According to the Federal Register (Vol.  40, No. 123, June 25, 1975), "no
   universally applicable methods are available  for field  testing  of pesticides
   because of the wide diversity of  mechanisms whereby  a pesticide may enter
   the environment, the diversity of habitats which may be  affected and the
   nature of the pesticide (solubility in water, degradability,  etc.)."  The
   U.S. Environmental Protection Agency has indicated field  methods as
   "developmental".  That means the  method  has been  proposed by one or a few
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   toxicologists and has been used to test only a few compounds by the original
   researcher.  "There is no concensus that the method provides appropriate
   data or that modifications of the method would not be appropriate.   Tech-
   niques involved may not be well known to other toxicologists, and therefore
   other toxicologists may require considerable experience with the method
   before they can obtain consistent results."

•  Other than the complexity of the environment and the unique nature of each
   chemical, the inherent difficulties of sampling and biological variability
   encountered in the field have hindered the progress of field chemical tests.

•  In spite of major difficulties, it is expected that as the theoretical  and
   practical aspects of environmental research improve, there will be a capa-
   bility to measure the effects of single and combined factors under field
   conditions.  The approaches to become "routine" methods are most likely
   applicable to cage-type (C) exposure or confined-area (CA) exposure.  See
   Table 3.2.12

Test Subjects

•  Some general considerations in the selection of test subjects are (U.S. EPA,
   1975a):

     o  Be realistic in choice of species.  Species collected locally will
        normally be easier to work with.

     o  Be aware of the possibility of induced resistance.

     o  Should caged animals be used, an adequate period of acclimation is
        necessary.

     o  Within the constraints of acceptable techniques, choose the most sen-
        sitive species and/or life stage inhabiting that ecosystem.
        The species must be readily available.
     o  Whether organisms are collected directly or purchased, every effort
        should be made to insure that they are healthy and are not subjected to
        unnecessary stress.  See Perkins (1972) on discussion of the importance
        of stresses such as collecting, handling and maintenance.

     o  Collection techniques described in "Biological Field and Laboratory
        Methods," U.S. EPA (1973), should be used.

   From Table 3.2.12, the most common species of fish used in cage-type field
   testing of toxic pollutants from the marine environment are as follows:
   sheepshead minnow (Cyproinondon variegatus), striped mullet (Mugil cephalus),
   mosquitofish (Gambusia affinis), sailfin molly (Mollienesia latiphinna) and
   killifish (Fundulus heteroclitus).  From the freshwater environment the
   representative ones are:  Largemouth bass (Micropterus salmoides), bluegill
                                      180

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   (Lepomis inacrochirus). fathead minnow (Pimephales promelas), rainbow trout
  (Salmo gairdneri). and brook trout (Salvelinus fontinalis).

   A similar list of invertebrates would include:

     Marine - Blue crab (Callinectes sapidus)
              Fiddler crab (Uca minax, U.  pufnax)
              Grass shrimp (Palaemonetes pugio)
              Eastern oysterTCrassostrea  virginica)

     Freshwater - The limited studies (cage-type) suggest no common species.
TABLE 3.2.12 AQUATIC SPECIES OR TAXA, FRESHWATER AND MARINE, USED IN FIELD
                CAGES (C) OR CONFINED-AREA (CA) TYPE STUDIES


Species                               Type of Study    References
	 (C or CA)	

FRESHWATER FISH:
  Brown trout (Salmo trutta)               C           Adams, 1975

  Bluegill (Lepomis macrochirus)           CA          Andrews et al., 1966

  Black crappie (Pomoxis vigro-maculatis)  C           Bridges, 1958
  Bluegill
  Black bullhead (Ictalurus melas)
  Carp (Cyprinus carpio)
  Flier sunfish (Centrachus macropterus)
  Golden shiner (Notemigonus crysoleucas)
  Goldfish (Carassius auratus)
  Green sunfish (Lepomis cyanellus)
  Grass pickerel (Esox americanus)
  Largemouth bass  (Micropterus salmoides)
  Longnose gar (Lepisosteus osseus)
  Mosquitofish (Gambusia affinis)
  Smallmouth buffalo (Ictiobus bubalus)
  Steel-colored minnow (Notropis whippli)
  Swamp darter (Ethoestoma gracile)
  Warmouth (Chaenobryltus gulosus)
  White crappie (Pomoxis annularis)
  Yellow bullhead  (Ictalurus netalis)

  Minnows                                  C           Carpenter,  1925
  Trout

  Brown trout                              CA          Dacre and  Scott,  1973
                                                             (continued)
                                     181

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                           TABLE 3.2.12 (Continued)
 Species                              Type of Study     References
	(C of CA)	

   Largemouth bass                          C           Eipper, 1959
   Bluegill
   Golden shiner
   Fathead minnow (Pimephales promelas)
   Rainbow trout (Salmo gairdneri)
   Brook trout (Salvelinus fontinalis)

   Java fish (Puntinus javanicus)           C           Gorbach et al., 1971

   Bluegill                                 CA          Hemphill, 1954
   Bonytail (Gila robusta elegans)
   Bullhead
   Brown trout
   Carp
   Largemouth bass

   Black bullhead                           CA/C        Kallman et al., 1962
   Channel catfish (Ictalurus punctatus)
   Greek chub (Semotilus atromaculatus)
   Green sunfish
   Fathead minnow
   Rainbow trout
   Smallmouth bass
   White sucker (Catostomus commersoni)
   Yellow bullhead

   Bluegill                                 CA          Lawrence, 1950
   Goldfish
   Largemouth bass fingerling

   Bigmouth buffalo (Ictiobus               CA          Mayhew, 1959
     cyprinellus)
   Bluegill
   Black bullhead
   Black crappie
   Carp
   Channel catfish
   Gizzard shad (Dorosoma cepedianum)
   Largemouth bass
   Guillback (Carpiodes cyprinus)
   Yellow bass (Roccus mississippiensis)

   Snakeskin gourami (Trichogaster          C           Moulton, 1974
     pectoralis)

   Mosquitofish                             CA          Mulla, 1962b

                                                         (continued)
                                      182

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                          TABLE 3.2.12  (Continued)
 Species
Type of Study
  (C of CA)
References
   Atlantic salmon                          CA

   Green sunfish                            CA
   Kwi Kwi (Haplosternum littorale)          CA
   Srieba (Astyanax bimaculatus)
   Krobia (Cichlasoma bimaculatum)

   Utah chub (Gila atravja)                 CA

   Leatherside chub (Synderichthys sp.)
   Dace (Rhinichtys sp.)
                  Sprague et al., 1965

                  Summerfelt and Lewis,
                     1967

                  Vermeer et al., 1974
                  Workman and Newhold,
                     1963
FRESHWATER INVERTEBRATES:
   Gastropod
   Diptera
   Odonata
   Ephemerop tera
   Coleoptera
   Hemiptera

   Copepod
   Cladocera
   Rotifera
      CA
      CA
Andrews et al., 1966
Coswell, 1965; Eipper,
   1959
   Louisiana red crawfish
                  Hendrick and Everett,
                     1965
   Protozoa
   Rotifera
   Entomostraca
                  Hoffman and Olive,
                     1961
   Aquatic insects
   Water mites
   Midges, etc.

   Mayflies (Ephemeroptera)
   Caddisflies (Trichoptera)
   Elmid beetles (Elmidae)
   Midges (Chironomidae)
      CA
      C/CA
May et al., 1973
Moye and Luckmann,
   1964
                                      183
                                                            (continued)

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                          TABLE 3.2.12 (Continued)
 Species                            Type of Study       References
	(C or CA)  	

   Texas Snails (Tropicorbis sp.)         CA            Nolan and Berry, 1949
   Plankton, Benthic Invertebrates        CA            Tarzwell, 1948
   Egyptian snails:                       C             Unrau et'al., 1965
     Balinus trunccatus
     Biomphalaria alexandrina
     Physa sp.

   South American Snail (Pomacea sp.)     CA            Vermeer et al., 1974

 OTHER FRESHWATER ORGANISMS

   Frog and Toad                          CA            Mulla, 1962a

   Bullfrog - Tadpoles (Rana              CA            Mulla, 1962b
     catesbeiana)

   Plants:                                              Eipper, 1959
     Lemna
     Alisma
     Sagittaria
     Chara
     Potamogeton
     Algae

   Frog (Pseudis paragoxa)                CA            Vermeer et al., 1974

 MARINE FISH

   Diamond killifish (Adenia xenica)      CA            Croker and Wilson,
   Darter goby  (Gobionellus bolcosoiria)                     1965
   Gulf killifish  (Fundulus grandius)
   Killifish (Fundulus sp.)
   Longnose killifish (Fundulus similis)
   Mosquitofish (Gambusia affinis)
   Rainwater killifish (Lucania parva)
   Sailfin molly (Mollinesia latipinna)
   Sheepshead minnow (Cyprinodon
     variegatus)
   Spot (Leiostomus xanthurus)
   Striped mullet  (Mugil cephalus)
   Tidewater silverside (Menidia
     beryllina)
   Mummichog (Fundulus heteroclitus)      C             George et al., 1957
   "Variegated  cyprinodon"  (Cyprinodon
     variegatus)
   Spot
   White mullet (M. curema)                                   (continued)
                                       184

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                       TABLE 3.2.12 (Continued)
Species Type of Study
(C or CA)
Mullet (M. Cephalus) = Striped C
mullet
Croakers (Leiostomus xanthurus)
Broad killifish (C. variegatus)
Gulf killifish
Cyprinodon sp. C/CA
Fundulus sp.
Sheepshead minnow C
Flounder (Paralichthys sp.) C
Mullet (M. cephalus)
Puffer (Sphaeroides sp.)
Sailfin molly
Pinfish C
Sheepshead
Drum
Mollies
Fundulus
MARINE INVERTEBRATES
Fiddler crab (Uca minax) C
Blue crab (Callinectes sapidus)
Marsh fiddler (Uca pugnax)
Red-jointed fiddler (II. minax)
Marsh crab (Sesarma reticulatum)
Blue crab (C. sapidus) CA
Marine mussel (Mytilus edulis) C
Reference
Ludwig et al . ,
1968
Springer and Webster,
1951
Tagatz et al., 1974
U. S.D.I., 1967
U. S.D.I., 1968
Croker and Wilson,
1965
George et al. , 1957
Koenig et al., 1976
Lee et al., 1972
Blue crab                              C

Soft shell clam (Mya arenaria)         C
Blue crab
Eastern oyster (Crassostrea
  virginica)
Blue crab                              CA
Marsh fiddler
"Bait" shrimp (Palaemonetes pugio)
Blue crab
Ludwig et al., 1968

Rawls, 1965
Springer and Webster,
 1951

Springer and Webster,
 1951
                                   185
(continued)

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  TABLE 3.2.12 (continued)
  Species                            Type of Study     References
         	 (C or CA)	
  Grass shrimp (Palaemonetes             C             Tagatz et al., 1974
    vulgaris)
  Pink shrimp (£. pugio)
  Snail (Littorina irrorata)

  White shrimp (Penaeus setiferus)       C             U.S.D.I., 1967
  Blue crab
  Fiddler crab (Uca sp.)
  Oysters (Crassostrea sp.)

  "Bait" shrimp (_P. pugio)               C             U.S.D.I., 1968
  Fiddler crab
  Blue crab
3.2.5.2.  Non-aquatic Field Tests—

    Dr. J. L. Lincer of the Mote Marine Laboratory has compiled some
protocols for wildlife toxicology and hazard evaluation for the Environmental
Protection Agency (U.S. EPA, 1975b).  These protocols include:

    •  Protocol for determination of the approximate maximum
       tolerated dose
    •  Protocol for laboratory acute oral toxicity - Birds
    •  Protocol for determining lethal dietary concentrations of
       chemicals to birds (5-day dietary LC50)
    •  Protocol for evaluation of reproductive effects of
       pesticides on the mallard
    •  Protocol for laboratory acute dermal toxicity test
    •  Protocol for small pen simulated field test to evaluate
       pesticide hazards to birds
    •  Protocol for large pen simulated field studies
    •  Protocol for full-scale field tests to evaluate pesticide
       hazards to wildlife.

    As toxicity tests move from the laboratory to full-scale field tests, it
becomes more necessary, but more difficult to control important variables.
Simulated field tests, both small-pen and large-pen, furnish intermediate
data to evaluate wildlife toxicity under semi-natural conditions.  Simulated
field tests should follow acute and subacute  toxicity studies.  Large-pen
simulated field tests have been used to measure chronic effects, including
those on reproduction.  Data from laboratory toxicity tests and simulated
field tests are serviceable in designing a full-scale (or unrestricted) field
test.  The unrestricted field test must necessarily follow both acute and
subacute toxicity tests and simulated field tests.  This test produces data
on actual commercially treated pesticide target areas where non-target wildlife

                                      186

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live unshackled in their feeding, reproductive and other activities (U.S.
EPA, 1975b)

    Examples of protocols for Small Pen Simulated Field Test,  Large Pen
Simulated Field Studies, and Full-scale Field Tests to Evaluate Pesticide
Hazards to Wildlife are given in the following pages.


                  EXAMPLE:  SMALL PEN SIMULATED FIELD  TEST

Purpose of Study

 .  Avian toxicity — To evaluate pesticide hazards to birds.

Materials

 •  Bobwhite (this protocol has been developed as an initial simulated field
    test for this bird).  With modifications, other species  could  be tested.

 •  The test subjects shall be obtained from pen-reared stock.

Design of Experiment

    QUALITY CONTROL — Use statistical consultation in the design  of the
    experiment.
    QUALITY CONTROL — Use good supervisory practices  to ensure that protocols
    are followed.

 •  Quarantine period.  All birds shall be maintained  in outdoor pens, in  the
    general area where the field test is to be conducted, for  at least 2 weeks
    prior to the test.

 •  Number of birds.  Each test should contain not less than six pairs of
    birds per control group and not less than six pairs of birds per test
    group* with one pair of birds per pen.
    QUALITY CONTROL — It is recommended that at least 12 additional birds
    be procured and held in outdoor pens for replacement purposes.

 •  Pens (size, construction, etc.).  Each pen shall contain approximately
    1.8 m2 (20 ft2).  Suitable pen dimensions might be 1.20 m  by 1.50 m
    (4 ft by 5 ft) or 0.90 m by 2.10 m (3 ft by 7 ft) and 0.30  m (1 ft) high.
    The pens should consist of a wooden frame made from 4 cm by 4  cm
    (2 in by 2 in) lumber and covered on the inside with 1 cm  (Jg in) mesh
    hardware cloth.  Pen height may be increased to a  height that  will
    accomodate vegetation growth through the test period.  Pens should have
    an opening through which birds can be removed or added.  Each  pen should
    contain a poultry waterer, preferably a 1-liter (1-quart)  chick fount
    and a small box, 30 cm by 30 cm by 25 cm (12 in by 12 in by 8  in), open
    on one side, to serve as a shelter for the birds.
    QUALITY CONTROL — To avoid possible contamination, scrub  wire and replace
    frames if pens have been used for previous testing.  Use of aluminum
    tubing for framing will make cleaning of pens easier.

                                    187

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    QUALITY  CONTROL  —  Do not cover pen  bottom.   Stake  pen  securely  to  the  ,
    ground to minimize  predation.
    QUALITY  CONTROL  --  Move pens daily, or as  required to  maintain  adequate
    vegetation cover.

 •   Test Conditions.  Conditions for evaluation  of each pesticidal formulation
    should approximate  those to be encountered in the routine  use  of the
    product.  For example, evaluation of a cotton insecticide  should be made
    in a cotton field,  and the timing, rate,  number, and  manner  of applica-
    tions should be  identical with those for  control of cotton insects.
    QUALITY  CONTROL  —  Handle (feeding,  watering and observation)  the
    control  birds the same as the test birds.
    QUALITY  CONTROL  —  Clearly mark all  pens  and all birds  to  assist accuracy
    in data  collection.
    QUALITY  CONTROL  —  Care should be taken at all times  to avoid  possible
    contamination through drift from adjacent areas or  from improper cleaning
    of equipment.

Conduct of Experiment

 •  Place pens and shelters in positions, and introduce birds  (1 male and  1
    female per pen)  prior to application of pesticide.
    QUALITY  CONTROL  —  Establish regular audits  of performance.
    QUALITY  CONTROL  —  Sufficient food and water are to be  available to the
    birds at all times, other than during the indicated 12-hour  period.

 •  Place filled waterers and about 100  g (3  to  4 ounces) of cracked corn,
    wheat or other grain in 1/3 of the total  test and control  pens used
    prior to test.  The remaining test and control pens are to be  left
    without feed and water for 12 hours  after the pesticide application, at
    which time feed  and water are to be  introduced to these pens,  as above.
    QUALITY  CONTROL  —  Follow all  safety precautions, as  specified on the
    product label, when entering the treated  field.

 •   Observation  of Test:   If  either member of the  pair dies, the survivor is
    to be removed, placed  in  an  individual holding  pen,  and a fresh pair
    placed in  the pen.   The survivor  should be observed until death or for
    14 days.   Sacrifice  survivors,  including "control" group and birds held
    for replacement at  the termination of the experiment.
    QUALITY  CONTROL --  Same individual should be assigned on the routine
    observation  job, and at the  same  time period each day the observations
    should be  made.

 •   Duration of  test: For  pesticides  which are to  be applied once per season,
    tests are to be continued for  not less than  14  days.  For pesticides
    which are to be applied more than once per season, tests are to be con-
    tinued for 14 days  after  the final application, with movement of pens
    immediately  prior to each application.
                                      188

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Reporting of Data

 •  Appropriate items to be considered are as  follows:
       -  Location of test
       -  Dates
       -  Weather data
       -  Species, sources, age, medical  and chemical  administration
          history, body weight, weight changes of birds;  individual
          identification
       -  Chemical formulation, rate of application,  manner  of
          application
       -  Vegetative cover, residue analysis
       -  Pen description, pen placement
       -  Diet, food and water supply schedule, feed  consumption
       -  Visual signs of intoxication,  accidential deaths,  or
          injuries
       -  Replacement schedule
       -  Gross pathological or histological examinations
       -  Statistical methods.
    QUALITY CONTROL —  Use statistical  expertise in  analysis of  results.
    QUALITY CONTROL —  Adopt a system for review and publication of data  and
    reports.

Reference

 •  U.S. EPA, 1975.  Guidelines for Registering Pesticides  in the United
    States, Appendix, Part VII - Hazard Evaluation, Subpart  C:  Wildlife
    Toxicology.  Federal Register, Vol.  40, No. 123 - Wednesday,  June 25,
    1975, pp. 26920-26921.


                EXAMPLE:  LARGE PEN SIMULATED  FIELD STUDIES

Purpose of Study

 •  Avian Toxicity - To determine pesticide effects on birds under semi-
    natural conditions and to assess the degree of hazard presented by the
    formulation and application rates of pesticides being considered for
    registration.

Materials

 •  Bobwhites, ring-necked pheasants or other  species.

Design of Experiment

    QUALITY CONTROL -- Use statistical consultation in design of experiment.

 •  Size of pens:  wire-covered pens should be constructed covering a minimum
    ground area of 45m2 (500 ft2) per pen.  Suitable  pen dimensions might be 3 m
    or 3.5 m by 15 m or 23 m (10 ft or 12 ft bv 50 ft or 75- ft), with the top
    cover at  a  height of about 2.0 m (6.5 ft).  Other dimensions covering

                                      189

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    45m2  (500  ft2)  or more per pen  may  be  used.
    QUALITY  CONTROL — Before pens  are  planned and  constructed,  the  designer
    and builder should consult wildlife agencies  and  successful  game farms
    to  learn practical consideration  such  as  prevention  of  disease and  para-
    sites, soil drainage requirements,  support of top cover to prevent
    collapse under the weight of snow,  types  of watering, etc.

 •   Number of cages:   24 to 36 pens are sufficient  to test  one chemical.
    This  would provide 6 to 9 control pens and 6  to 9 pens  for each  of  3
    treatment levels (the proposed treatment rate  and  2 multiples of  that  rate
    such  as  3 or 5X and 5 or 10X).  An  independent  water supply  and  a small
    shelter  should be furnished in  each pen.  Metal flashing should  be  placed
    around  all pens to a height of  about 45 cm (18  in) above ground  and to
    a depth  of about 15 cm (6 in) below the ground  surface.
    QUALITY  CONTROL -- Double the size  of  pens used when pheasants are
    utilized in this experiment. For example, two  3.7-by 22.9-m pens could
    be converted to one 3.7-by 45.7-m pen.

Birds

 •  If bobwhites  are used, pens may  be stocked with  1 mated pair per pen.
    One-year-old birds of known history, not  previously  exposed  to pesticides,
    shall be placed in the pen at least 2  weeks prior to the pesticide
    applications.  If pheasants are used a pen should be stocked with 1 male
    and 5 females per larger pen.
    QUALITY  CONTROL — A supply of  replacement birds  should be maintained in
    outdoor  pens near the control pens.
    QUALITY  CONTROL — All birds must be in healthy condition prior  to  the
    test.

Test Conditions

 •  Pen position:  Keep pens under  conditions as  natural as possible.  Use
    movable  pens that can be set up over the  crop or  vegetation  on which  the
    pesticide will be applied.  If  nonportable pens are  used, then soil
    should  be suitable for growing  the  pertinent  crop or vegetation.
    QUALITY  CONTROL — For statistical  purposes,  randomize  the test  pens.
    Before  randomization, stratify  the  treatment  pen  locations first because
    of the  drift problem of pesticides.

 .  Pesticide administration:  Handspray the  pesticide at the same rate,
    timing,  number of applications, and formulation as outlined  in the
    proposed registration.  Replicate pens should also be treated at two
    multiples (such as 3 or 5Xand5 or  10X) of the  rate  requested in the
    petition.
    QUALITY  CONTROL — Spraying should  be  done under  minimum wind conditions
    and with protective shielding to  prevent  contamination  of adjacent
    sprayed pens and/or control pens.
                                     190

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                  r:  IheSe Should also be treated at  t*e  3  rates.   The     .
              ^ rates can be based on results  of residue studies
               nL0?1^ pWsef in the registration procedure.  Treated  food
     Trp      nPH Pare?7Zthin ] ** of the ttme environments  are sprayed.    -
     Treated food should be supplied daily or every other  day  to the test
     birds in feeders protected from the weather.
       Another desirable phase of the test would be to provide treated animal
     foods such as grasshoppers or other invertebrates (earthworms,  etc.) to
     the penned birds simultaneously with the pen  environment  application.
       Various combinations of treatments can be made  to determine the major
     route of pesticide exposure to the test birds as  follows:
       (1)  Pen environment only with "clean" food and water.
       (2)  "Clean" pen environment and water and  treated  food only.
       (3)  "Clean" pen environment and food with  treated  water only.
     or(4)  Other combinations.
       Birds in half the pens at a given treatment rate may  be fasted and
     water withheld for 12 hours prior to the pesticide applications.  If so,
     half the control pens should also be fasted and water withheld  for the
     same 12-hour period.
     QUALITY CONTROL — Toxicants should be carried in a table-grade corn oil.
     QUALITY CONTROL — Food and water treatments  should be  made with pro-
     cedures and rates that are consistent with the characteristic of the
     chemical and the usage being tested.
     QUALITY CONTROL — Adequate replicates must be used if  various  treatment
     combinations are tested.

Conduct of Experiment

     QUALITY CONTROL — Establish regular audits of performance.

  •  Place pens, shelters  and feeders in position, and introduce birds into
     pens as described above prior to the pesticide administration.
     QUALITY CONTROL — All pens should be numbered and locations mapped  or
     charted.
     QUALITY CONTROL — All birds should be marked to  facilitate accurate data
     collection.

  •  Administer the chemical as indicated above and as desired in various
     treatment combinations.

  •  Provide sufficient food and water to the test birds at  all times, except
     during the specified  periods of fasting and water withholding.

  •  Test Observations:
       Mortality.   In case of bobwhite,  if either  member of  test pair dies,
     the survivor should be removed and  held for observation and a fresh  pair
     placed in the test pen.  All survivors are held for the observation  of
     possible toxic signs.
     QUALITY CONTROL —  The same individual  should be  at the post to observe
     the toxic signs of the intoxicated  birds.
                                     191

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    Reproduction.   Reproductive success  of the test birds  should be
    observed during the year of the test.   Eggs may be picked up period-
    ically for artificial  incubation and rearing of young  in the first
    half of the breeding season but eggs may be left for the hen(s)  to
    incubate in the last half of the normal  breeding season.  Hens should
    be allowed to rear the young to 14 days  of age in test pens.
    Test duration.   This should be a minimum of 21 days after the final pesti-
    cide application.   It must be longer if any birds are  showing toxic
    signs or other effects. Reproductive test would certainly continue beyond
    21 days post-treatment.
    Residue analysis.   Confirm diet and water levels of the test chemicals.
    Analyze vegetation, soil and other environmental samples for residues
    in accordance with other label requirements and determine the
    persistence and bioaccumulation.  Analyze the dead and surviving birds
    for residues in selected organs and/or tissues.   Determine the gross
    pathology at the same time.
    QUALITY CONTROL — For pesticide residue analyses, use the two following
    standard manuals:   (1) Manual of Analytical Methods for the Analysis of
    Pesticide Residues in Human and Environmental Samples.   U.S.  EPA, HERL-RTP,
    Environmental Toxicology Division, Rev.  in June, 1974.   (2)  Manual of
    Analytical Quality Control for Pesticides in Human and Environmental
    Media.  U.S. EPA,  HERL-RTP, Environmental Toxicology Division. J.F.
    Thompson (ed.).  EPA-600/1-76-017.  February, 1977.
    QUALITY CONTROL — Train personnel for quality.   See "Pesticide  Residue
    Analysis in Water-Training Manual.  U.S. Environmental  Protection Agency,
    Office of Water Programs.   EPA-430/1-74-012.  September, 1974.

Collecting and Reporting of Data

 •  Collect the data on mortality - number,  dates, etc.; toxic signs; weight
    changes; food consumption; clinical  observations; necropsy observations;
    residue analysis results;  weather data of tests; reproduction test(s)
    results.
    QUALITY CONTROL — Signing and witnessing of data collection.
    QUALITY CONTROL — Use statistical expertise in analysis of results.

 •  Report all the data collected above  and  test methods and materials used.
    QUALITY CONTROL — Adopt a system for review and publication of  data and
    reports.

Reference

 •  U.S. EPA, 1975.  Guidelines for Registering Pesticides in the United
    States, Appendix,  Part VII - Hazard  Evaluation,  Subpart C:  Wildlife
    Toxicology.  Federal Register, Vol.  40,  No. 123 - Wednesday, June 25,
    1975, pp. 26921-26922.
                                     192

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EXAMPLE 1:  FULL-SCALE FIELD TESTS TO EVALUATE PESTICIDE  HAZARDS  TO  WILDLIFE

Purpose of Study

 •  To determine the total impact of pesticide applications  on wildlife
    populations.

Materials

 •  All wildlife including arthropods on sprayed  and  unsprayed  (control)
    areas.

Design of Experiment

 •  A thorough pesticide-wildlife ecology study should  include collection of
    data on wild birds and mammal populations  (resident and  nonresident
    species), climate, soil, vegetation biomass by  species,  numbers  and bio-
    mass of arthropods, food habits of the most abundant  wildlife species,
    and distribution and fate of pesticide residues in  animals, plants, and
    environment.  Each parameter would require a  separate sampling method.
    These data should be collected on sprayed  and unsprayed  areas before and
    after the treatment dates.

Treatment Areas

 •  Treatment areas should be a minimum of 130 ha (320  acres) in  size  for a
    given chemical and rate of application. Cropland or  right-of-way  study
    areas may be smaller if the typical field  or  area sprayed would  be
    smaller.
    QUALITY CONTROL — All treatment areas should be  sufficiently large to
    accommodate a minimum of 2 replicates of 8 to 16  ha (20  to 40 acres)
    census plots with a sprayed buffer zone of at least 45 m (150 ft)
    around all plot boundaries.

 •  The experimental applications should be made  at the proposed  registra-
    tion rate and at two multiples of that rate,  e.g.,  3x or 5x.
    QUALITY CONTROL — Control  areas should be studied  simultaneously  on
    replicated plots in the same manner as the sprayed  areas.

Conduct of Experiment

 •  Strip census is generally used for censusing  cropland or rangeland birds,
    The basic procedure in this census technique  is to  walk  a straight line
    transect, usually within a given time period, and identify, record and
    plot locations of all birds seen within a  predetermined  width of strip,
    e.g., 50 m to either side of the line to travel,  i.e., a width of  100 m.
    Transects are marked in some manner so that the same  routes can  be
    repeated daily, weekly, monthly, seasonally or  yearly.
    QUALITY CONTROL — Strip censuses should be run in  the early  morning
    hours to coincide with a major activity period  of the birds.
                                    193

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QUALITY CONTROL —  Frequent counts within the breeding season on
replicated transects will provide statistically adequate data for
comparing pre- and postspray populations and sprayed plots with
unsprayed plots.
QUALITY CONTROL — Experienced personnel must go on the trip.

Plot census is usually used for censusing birds in forested or mixed
habitats.  The general approach for this census is similar to the strip
census.  The basic difference is that birds are observed, identified and
plotted on a map of a square or rectangular plot, usually approximately
about 16 ha (40 acres) in size.  The observer walks a more or less fixed
route taking him to all portions of the census plot within a given time
period.
QUALITY CONTROL — Same as for strip census.

Mark and recapture method for small mammals using grids of Sherman-type
live traps:  There are various systems of trap layouts, length of
trapping period, and data treatment.  The International Biological
Program (IBP), Grassland Biome (Swift and French 1972) has recommended
the system of  utilizing a square grid of 12x12 stations (or 144 trap
sites) (15 m between stations) with 1 or 2 live traps per station.
Animals captured are marked and released over a trapping period of 5
consecutive days.  Data are analyzed by the Jolly (1965) method.
QUALITY CONTROL — The mark and recapture procedure must be repeated
in the same manner in a pre- and postsprayed period on marked, replicated
grids.
QUALITY CONTROL — A control must be used.

The effects on target insects and total arthropod numbers and biomass
should be measured by standard entomological methods.  Particular
attention should be paid to arthropod species known to be important for
wildlife food.
QUALITY CONTROL —  The limitations of the arthropod sampling techniques
used should be noted and reported.
QUALITY CONTROL --  Use an adequate manual for arthropod population
analysis, e.g., Methods of Study in Quantitative Soil Ecology:  Popula-
tion, Production and Energy Flow (Phillipson, 1972).

Residue analyses should be done on the following types of samples:
  o  tissues of one or more species of common resident omnivorous
     mammals,
  o  tissues of one or more species of common resident omnivorous birds,
  o  common arthropods,
  o  vegetation including entire above-ground parts,
  o  plant litter,
  o  soil  (to a depth of about 2% cm),
  o  water (if any) from the sprayed area.
QUALITY CONTROL —  Use the best available technique for residue analysis
in  replicated aliquots.
QUALITY CONTROL —  Delayed analysis will invalidate the data.
QUALITY CONTROL —  All samples should be collected periodically in
duplicates until  residue levels fall below 0.01 ppm.

                                 194

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•V

3
d
Data Collection and Handling

    Collect the data on mortality with dates,  signs  of intoxication, wild-
    life census results, arthropod numbers  and biomass,  pathology,  residue
    analysis, nest studies, weather conditions during  the  study  period,
    fledgling observations, and other studies  on  reproduction  of resident
    wildlife.
     lUALITY CONTROL— Signing  and witnessing  of  data.  Integrate all
     ata into a picture of the total ecology of the  introduction of the
    pesticide.
    QUALITY CONTROL —  Use available statistical  methods  of analysis  and
    statistical expertise in data analysis.

References:

    The discussion here is principally derived from  the following report:
    U.S. EPA, 1975.  Guidelines for Registering Pesticides in  the United
    States, Appendix, Part VII - Hazard Evaluation,  Subpart C:   Wildlife
    Toxicology.  Federal Register, Vol. 40,  No. 123  -  Wednesday, June  25,
    1975, pp. 26926-26928. Other references are:

    Jolly, G.M 1965.  Explicit estimates from  capture-recapture  data with
    both death and immigration-stochastic model.   Biometrika.  52:   225-247.

    Swift, D.M., and N.R. French.  1972.  Vertebrates  - small  mammals. Pages
    24-28 in: Basic Field Data Collection Procedures  for the Grassland  Biome.
    IBP, Nat. Res. Ecol. Lab., Ft. Collins,  Colorado,  86 p. (Tech.  Rpt.  No.
    145).
                                 195

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Croker, R. A., and J. Wilson.  1965.  Kinetics and effects of DDT in a
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Weber, C. I. 1977.  Personal communication.

Westlake, D. F. 1966.   The biomass and productivity of Glyceria Maxima.   I.
     Seasonal changes  in biomass. J. Ecol.  54:   745-753.

Westlake, D. F. 1968.   Methods used to determine the  annual production of
     reedswamp plants  with extensive rhizomes.   In:   Methods of Productivity
     Studies in Rootsystems and  Rhizosphere Organisms.   IBP Root Symposium,
     Moscow, 1968, 226-234.  NAUKA, Moscow, USSR.

Westlake, D. F. 1974.   Macrophytes.  In:  A Manual on Methods  for  Measuring
     Production in Aquatic Environments.  R. A.  Vollenweider  (ed.).  IBP
     Handbook No. 12,  pp. 32-42.  Blackwell Scientific Publications, Oxford
     and Edinburgh, England.

Wetzel,  R.  G., and D.  F. Westlake. 1974.  Perpiphyton.   In:  A Manual on
     Methods for Measuring Primary Production in Aquatic Environments. R.
     A.  Vollenweider  (ed.). IBP  Handbook No. 12, pp.  42-50.  Blackwell
     Scientific Publications, Oxford and Edinburgh, England.

Workman,  G. W., and J. M. Newhold. 1963.  Lethal concentrations of toxaphene
      for goldfish, mosquito fish, and rainbow trout,  with notes on
      detoxification. Progr. Fish-Cult. 25(1):  23-30.

Zillich,  J. 1969.  The simultaneous use of  continuous flow bioassays and
     automatic water quality monitoring equipment to  evaluate  the toxicity
     of  waste water discharges.  Presented  at the 44th Annual  Conf.  of
     Mich.  Water Poll. Cont. Assoc., Boyne  Falls, Mich., June  16,  1969.

Zillioux, E. J., and D. F. Wilson. 1966.  Culture of  a planktonic calanoid
      copepod through multiple generations.  Science 151: 996.

                 General Bibliography on Identification  of
                        Organisms for Bioassay Tests

                                  Viruses

 Andrewes, C. H.,  and H. G. Pereira.  1972.   Viruses of Vertebrates. 3rd  Ed.,
      Bailliere,  Tindall and  Cox, London,  England.
                                      204

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Gibbs, A. J., et al. 1966.  What's in a virus name?  Nature 209:  450.

Melnick, J. L. 1971, 1972.  Classification and nomenclature of animal
     viruses, 1971 and 1972.  Progr. Med. Virol. 13:  462-484, 1971; 14:
     321-332, 1972.

Newman, J. F. E., et al. 1973.  A physico-chemical sub-grouping of the
     mammalian picornaviruses.  J. Gen. Virol. 18:  171.

Rosen, L., et al. 1970. Subclassification of enteroviruses and ECHO virus
     type 34.  Arch. Gesam. Virusforsch. 30:  39.

Wildy, P. 1971.  Classification and nomenclature of viruses.  International
     Committee on Nomenclature of Viruses.  Monogr. Virol. 5:  1.

World Health Organization. 1971.  A revised system of nomenclature for
     influenza viruses.  Bull. World Health Organ. 45:  119.
                                   Funei
Barnett, H. L. 1960.  Illustrated Genera of Imperfect Fungi.  Burgess
     Publishing Company, Minneapolis, Minn.

Barren, G. L. 1968.  The Genera of Hyphomycetes from Soil.  Williams and
     Wilkins, Baltimore, Md.

Cooke, W. B. 1963.  A Laboratory Guide to Fungi in Polluted Waters, Sewage,
     and Sewage Treatment Systems.  Their Identification and Culture.  U.S.
     Public Health Service, Publ. 999-WP-l, Cincinnati, Ohio.

Gillman, J. C. 1957.  Manual of Soil Fungi.  Iowa State University Press,
     Ames, Iowa.

Grove, W. B. 1937.  British Stem and Leaf Fungi.  University Press,
     Cambridge, England.

Raper, K. B., and D. I. Fennel. 1965.  The Genus Aspergillus.  Williams
     and Wilkins, Baltimore, Md.
                        Bacteria and Actinomycetes
Breed, R. S., et al. 1957.  Sergey's Manual of Determinative Bacteriology.
     8th Ed. Bailliere, Tindall and Cox, London, England.

Buchanan, R. E., et al. 1966.  Index Bergeyana.  E. & S. Livingston,
     Edinburgh and London, England.

Cowan, S. T., and K. J. Steel. 1961.  Diagnostic tables for the  common
     medical bacteria. J. Hyg. 59:  357.
                                    205

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Cowan, S. T., and K. J. Steel.  1965.  Manual  for the  Identification of
     Medical Bacteria.  University Press, Cambridge,  England.

Gordan, R. E. 1968.  The  taxonomy of soil bacteria.   In:  The  Ecology of
     Soil Bacteria.  T. R. G. Gray and D. L.  Parkinson  (eds.).  University
     Press, Liverpool, England.

Skerman, V. B. D. 1967.   A Guide to the  Identification  of the  Genera of
     Bacteria.   2nd  Ed. Williams and Wilkins, Baltimore, Md.

Smith, N. R., et al. 1952.  Aerobic Spore-forming Bacteria.  U.S.  Department
     of Agriculture, Monograph  16, Washington, D.C.

Williams, S. T., et  al. 1968.   Identification of genera of  the Actinomyce-
      tales.  In:  Identification Methods for  Microbiologists.  B.  M. Gibbs
      and D. A. Shapton  (eds.)-  Academic Press, London, New York.
                                 Protozoa


 Bick,  H.  1967-69.  An  Illustrated  Guide  to  Ciliated Protozoa  (used  as
     biological  indicators  in  freshwater ecology).  Parts  1-9.  World
     Health  Organization, Geneva,  Switzerland.

 Kudo,  R.  R.  1963.  Protozoology.   Charles Thomas,  Springfield,  111.


                         Other Microinvertebrates


 Donner, J. 1966.   Rotifers.  Butler  and  Tanner,  Ltd.,  London,  England.

 Edmundson, W. T. 1959.   Freshwater Biology.   John  Wiley  and Sons, New York,
     N.Y.

 Pennak, R. W. 1953.  Freshwater  Invertebrates of the United States.  Ronald
     Press,  New  York,  N.Y.
                                  Fishes
      See  Section 6.4, Fish  Identification,  in: Biological  Field  and  Labora-
 tory  Methods  for Measuring  the Quality of Surface Waters and  Effluents
 (U.S.  EPA,  1973).  This section  gives general references for  fish identi-
 fication, as  well as  references  for  regional fish identifications, such as;

                      o  Marine:   Costal Pacific
                      o  Marine:   Atlantic  and Gulf of Mexico
                      o  Freshwater:  Northeast
                      o  Freshwater:  Southeast

                                     206

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                      o  Freshwater:  Midwest
                      o  Freshwater:  Southwest
                      o  Freshwater:  Northwest


                                 Amphibia
Bishop, S. C. 1943.  Handbook of Salamanders.  Corns tock Publishing Company,
     Ithaca, N.Y.
        $.^19.58.  4vfi*W Guide Co Reptiles and Amphibians of Eastern North
     America.  Houghton Mifflin Company, Boston, Mass.

Cochran, D. M. 1961.  Living Amphibians of the World.  Doubleday & Company,
     New York, N.Y.

Dunn, E. R. 1926.  The Salamanders of the Family Plethodontidae.  Smith
     College, Northampton, Maine.

Noble, G. K. 1931.  Biology of the Amphibia.  McGraw-Hill Book Company,
     Inc., New York, N.Y.

Stebbins, R. C. 1951.  Amphibians of Western North America.  University of
     California Press, Berkeley, Calif.

Wright, A. H., and A. A. Wright. 1949.  Handbook of Frogs and Toads of the
     United States and Canada.  Comstock Publishing Company, Ithaca, N.Y.
                                 Reptilia
Barbour, T. 1934.  Reptiles and Amphibians.  2nd Ed. Houghton Mifflin Co.,
     Boston, Mass.

Carr, A. F., Jr. 1952.  Handbook of Turtles.  Cornell University Press,
     Ithaca, N.Y.

Conant, R. 1951.  The Reptiles of Ohio.  2nd Ed.  University of Notre Dame,
     Notre Dame, Ind.

Ditmars, R. L. 1944.  Snakes of the World.  Macmillan Publ. Co., New York,
     N.Y.

Gadow, H. 1901.  Amphibia and Reptiles  (Cambridge Natural History).
     MacMillan & Co., London, England.

Coin, C. J., and 0. B. Coin. 1962.  Introduction to Herpetology.   W. H.
     Freeman & Co., Publ., San Francisco, Calif.
                                     207

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Klauber, L. M. 1956.  Rattlesnakes, 2 vols.  University of California
     Press, Berkeley, Calif.

Pope, C. H. 1955.  The Reptile World.  Alfred A. Knopf, Inc., New York, N.Y.

Schmidt, K. P., and D. D. Davis. 1941.  Field Book of Snakes of  the United
     States and Canada.  G. P. Putnam's Sons, New York, N.Y.

Smith, H. M. 1946.  Handbook of Lizards of the United States and Canada.
     Comstock Publ. Co., Ithaca, N.Y.

Wright, A. M., and A. A. Wright. 1957.  Handbook of Snakes of the United
     States and Canada, 2 vols. Comstock Publishing Co.,  Ithaca, N.Y.
                                   Birds
Allen, A. A. 1936.  Ornithology Laboratory Notebook.  Comstock Publishing
     Co., Ithaca, N.Y.

American Ornithologists' Union. 1957.  Check List of North American Birds.
     5th Ed. Amer. Ornith. Union, Baltimore, Md.

Audubon, J. J. 1937.  The Birds of America.  Macmillan Publ. Co., New York,
     N.Y.

Bent, A. C. 1961-68.  Life Histories of North American Birds.  Dover Publica-
     tions, New York, N.Y.

Forbush, E. H. 1939.  Natural History of the Birds of Eastern and Central
     North America.  Houghton Mifflin Co., Boston, Mass.

Gilliard, E. T. 1958.  Living Birds of the World.  Doubleday & Co., Inc.,
     New York, N.Y.

Peters, J. L. 1931.  Checklist of Birds of the World.  Harvard University
     Press, Cambridge, Mass.

Peterson, R. T. 1947.  Field Guide to the Birds.  2nd Ed. Houghton Mifflin
     Co., Boston, Mass.

Peterson, R. T. 1961.  A Field Guide to Western Birds.  Revised Ed.  Houghton
     Mifflin Co., Boston, Mass.

Pettingill, 0. S., Jr. 1956.  A Laboratory and Field Manual of Ornithology.
     3rd Ed. Burgess Publishing Co., Minneapolis, Minn.

Rand, A. L. 1956.  American Water and Game Birds.  E. P. Dutton & Co., New
     York, N.Y.
                                     208

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Van Tyne, J., and A. J. Berger. 1971.  Fundamentals of Ornithology.  Dover
     Publications, New York, N.Y.

Wood, N. A. 1951.  The Birds of Michigan.  University of Michigan Press,
     Ann Arbor, Mich.
                                  Mammals
Anthony, H. E. 1928.  Field Book of North American Mammals.  6. P. Putnam's
     Sons, New York, N.Y.

Blair, W. F., et al. 1957.  Vertebrates of the United States.  McGraw-Hill
     Book  Co., Inc., New York, N.Y.

Burt, W. H., and R. P. Grossenheider. 1952.  A Field Guide to the Mammals.
     Houghton Mifflin Co., Boston, Mass.

Burt, W. H. 1957.  Mammals, of the Great Lakes Region.  University of
     Michigan Press, Ann Arbor,. Mich.

Cockrum, E. L. 1955.  Manual of Mammalogy.  Burgess Publishing Co.,
     Minneapolis, Minn.

Collins, H. H. 1959.  Complete Field Guide to American Wildlife.  Harper &
     Brothers, New York, N.Y.

Davis, E. E., and F. B. Golley. 1964.  Principles of Mammalogy.  Reinhold
     Publishing Corporation, New York, N.Y.

Hall, E. R., and K. R. Kelson. 1959.  The Mammals of North America, 2 vols.
     The Ronald Press Co., New York, N.Y.

Hamilton, W. J., Jr. 1939.  American Mammals.  McGraw-Hill Book Co., Inc.,
     New York, N.Y.

Hartman, C. G. 1952.  Possums.  University of Texas Press, Austin, Tex.
                                   •
Leopold, A. S. 1959.  Wildlife of Mexico.  University of California Press,
     Berkeley, Calif.

Littlewood, C. 1970.  World's Vanishing Animals:  The Mammals.  Arco Publ.
     Co., New York, N.Y.

Sanderson, I. T. 1955.  Living Mammals of the World.  Doubleday &  Co.,  Inc.,
     New York, N.Y.

Scheffer, V. B. 1958.  Seals, Sea Lions, and Walruses.  A  Review  of  the
     Pennipedia.  Stanford University Press, Stanford, Calif.
                                     209

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Schmidt-Nielsen, K. 1964.  Desert Animals.  Clarendon Press, Oxford,  England.

Simpson, G. G. 1945.  The principles of  classification and a classification
     of mammals.  Bull. Amer. Mus. Nat.  Hist. 85:   1-350.

Tomich, P. Q. 1969.  Mammals of  Hawaii - A  Synopsis and Notational Biblio-
     graphy.  Bernice P. Bishop  Mus. Spec.  Publ. 57, Bishop Mus. Press,
     Honolulu, Hawaii.

Vaughan, T. A. 1972.  Mammalogy. W. B. Saunders Co., Philadelphia, London
     and Toronto.

Walker, E. P. 1968.  Mammals of  the World,  2 vols.  2nd Ed.  Johns Hopkins
     Press, Baltimore, Md.
                                  Plants
Britton, N. L. 1907.  Manual of the Flora of the Northern States and Canada.
     3rd Ed.,  revised and enlarged.  New York, N.Y.

Britton, N. L., and A. Brown. 1913.  An Illustrated Flora of the Northern
     United States, Canada, and British Possessions from Newfoundland to
     the Parallel of the Southern Boundary of Southern Virginia and from
     the Atlantic Westward to the 102nd Meridian.  2nd Ed., revised and
     enlarged.  3 vol. New York, N.Y.

Coulter, J. M. 1909.  New Manual of Botany of the Central Rocky Mountains
     (Vascular Plants), revised by Aven Nelson.  New York.

Dean, C. C. 1940.  Flora of Indiana. Indianapolis.

Fernald, M. L. 1950.  Gray's Manual of Botany, 8th Ed. Boston and New York.

Jepson, W. L. 1923-1925.  A Manual of the Flowering Plants of California.
     Berkeley.

Jepson, W. L. 1909-1943.  A Flora of California.  3 vols (incomplete).  San
     Francisco, Berkeley.

Kearney, T. H., and R. H. Peebles. 1942.  Flowering Plants and Ferns of
     Arizona.  U.S. Dept. Agr. Misc. Publ. 423.

Munz, P. A. 1935-  A Manual of Southern California Botany.  Claremont, Calif.

Peck, M. E. 1941.  A Manual of the Higher Plants of Oregon.  Portland,
     Oregon.

Piper,  C. V. 1906.  Flora of the State of Washington.   Contr.  U.S.  Nat.
     Herb.,  vol. 11.
                                    210

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Reeve, R. G., and D. C. Bain. 1946.  A Flora of South Central Texas.  College
     Station, Texas.

Rydberg, P. A. 1922.  Flora of the Rocky Mountains and Adjacent Plains,
     Colorado, Utah, Wyoming, Idaho, Montana, Saskatchewan, Alberta and
     Neighboring Parts of Nebraska, South Dakota, North Dakota and British
     Columbia.  2nd Ed. New York.

Rydberg, P. A. 1932.  Flora of the Prairies and Plains of Central North
     America.  New York.

Schaffner, J. H. 1928.  Field Manual of the Flora of Ohio and Adjacent
     Territory. Columbus.

Small, J. K. 1933.  Manual of the Southern Flora, Giving Descriptions of
     the Seed Plants Growing Naturally in Florida, Alabama, Mississippi,
     Eastern Louisiana, Tennessee, North Carolina, South Carolina, and
     Georgia.  New York.

Steman, T. R., and W. S. Myers. 1937.  Oklahoma Flora.  Oklahoma City.

Tidestrom, I. 1925.  Flora of Utah and Nevada.  Contr. U.S. Nat. Herb., vol.
     25. p. 665.

Wooton, E. 0., and P. C. Stanley. 1915.  Flora of New Mexico.  Contr. U.S.
     Nat. Herb., vol. 19.
                              Aquatic Plants
Beal, E. 0., and P. H. Monson. 1954.  Marsh and aquatic angiosperms of Iowa.
     State University, Iowa.  Studies Nat. Hist. 19(5):  1-195.

Correll, D. S., and H. B. Correll. 1973.  Aquatic and Wetland Plants of the
     Southwestern United States.  Washington, D.C.

Eyles, D. E., and J. L. Robertson, Jr. 1944.  A Guide and Key to the Aquatic
     Plants of the Southeastern United States.  Public Health Bull. No. 286.,
     U.S. Gov. Printing Office, Washington, D.C.

Fassett, N. C. 1967.  A Manual of Aquatic Plants.  2nd Ed. Madison, Wis.

Hotchkiss, N. 1972.  Common Marsh, Underwater and Floating-leaved Plants of
     the United States and Canada.  New York, N.Y.

Mason, H. L. 1957.  A Flora of the Marshes of California.  Berkeley and Los
     Angeles, Calif.

Mitchell, D. S. (ed.). 1974.  Aquatic vegetation and its use  and control.
     A contribution to the Intern. Hydrol. Decade. UNESCO-Paris:  pp.  1-135.
     ISBN 92-3-101082-4.  Printed in France.

                                     211

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Muenscher, W. C. 1944.  Aquatic Plants of the United States. Cornstock
     Publishing Company, Ithaca, N.Y.

Otto, N. £., and T. R. Hartley. 1965.  Aquatic Pests on Irrigation Systems,
     Identification Guide.  Washington, D.C.

Prescott, G. W. 1969.  The Aquatic Plants.  Dubuque, Iowa.

Stewart, A.  N., J. D. LaRea, and H. M. Gilkey. 1963.  Aquatic Plants of the
     Pacific Northwest Oregon. 2nd Ed.

Stodola, J.  1967.  Encyclopedia of Water Plants.  New Jersey.

Winterringer, G. S., and A. C. Lopinot. 1966.  Aquatic Plants of Illinois.
     III.   State Museum Popular Ser. Vol. VI, ILL. State Museum Division.
                                     212

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3.3 AQUATIC BIOASSAY

3.3.1  Basic Requirements of Aquatic Bioassay

3.3.1.1  Personnel—

     Almost without exception, activities in both experimental and applied
toxicology have biological, chemical, and statistical aspects.  Because of
the interdisciplinary character of toxicological activities and because few
people are really competent in more than one field,  biologists, chemists,
and statisticians must work together (Stephan, 1973).  In aquatic biological
laboratories the discipline of toxicology may also be represented but in
smaller laboratories in particular, the toxicological aspects may be handled
by the biologist with the support of the chemist.

     The biologist, in any event, is required to maintain a broad overview
of the basic toxicological questions and guides the whole experimental effort,

     Chemists may be full members of the biological team or may play a
supporting role by supplying analytical laboratory capability for one or
more teams.  The chemist can contribute to experimental toxicology by:

          • aiding in the selection of toxicants that should be tested
          • helping design toxicity tests
          • measuring and characterizing the level of the toxicant
            to which the subjects are actually exposed
          • determining the fate of the toxicant after it comes in
            contact with the subjects
          • helping determine the mode of action of the toxicant
          • aiding in detecting some of the effects of the toxicant
            on the subjects
          • recommending good sample collection and dosage techniques
          • devising ways to prepare special materials and toxic
            agents that have been designed by toxicologists.

     Chemists are in a good position to identify actual and potential
environmental contaminants because many of these are used or produced by
the chemical industry.

     Statisticians usually play a supporting role by:

          • aiding in the design of bioassay
          • providing good sampling plans
          • helping ensure the validity of chemical and biological
            test results by calling for duplicate samples, standard
            samples, and interlaboratory samples
          • suggesting methods for data analysis and assisting in
            the analysis and interpretation of data.
                                      213

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     Fish bioassay techniques usually involve exposure of the experimental
species to toxic agents in water rather than the direct application of agents
to the animals.  Thus, information regarding the chemical reaction between
the toxic agent and the media (water) is very essential for designing and
interpreting of toxicity tests conducted on fish.

     Generally, biological tests will be better measures of biological prop-
erties than chemical tests, even if the biological tests are not as well
developed as many of the chemical tests.  Environmental protection needs
toxicological accuracy as much as it needs statistical precision.

     Training is available in the form of courses provided at Federal or
Academic institutions.  Laboratory personnel should be encouraged to attend
professional meetings to help the individual keep abreast of the state of the
art within his particular professional interest.  Overall the biology labora-
tory as a unit benefits from individual training and self-enrichment programs
(U.S. EPA, 1975b).

3.3.1.2 Facilities and Equipment—

     • Facilities

     For maximum convenience and versatility, the facilities should include:

     o tanks for holding and acclimating test organisms
     o a constant temperature area or recirculating water bath for the test
       chambers
     o a dilution water tank that may be used to prepare reconstituted water
       and which is elevated, if possible, so dilution water can flow by
       gravity into holding and acclimation tanks and test chambers.

     Ceilings should be at least 10 feet high to accommodate proportional
diluters and strainers, and air traps should be included in the water supply
system.  Holding, acclimation, and dilution water tanks should be equipped
for  temperature control and aeration.   The test facilities should be well
ventilated and free of fumes (U.S. EPA, 1975a).

     • Construction material

     Construction materials and commercially purchased equipment that may
contact any water into which test organisms are placed should not contain
any  substances that can be leached or dissolved by the water.  In addition,
materials and equipment that contact stock solutions or test solutions should
be chosen to minimize sorption of toxicants from water.  Glass, #316 stainless
steel, and fluorocarbon plastics must be used whenever possible.  Rubber,
copper, brass and lead must not come in contact with dilution water, effluent
samples, or test solutions (U.S. EPA, 1975a).
                                      214

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     • Test chambers

     Test chambers can be made by welding, not soldering,  stainless steel or
by gluing double-strength or stronger window glass with clear silicone ad-
hesive.  As little of adhesive as possible should be in contact with water;
extra beads of adhesive should be on the outside of the chamber rather than
on the inside (U.S. EPA, 1975a).

     • Embryo and fry chambers

     Embryo and fry chambers should be constructed to allow for adequate
exchange of water and to ensure that the proper quantity of test material is
entering the chambers.  These chambers must be brushed daily to prevent
clogging.  Embryo and fry chambers should be designed so that water can be
drained down to 2.5 cm  (1 inch) in order to facilitate growth measurements
of fry.  These chambers may be supplied with the test water by:

     o separate delivery tubes from the mixing chamber,
     o splitting the flow from the aquaria,
     o or "egg" cups on a "rocker" arm (U.S. EPA, 1976).

     • Toxicant mixing chambers

     A mixing chamber is necessary to assure adequate mixing of the test
material.  Aeration should not be used for mixing.  Mixing is extremely
important because if the test materials are not adequately mixed with water,
toxicity cannot be properly assessed.  Improper mixing can either expose the
animal to too much or too little of the material, and toxicity would be over-
or underestimated  (ASTM, 1974).

     • Calibration and  standardization of test containers

     Before filling the test containers,  it is necessary to determine a suit-
able aeration rate so that the loss of any dissolved volatile substances
from the liquid in the  test container will be excessive.  This involves
determining the total number of bubbles of air or oxygen or both released
per minute in a given test container filled with the test solution up to a
given level.  The dissolved oxygen content of the test  solution shall not
fall below 4 ppm when warm-water fish are used as test  animals, or below 5ppm
when cold-water fish are used and it should not exceed  the saturation value
at the experimental temperature.

     Calibration method is as follows:

     Fill the test container to the fixed level with clean soft water having
an alkalinity to methyl orange indicator  not in excess  of 40 ppm as  CaC03.
Dissolve C0a gas in the water to obtain a concentration rate  (in terms  of
the number of bubbles of air or oxygen released per minute)  such that  the
amount of C02 lost from the solution in 24 hours under  these experimental
conditions will not exceed 67 percent of  the initial  free C02  (ASTM, 1974a)
                                      215

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     • Toxicant delivery system

     Although many toxicant delivery systems can be used  (Lowe, 1964:
Sprague, 1969; Freeman, 1971; Cline and Post, 1972; Granmo and Kollberg, 1972;
Bengtsson, 1972; Lichatowich et al., 1973; Shumway and Plaensky, 1973; Abram,
1973; Schiflmel et al., 1974; DeFoe, 1975; National Water Quality Laboratory,
Duluth, Minnesota, personal communication; Carton, R., Western Fish Toxicol-
ogy Station, Corvallis, Oregon, personal communication), the proportional
diluter (Mount and Brungs, 1967) is considered to be the best for routine
use.  One disadvantage of the Mount and Brungs diluter is that it is imprac-
tical when the dilution factors between concentrations exceed fifty percent
and the logarithmic gradient frequently exceeds a fifty percent dilution
factor when testing with chemicals such as pesticides.  The mechanical multi-
channel injection apparatus designed by Ozburn and Alasdair (1976) overcomes
this problem, but its reproducibility and reliability depend heavily upon
smooth operation of the mechanical components.  For this reason the system
is not recommended for use in chroi ic toxicity tests employing salt water
as the diluent because excessive exposure to salt water may result in de-
terioration of the metal by corrotion (Ozburn, 1976).

     The calibration of the toxicant delivery system should be checked care-
fully before, during, and after each test.  This should include determining
the volume of stock solution and dilution of water used in each portion of
the toxicant delivery system and the flow-rate through each test container.
The general operation of the toxicant delivery system should be checked
daily during the test (U.S. EPA, 1975a).

     • Dilution water

     A minimal criterion for an acceptable dilution water is that healthy
test organisms will survive in it for the duration of acclimation and testing
without showing signs of stress such as discoloration or unusual behavior.

          o Freshwater

          Water in which Daphnids  (which are more sensitive to many toxi-
cants than most other freshwater animals) will survive and reproduce satis-
factorily should be an acceptable water for most tests with freshwater
animals.

          o Estuarine and marine v.  iter

          Water in which Acartia lonsa or Mysid shrimp (which are more
sensitive to many toxicants than raost other estuarine and marine aquatic
animals) will survive, grow, and reproduce satisfactorily should be an
acceptable dilution water for most  tests with estuarine and marine animals.
If a dilution water is prepared from a dechlorinated water, it must be shown
that in fresh samples of the dilution water taken daily during flow-through
tests, the concentration of residual chlorine is less than 3ug per liter or
that Acartia Tonsa. Mysid shrimp, oyster larvae or first instar Daphnids can
survive for 48 hours without food.
                                     216

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          o  Reconstituted water

          The recommended reconstituted waters (Table 3.3.1,  3.3.2 and 3.3.3)
should be used as dilution water for as many tests as possible to maximize
the number of reliable comparisons that can be made concerning relative
toxicity and relative sensitivity.  Reconstituted water is  prepared by adding
a known amount of specified reagent-grade chemicals to water  which meets the
specifications in Tables 3.3.1, 3.3.2, and 3.3.3.

     •  Alternative water

     Alternative dilution water should be uncontaminated and  of constant
quality and should meet the following specifications:

          Suspended solids                    20 mg/1
          TOC                                 10 mg/1
          Un-ionized ammonia                  20 yg/1
          Residual chlorine                    3 yg/1
          Total organophosphorus pesticides   50 ng/1
          Total organochlorine pesticides     50 ng/1
            plus PCB's

     For effluent tests, the dilution water must be a representative
sample of the receiving water obtained as close to the point  of discharge as
possible, but upstream of or outside the zone of influence of the effluent.
For tests with freshwater organisms, municipal water supplies often contain
unacceptable concentrations of copper, lead, zinc, fluoride,  and chlorine or
chloramine.  Metals can be remonved by chelating resins.  Sodium bisulfite
is better for dechlorinating water than sodium sulfite, and both are much
more reliable that a carbon filter, especially for removing chloramine
(U.S. EPA,  1975a).

     •  Cleaning of test chambers, delivery systems, holding tanks, etc.

     Toxicant delivery systems and test chambers must be cleaned before
use.  New ones must be washed with detergent and rinsed with fresh tap water.
At the end  of every test, if the  toxicant delivery systems or test chambers
are to be used again, they should be:

          o emptied
          o cleaned by a procedure appropriate  for  removing the toxicant
             tested  (e.g., acid to remove metals and bases; detergent,  organic
             solvent, or activated carbon to remove  organic compounds)
          o rinsed twice with water

Acid is useful for removing mineral   deposits, and 200 mg  of hypochlorite  per
liter or 30% formalin plus 1% benzalkonium chloride  are useful  for removing
organic matter and for disinfection.  However, acid  and hypochlorite  must
not be used together.  Test chambers  and toxicant delivery systems must be
rinsed with dilution water just before use.  Holding and acclimation  tanks
should be sterilized with an lodophor or with  200 mg of hypochlorite  per liter
                                      217

-------
for  1 hour,  scrubbed well  once  during  the  hour  and  rinsed will between groups
of test  organisms  (U.S.  EPA,  1975a).
   TABLE 3.3.1  QUANTITIES OF REAGENT-GRADE CHEMICALS REQUIRED TO PREPARE
  RECOMMENDED RECONSTITUTED FRESH WATERS AND THE RESULTING WATER QUALITIES
                          (Marking and Dawson, 1973)
Name
Very soft
Soft
Hard
Verv nard
Salts Required (mg/1) ,,a
NaHC03
12
48
192
334
CaSOit-2H20 MgSOi«
7
30
120
240
.5
.0
.0
.0
7.5
30.0
120.0
240.0
H"
KC1
0
2
8
16
.5
.0
.0
.0
6
7
7
8
.4-6
.2-7
.6-8
.0-8
.8
.6
.0
.4
Hardness0
10-13
40-48
160-180
280-320
Alkalinityb
10-13
30-35
110-120
225-245
 Approximate equilibrium pH after aeration and with fish in water.
^Expressed in mg/1 as CaCQ$.
  TABLE 3.3.2  QUANTITIES OF REAGENT-GRADE CHEMICALS TO BE ADDED TO AERATED
  SOFT RECONSTITUTED FRESH WATER FOR BUFFERING pH (Marking and Dawson, 1973)
   (The solutions should not be aerated after addition of these chemicals.)
pH*
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
Milliliters
l.ON HaOH
1.3
5.0
19.0
	
19.0
6.5
8.8
11.0
16.0
of Solution for
O.OM KH2PO»t
80.0
30.0
30.0
	
20.0
	
	
	
	
15 Liters of Water
0.5M H3B03
	
	
	
, 	

40.0
30.0
20.0
18.0
1Approximate equilibrium pH with fish in water,
                                     218

-------
   TABLE 3.3.3  RECOMMENDED PROCEDURE FOR PREPARING RECONSTITUTED SEA WATER
     (Kester et al., 1967; Zaroogian et al., 1969; Zillloux et al., 1973)

 (Add the following reagent-grade chemicals in the amounts and order listed to
    890 ml water.  Each chemical must be dissolved before another is added.)
Chemical
NaF
SrCl2-6H20
H3B03
KBr
KC1
CaCl2*2H20
Amount
3 mg
20 mg
30 mg
100 mg
700 mg
1.47 g
Chemical
Na2SOi4
MgCl2-6H20
NaCl
Na2Si03-9H20
Na^EDTA
NaHCO
Amount
4.00 g
10.78 g
23.50 g
20 mg
1 mg
200 mg
     • Laboratory instrumentation calibration

     All calibration of instruments used for water quality analyses must be
documented on an appropriate laboratory data sheet.  This is accomplished by
recording the following information:

     o Date
     o True value of standards and calibration value
     o Factor, if any, required to correct reading from meter
     o Amount of drift
     o Initials of person performing calibration

     The following is a list of instruments that require calibration:

     ° Laboratory pH meter
     Calibrate with two standard buffer solutions that cover the pH range of
the samples being analyzed.  Calibrate at start of testing (daily) and check
for drift with one buffer solution periodically during laboratory use.

     o Laboratory dissolved oxygen meter

     Calibrate by running modified Wlnkler Full Bottle Technique (U.S. EPA,
1973) on three samples.  Average and calibrate to this value.  Run a final
Winkler daily to check for drift upon completion of analysis.

     o Temperature meter (Dissolved oxygen meter)

     Calibrate with NBS thermometer semiannually.

     o Conductivity meter

     Standarize monthly with standard potassium chloride (0.01M) as stated in
"Standard Methods",  14th Edition (Rand et al, 1975).

                                     219

-------
     o Refractometer

     Calibrate with water (U.S. EPA, 1975b).

3.3.1.3.  Test substance—

     The test substance can be one or more pure chemicals, a complex mixture
such as formulation, or an effluent.  Sometimes, the test solutions are
not true solutions because they contain undissolved toxic agents.

     o Basic test

     The toxicant should be added to the dilution water or the toxicant
delivery system without the use of any solvent or other additive, except
water, if possible (U.S. EPA, 1975a).
                                     220

-------
     If a carrier or vehicle ia used to dissolve or dilute the test sub-
Stance, it fthduld possess as many of the following characteristics as
possible:         ;

           o  it should not interfere with absorption, distribution,
              metabolism, or retention of the test substance
           0  it should not alter the chemical properties of the
              test substance or enhance, reduce, or alter the toxic
              characteristic of the test substance
           o  It should not affect the food and water consumption of
              the test organism
           6  at the levels used in the study, it should not produce
              physiological effects or have local or systemic
              toxlcity (Anon., 1977).
     Ifl addition, such a catrler or vehicle should, if possible, closely
resemble the substance to be used under expected conditions of use
(Anon., 1977).

     the calculated concentrations of the additives to which any test
organism are exposed must never exceed one twentieth of the concentra-
tion of the toxicant and must never exceed one-tenth gram per liter of
water.  Two sets of controls must be used, one exposed to no additive
and one exposed to the highest level of additive to which any other
organism* in the test are exposed (U.S. EPA, 1973).

     The test substance should be of technical-grade.  The lot and purity
of the test substance should be known and recorded.  The stability of the
test substance in the stock solution should be determined.  For long-term
studies, when the test substance is incorporated into the dilution water,
thfe concentration of the test Substance in the dilution water should be
determined at the start of the study and samples collected periodically
to verify the concentration (Anon., 1977).
                                   221

-------
     • Effluent Test

     The test substance may be a sample of an effluent.  Such a sample must not
be aerated or altered in any way except that it may be filtered through a
sieve or screen with 2-mm or larger holes.  Samples must be covered at all
times and violent agitation must be avoided.   The collection of samples
should be based on an understanding of the short-and long-term operations
and schedules of the discharger if possible.

         o  For effluent static tests, separate tests generally should be
conducted on at least two grab samples and more tests may often be desirable,
especially if there are known sources of variability such as process  changes.
Tests should be begun as soon as possible, but must be begun within 8 hours,
after the sample is obtained.   The temperature of the sample should be ad-
justed to the test temperature (*2 C) and maintained at that temperature
until portions are added to the dilution water.  Often it is convenient to
store the sample in the constant temperature water bath or area in which the
test chambers are placed during the test.

         o  For effluent flow-through tests, the sample of the effluent must
be taken continuously from the discharge line and introduced directly into a
small effluent headbox that feeds the toxicant delivery system.  If the dis-
charge rate is not reasonably constant, flow-proportional continuous  sampling
may be desirable.   For effluents that are only discharged in batches, a grab
sample must be used and the test must begin within 8 hours after the  sample
is obtained.   The temperature of the sample should be adjusted to be within
the allowable test temperature range before it is added to the dilution water.

         0  Special effluent tests may be conducted on altered or treated
samples of the effluent or on other samples to obtain additional information
concerning the toxicity of the effluent.  When special tests are conducted,
the exact methodology must be described in all test reports (U.S. EPA, 1975a).

      • Periodic Check of Concentration

     During the test, it is desirable to measure the concentration of the
test substance in the test chambers as often as practical.  At a minimum,
the concentration of the test substance must be measured in:

          o each test chamber at least once during the test
          o at least one test chamber at the next to the
            lowest test substance concentration at least once
            every 24 hours during the test
          o at least one appropriate test chamber whenever
            malfunction is detected in any part of the
            toxicant delivery system
                                     222

-------
     For replicate test chambers at the same test substance concentration,
the highest measured concentration divided by the lowest measured concen-
tration must be less than 1.2.  If it is not, the toxicant delivery system
should be checked and additional samples from the proper test chambers should
be analyzed to determine if the sampling or analytical methods are precise
enough.  In addition, the measured concentration of the test substance in any
test chamber must be no more than 30% higher or lower than the concentration
calculated from the composition of the stock solution and the calibration
of the toxicant delivery system.  Measurement of degradation products of the
test substance is desirable (U.S. EPA, 1975).

     Whenever samples from a toxicity test are analyzed, at least one reagent
blank must also be analyzed, if appropriate.  Also, at least one sample for
the method of known additions must be prepared by adding test substance to
water from a control test chamber to match the next to the lowest test
substance concentration used in the toxicity test.  Methods used for
analysis of test substances must be those specified in the latest edition of
the Annual Book of Standards, Part 31  (American Society for Testing Materials,
1974) or methods for Chemical Analysis of Water and Wastes (U.S. EPA, 1974a).
The accuracy of standard solutions should be checked against other standard
solutions whenever possible.  Atomic absorption spectrophotometric methods
for metals and gas chromatographic methods for organic compounds are
generally preferable to colorimetric methods (U.S. EPA, 1975a).
                                      223

-------
     Reagent grade chemicals should be used in all tests.  All reagents
should conform to the specifications of the Committee on Analytical Reagents
of the American Chemical Society, where such specifications are available.
Other grades may be used, provided it is first ascertained that the reagent
is of sufficiently high purity to permit its use without lessening the
accuracy of the determination (ASTM, 1974b).

     • Standard toxicant

     To insure that the technical aspects of the bioassay are properly per-
formed, an internal standard is recommended (LaRoche et al., 1970).  The
compound used routinely is sodium dodecyl sulfate (SDS), a surfactant and
membrane lytic agent.   This compound produces a very sharp response curve
indicating an almost "all or none" effect at concentrations of 1 to 2 mg/1.
While the use of an internal standard can serve as a quality assurance moni-
tor, it does not, in itself, validate an experiment.  Adequate control
survival (>^ 85%) is the primary criterion for the success or failure of a
bioassay.

     • Toxicant concentration selection

     Generally a broad range of concentrations covering at least four orders
of magnitude is chosen initially.  This is followed by a progressive bisection
of intervals on a logarithmic scale (Table 3.3.4) or decilog intervals (Table
3.3.5)  (Rand et al., 1975).

TABLE 3.3.4 GUIDE TO SELECTION OF EXPERIMENTAL CONCENTRATIONS, BASED ON
PROGRESSIVE BISECTION OF INTERVALS ON LOGARITHMIC SCALE  (Rand et al., 1975)
Column 1 Column 2 Column 3
10.0
	 	 	
	 	 	
5.6
	 	 	
	 	 	
	 	 	
3.2
	 	 	
	 	 	
	 	 	
1.8
	 	 	
	 	 	
	 	 	
Column 4
_— —
7.5
	
	
	
4.2
	
	
	
2.4
	
	
	
1.35
	
Column 5
8.7
	
6.5
	
4.9
	
3.7
	
2.8
	
2.1
	
1.55
	
1.15
        1.0
                                     224

-------
              .TABLE 3.3.5 GUIDE TO SELECTION OF EXPERIMENTAL
                     CONCENTRATIONS, BASED ON DECILOG
                      INTERVALS (Rand et al., 1975)
Concentrations
Column 1
10

6.

3.

2.

1.

1.
.0

31

98

51

58

00


(or

(or

(or

(or




6

4

2

1




.3)

.0)

.5)

.6)


Column 2

7

5

3

1

1


.94

.01

.16

.99

.26


(or

(or

(or

(or

(or


7

5

3

2

1


.9)

.0)

.15)

.0)

.25)

Log of Concentration
1
0
0
0
0
0
0
0
0
0
0
.00
.90
.80
.70
.60
.50
.40
.30
.20
.10
.00
     • Sample collection and handling

     All effluent samples collected in the field should be accompanied by a
complete Field Data Sheet (Figure 3.3.1).  Also, the sample containers used
should be labelled with the following information, using a waterproof marker:

          o Name of water body
          o Station number
          o Number of subsamples of sample
          o Date
          o Time
          o Name of collector.

     A chain of custody form (Figure 3.3.2) should also be completed.  The
samples during the transit stage must be at all times either under personal
care or in locked containers.  Upon arrival at the laboratory the samples
are kept in a locked cabinet (e.g., preserved sample - benthic) or locked
refrigerator (e.g., bioassay samples) until analyses of such samples are
initiated.  At the start of a project a professional level biologist is
assigned as project officer with the responsibility to keep a complete
project file, including all record sheets.  It is also his or her respon-
sibility to be aware of the location of the samples in the laboratory and
their analytical status (U.S. EPA, 1975b).

     • Safety precautions

     Many toxicant agents can adversely affect human beings if adequate pre-
cautions are not taken.  Therefore, contact with all toxic agents and test
solutions should be minimized, and special precautions should be taken with
volatile toxicants.  Recommended handling procedures should be studied before
tests are begun with any toxic agent.  Because many effluents contain
sanitary wastes, the investigators should be inoculated for typhoid, polio,
and tetanus before effluent tests are begun.

                                    225

-------
    Location _
    Collector
                  Sta.  Depth (Ft.)
                                 Air Temp.  (°F)
    SAMPLING METHOD  (Circle)
     Kemmerer Petersen Surber  Manual
     Plankton Net  Seine Trawl  Bucket
     Other
                              COMPOSITE DATA (Circle)
                                  Flow   Space
                              Observed Flow 	
                              Avg. Daily Flow
                                      Time
    OBSERVATIONS  (Circle)
     Weather
         Ft. Wave
         Surface
              Bottom %
             TIDE CONDITION
      Clear
North
         Clean
              Ooze
    P.  Cloudy
East
           Oil
              Sand
     Overcast
South
         Garbage
              Jravel
       Fog
West
          Trash
              Clay
     Drizzle
      Rain
0-5
MPH
       Gas Bubbles
              Rubble
       Dead Fish
              Rock
      Snow
5-15
MPH
5+
Sewage
Shell
                                 Ind.  Waste
                      Organic
               Over 15
               MPH
                  Float Solids
 LW      HW    LW
Slack  Slack  Slack

     FloodEbb

 Tide Stage (Height)
        Low
       Normal
        High
    WATER-Color
           Odor
                 From Plankton, Waste, Sediment, Other
                          Fresh/Brackish/Salt
    STREAM-Width (Ft.)
           Rapids 	
                  Depth (Ft.)
            % Pools
                % Riffles
                  Low/Normal/Flood
                           %
    ANIMALS-

    PLANTS-Floating
         Fish:  Adults, Fry
         	 % Emergent
                     Periphyton
                       Insects:  Adults, Larvae
                      	% Sumberged 	%
                                    Algae
Samples to;
 Bact  Bio  Chem  Other
              Collection (Ending) Date
                                 Sample Temp. (°C)
7
Mo
1
Day
1
Station No.
              Ending Time (24 Hr)
                                 DO (mg/1)
              1J
Sample Depth (Ft.)
              Beginning Date
Y
Mo
1
Day
1
                                 Cond. (uMHOS/CM)
Lab Number
              Beginning Time (24 Hr)
                                 Salinity (Z.)
Type of Sample
  Grab  Composite  Sediment
              pH
                                 Other
  Remarks
                                                             (EPA, Region II)
              Figure 3.3.1 Field data sheet.  (U.S. EPA, 1975b)

                                     226

-------
to
NaM of Unit and Address:
Nuaber Unit


Description of Samples

Person Assuming Reaponaiblllty for Samples! Time Date
Number
Number
Number
Relinqulahed 87:
Relinquished By:
Relinquished By:
TiM
TiM
TiM
Date
Date
Date
Received By:
Received By:
Received By:
TiM
TiM
TiM
Date
Date
Date
Reason for Change of Custody
Reason for Change of Custody
Reason for Change of Custody
                                                                               (EPA. REGION II)
                                 Figure 3.3.2  Chain of custody  form (EPA, Region  II)

-------
     Although disposal of test solutions and test organisms poses no special
problem in most cases, health and safety precautions should be considered
before the beginning of a test.

     Rinsing with acetone and other volatile solvents should be performed
only in well-ventilated areas.

3.3.1.4 Test  subject—

     An organism suitable as a test subject for Aquatic Bioassay must
possess a number of characteristics (Rand et al. , 1975):

                o  Sensitivity to the material or environmental factors
                   under consideration
                o  Wide geographical distribution, abundance and avail-
                   ability  throughout the year to allow comparative
                   studies  of control and exposed organisms under
                   different environmental conditions and different
                   locations
                o  Availability of culture methods for its rearing in
                   the laboratory and knowledge of its environmental
                   requirements
                o  Known recreational, economic, and ecological impor-
                   tance locally and nationally
                o  Good general physical condition and freedom from
                   parasites and diseases.

     The susceptibility of  the test organisms to particular test substances
 is  an  important factor to consider prior to choosing the test species.
 Ideally, the most sensitive resident species should be bioassayed.  Then,
 the distribution of the test organism within the system being assayed should
 be  considered,-because ideally the organisms selected should be among the
 representative species of the natural population (Martin, 1973).

     The following sections discuss the species most sensitive to selected
 chemicals and having other  desirable characteristics as test subjects.

     •  Fish, Macroinvertebrates, and Amphibians

     For acute toxicity studies, the following species have been found.
 suitable as test organisms, because they are extremely sensitive to the  test
 chemicals (See Table 3.3.6), they have wide geographic distribution, abun-
 dance, and availability throughout the year, and they adapt easily to
 laboratory conditions:
                                      228

-------
fO
K>
NO
        TABLE 3.3.6   24-,  40-»  and  96-HOUR LC50  VALUES  FOR THE SPECIES  OF FRESHWATER AND ESTUARINE  ORGANISMS
                                                       MOST SENSITIVE  TO SELECTED CHEMICALS
         Clu-mlralH

        .M.ORIX


        AKOCHI.OK  I01f>

        H.H.C.

        CHI.ORIUXK

        DDT

        Dt'RSBAN

        nm.DRix

        F.XDOSn.FA-\

        F.NDRIN


        HEPTACHI.OR  657
                                     24-Hour  (mg/1)
                     S<.Mis_i_t_ivi- Sju'cii's           I.C50   _   Most Sensitive Species ___ _
                                                                                             LC50
                Onco_rhviu*hus t sli.iwvt srlia
                 (fliinook salmon)
                                                 0.0124
                     ^s miUTiu-Jii r»s (hint-gill)  10.0

                l.i'pomls murnirhiriis (hlucg(ll)   0.016

                Papacmonetcs sp.  (grass shrimp)  0.0007

                Palaemonctes sp.  (grass shrimp)  0.0012

                l.epomls macrorhlrus (hluoglll)   0.014

                U-pomIs macrorliIrus (blueglll)   0.0036

                Sajmo ga 1 rdncrl (rainbow trout)  0.00079
HEPTACHLOR 74Z  Plmcphalcs pronelas (fathead
                 minnow)
        HEPTACHLOR 99?

        I.IXDAXE


        MALATHTOS

        METHOXYCHI.OR

        PARATHIOX

        TOXAPHF.XK


        .',4-1)

        CARBARYI.


        :-K)i.ix.\rr

        I'ROPAXIf.
                                 0.013
                                                 n.so
                rAlib-tinx'J-S SP- (prnss shrimp)  0.012
                C_as_t_c_ros^t_(/ti^ acii
                 (stTcVlVhnck)"
                                                82.0

                        marrorhj rus (hlm-glll)   O.OOfi
                (..imhii^i In iiffiuls (mosqni ti< llsh) 7.0

                OncojMivnrluis kisutrli (roho       0.001
                 salmon)
l'a^laemonc-Jj.-s sp.
()amh_usia af finis (mosquito
 fish)
                                    nss shrimp) J2.0

                                                II.)
                                            Oncorliyncluis tshawytscha
                                             (ohI nook salmon)
                                                                                             O.OlOf.
                                            l.i'juim_is mm rpchirus (hlueglll)   7.8

                                            l.epomls macrochtrus (blueglll)   0.032

                                            Salmo clarkl (cutthroat trout)   0.0016
                                                  is azte£us_ (brown shrimp)   0.0004
                                            Fundulus slmilts (longnose       0.00023
                                             ktllflsh)
Oncorhvnchus tshawytscha         0.026
 (rhlnook salmon)
                                            Pimcpjiale_s p_romel_as (fathead     0.070
                                             minnow)

                                            Panai-us diiorarum (pink shrimp)   0.0125
       Li ri-t irujjita (suppy)

       ijJ affijilji (mosquito
 "rso

l.i'juinils macroi-tu riis (hliR'gl 1 1 )

Carasshis aurntus (noldflsh)
                                                                            68.0

                                                                             O.OL'4


                                                                             I.I

                                                                             O.I I
           s sp. (urass shrimp) 20.0

                                1 1.0
                                                                     (iamhus^la  »i_f_r i n I s  (mosquit
                                                                      flsli)
                                                                                        	gft-Hour  (rag/1)	
                                                                                        Most Sensitive Species	LC50
Morone saxatilis  (striped  bass)   0.0072


Grassestrea virginica  (oyster)    0.102

Panaeus duorarun  (pink shrimp)    0.00034

Panaeus duorarun  (pink shrimp)    0.0004

Panaeus aztecus (brown shrimp)    0.0001



Panaeus duorarun  (pink shrimp)    0.0007

Panaeus duorarum  (pink shrimp)    0.00004

Panaeus duorarun  (pink shrimp)    0.0001


Panaeus duorarun  (pink shrimp)    0.0001

Panaeus duorarun  (pink shrimp)    0.0001


Panaeus duorarun  (pink shrimp)   0.00003

Panaeus duorarun  (pink shrimp)   0.0002


Panaeus duorarun  (pink shrimp)   0.0125

Panaeus duorarun  (pink shrimp)   0.0035

I'oecJJJjj ret Iculata (guppy)     56.0

Cyprlnodon vartcgatus (sheeps-   0.0011
 head minnow)



Oncorhvnchus kisutch (echo       0.0013
 salmon)

p-ilLaJLJE0-n£?.££ 2E-  (grass shrimp) 16.0

                                  9.46

                             (continued)
                                                                                                        (^i_m_hi^s_ia af T in is (mosquito
                                                                                                         fish)

-------
                                                       TABLE 3.3.6    (Continued)
 Chemicals
                            24-Hour (mg/I)
                                                        40-Hour (mg/1)
TRIFLURALIN

KEPONE

L.A.S.


PHENOL

CADMIUM


COPPER


CHROMIUM


LEAD


MERCURY

NICKF.L

ZINC
                Most Sensitive Species
Crangon soptemsplnosa (sand
 shrimp)
                                                 LCSO
                                                                                                   96-Hour  (mg/1)
Pimephales promelas (fathead     1.9
 minnow)
                                 2.4
Pimephales promelas (fathead     0.04
 minnow)

Pimephales promelas (fathead    19.6
 minnow)

PLmcphalcs proroelas (fathead     8.18
 minnow)

Morone saxat11 Is (striped bass)   0.22

Horone saxatills (striped bass)  10.0

Horone saxat ills (striped bass)  11.2
                                            Host Sensitive Species
                                                                                             LCSO
                                                                                       Most Sensitive Species
                                            Lepomls macrochirus  (blueglll)    0.019
                                            Pimephales  promelas  (fathead
                                             minnow)
                                 1.7
Lepomis macrochirus (blueglll)  20.5

Crangon septemspinosa (sand      0.50
 shrimp)

Pimephales promelas (fathead     0.023
 minnow)

Pimephales promelas (fathead    19.7
 minnow)

Pimephales promelas (fathead     5.9
 minnow)

Horone saxatilts (striped bass)   0.14

Poecilla rcttculata (guppy)      6.7

Cyprinus carpio (carp)           9.3
                                                                                        Leiostomus xanthurus  (spot)
                                                                             LCSO
                                                                                                                        0.0066
Lepomis macrochirus (bluegill)   19.3

Salmo galrdneri (rainbow         0.0010
 trout)

Pimephales promelas (fathead     0.022
 minnow)

Pimephales promelas (fathead    17.6
 minnow)

Salmo gairdneri (rainbow         1.17
 trout)

Morone saxatilis (striped bass)   0.09

Poecilia reticulata (guppy)       4.45

Salmo gairdneri (rainbow trout)   0.430

-------
          o  Grass Shrimp - Palaemonetes sp.
          o  Pink Shrimp - Panaeus duorarum
          o  Brown Shrimp - Panaeus aztecus
          o  Sand Shrimp - Crangon septemspinosa
          o  Fathead Minnow - Pimephales promelas
          o  Sheepshead Minnow - Cyprinodon variegatus
          o  Rainbow Trout - Salmo gairdneri
          o  Cutthroat Trout - Salmo clarki
          o  Coho Salmon - Oncorhynchus kisutch
          o  Chinook Salmon - Oncorhynchus tshawytscha
          o  Bluegill Sunfish - Lepomis macrochirus
          o  Stickleback - Gasterosteus aculeatus
          o  Killifish - Fundulus similis
          o  Mosquito Fish - Gambusia affinis
          o  Guppy - Poecilia reticulata
          o  Striped Bass - Morone saxatilis
          o  Gold Fish - Carassius auratus
          o  Carp - Cyprinus carpio
          o  Spot - Leiostomus xanthurus

     •  Macroinvertebrates

     Daphnia magna was found to be the most senitive animal to herbicides
(Table 3.3.7) followed in descending order of sensitivity by seed shrimp,
scud, glass shrimp, sowbug and crayfish (Sanders, 1970).  In a study of
acute toxicity of various metals to freshwater zooplankton (Table 3.3.8),
Daphnia hyalina was more sensitive than either Cyclop abyssorum and
Eudiaptomus padanus.  The high sensitivity of Daphnia makes this inverte-
brate a useful test organism for heavy metal pollutants.

     In addition, Daphnia fulfills a whole series of requirements for
an animal to be used in water pollution tests:

          o  it is easy to find everywhere
          o  it is of small size but not miscroscopic
          o  it has a simple level of organization thus
             avoiding secondary effects of toxic chemicals
          o  it is of rapid reproduction and easy to breed
             in the laboratory (Baudouin and Scoppa, 1974).

     •  Aquatic Insects

     Aquatic insects to be used as biological monitors of heavy metal
fishkills must fulfill three prerequisites (Nehring, 1976):

          o  The insect should be more tolerant of
             the heavy metals than the fish in question
                                     231

-------
                 TABLE 3.3.7  THE 48-HR TL50 (mg/1)  OF SOME HERBICIDES TO SIX SPECIES OF
                              FRESHWATER CRUSTACEANS AT TWO DIFFERENT TEMPERATURES (SANDERS, 1970)
to
HERBICIDE
Diclone
2,4.D
oilvex
(P.G. BE)
Trifuralin
Molinate
Simazine
Vernolate
Silvex
(B.E.E)
2,4.0 (Bi-
ne thyi-
anine
salt)
2.4..D
(B.E.E.)
Oichlobenil
Amitrol - T
Diphenanid .
Waterflea
Daphnia
magna
21°C
0.025
0.10

0.18
0.56
0.60
1.0
1.1

2.1



4.0
5.6
10.0
30.0
56.0
Seed
Shrimp
Cypridopsis
vidua
21°C
0.12
0.32

0.20
0.25
0.18
3.2
0.24

4.9


-
8.0 i "•
1.8
7..S
32,0
S0,0
Scud
Gamma r us
fasciatus
15.5°C
0.24
2.6

1.0
1.8
0.39
100.0
! 20,6

0.74



{ I**:
.: -5.*
w-:«
; 106.O
Sowbug
Asellus
brevicaudus
15.5°C
0.20
2.2

0.50
2.0
€.40
100.0
5.6

40.0



100.0
3.2
34.0
100.0
100.0
Glass
Shrimp
Palaemonetes
kadiakensis
21°C
0.45
2.7

3.2
1.2
1.0
100.0
1.9

8.0



aoo.o
1.4
9.O
1OO.O
58.0
Crayfish
Orconectes
Snails
15.5°C
3.2
100.0

100.0
50.0
5.6
100.0
24.0

€0.0



100,0

*
UH3.0
100.0

-------
      TABLE 3.3.8  ACUTE TOXICITY OF VARIOUS METALS (mg/1, 48 hour TL50)
            TO FRESH WATER ZOOPLANKTON (Baudouin and Scoppa, 1974)
 Metal
Cyclops abyssorum
Eudiaptomus padanus   Daphnia hyalina
Calcium
Magnesium
Strontium
Cesium
Chromium VI
Cobalt
Nickel
Lead
Mercury
Zinc
Cadmium
Copper
7000
280.0
300.0
400.0
10.0
15.5
15.0
5.5
2.2
5.5
3.8
2.5
4000
180.0
180.0
135.0
10.1
4.0
3.6
4.0
0.85
0.50
0.55
0.50
3000.0
32.0
75.0
7.4
0.022
1.32
1.90
0.60
0.0055
0.040
0.055
0.005
          o The insects must concentrate the toxic metal in relative
            proportion to the metal content of the water

          o The insects must concentrate the metal pollutant by some
            predictable factor over a short time period

     In this kind of experimentation a good bio-accumulator is desirable.

     Toxicity data for three aquatic insects are given in Table 3.3.9
(Warnick and Bell,1969).  A comparison of the TL50 values of lead, zinc,
copper, nickel and cadmium to toxicity in fish, i.e., stickleback (TL50
mg/1 for Zn = 0.01-10.0; for Cu - 0.01-0.02; for Ni = 0.08-1.0; for Pb =
0.1-0.4; for Cd = 0.03), reveals aquatic insects to be more tolerant of all
heavy metals tested.  The Mayfly, however, was less tolerant of silver than
rainbow trout (Jones, 1938).  Tables 3.3.10 - 3.3.13 (Nehring, 1976) compare
the levels of accumulation in the insect with the levels of exposure.  In
each test, the average level of exposure was paired with the corresponding
average accumulation level in the insect.  The correlation coefficients in
seven of the fourteen bioassays were 0.97 or greater (Table 3.3.14).  These
correlation coefficients indicate that aquatic insects accumulate heavy
metals in relative proportion to the metal concentration in the water.
                                      233

-------
TABLE 3.3.9  THE ACUTE TOXICITY OF SOME HEAVY METALS TO AQUATIC INSECTS
                                  (Warnick and Bell,1969)
Metal
Cu4"4" from CuSO^* 5H20


•7 ++
^n from ZnSO. *7H. 0
^ ^

Cd4"1" from CdSQtt«8H20


Pb"*"1" from PbSOit


^ 1
Fe from FeSOi+


Ni4* from NiSOtt«6H20


1 |
Co from CoSOi^ 7H£0


Insect
acroneuria
ephemerella
hydropsyche
acroneuria
ephemerella
hydropsyche
acroneuria
ephemerella
hydropsyche
acroneuria
ephemerella
hydropsyche
acroneuria
ephemerella
hydropsyche
acroneuria
ephemerella
hydropsyche
acroneuria
ephemerella
hydropsyche
96-hr
TL50
(mg/1)
8.3 (0.32
48-hr)





2.0





0.32

33.5
4.0


16.0

50%
(days)


14
14
10
11
14


14
7
7
>14
7
7


>14
8

7
Survival
(mg/1)


32.0
32.0
16.0
32.0
32.0


64.0
16.0
32.0
64.0
16.0
32.0


64.0
32.0

32.0
                               234

-------
      TABLE 3.3.10  COPPER BIOASSAYS, AVERAGE EXPOSURE vs.
                    AVERAGE ACCUMULATION  (Nehrlng, 1976)
     Mayfly  (2 Replications)
Exposure            Accumulation
 (mg/1)                 (ug/g)
10.0
4.82
2.51
1.22
0.63
0.00
9,125
5,787
3,882
1,933
1,240
94.7
                            Stonefly (3 Replications)
                       Exposure            Accumulation
                        (mg/1)                (yg/g)
                                             12.2
                                             10.4
                                              8.13
                                              6.47

                                              0.00
                                             2,540
                                             2,096
                                             1,767
                                             1,199

                                               122.3
      TABLE 3.3.11
LEAD BIOASSAYS, EXPOSURE vs.
ACCUMULATION (Nehring, 1976)
Exposure
 (mg/1)
   9.24
   4.90
   2.34
   1.32
   0.69
   0.00
            Mayfly
 Accumulation
     (ug/g)
  104,700
   73,200
   31,780
   14,560
    5,702
      126.6
                                  Stonefly
Exposure
(mg/1)
19.2
7.44
4.43
1.96
1.08
0.00
Accumulation
(ug/g)
8,172
2,249
1,666
736.6
716.7
8.18
      TABLE 3.3.12  SILVER BIOASSAYS, AVERAGE EXPOSURE
                    vs. AVERAGE ACCUMULATION (Nehring, 1976)
     Mayfly (2 Replications)
Exposure             Accumulation
 (mg/1)
    (ug/g)
0.75
0.40
0.23
 •M
 .00
                                             Stonefly  (3 Replications)
                                                            Accumulation
                                                                (yg/g)
Exposure
 (mg/1)
                                             0.738
                                             0.399
                                             0.217
                                             8:i§S
                                             0.000
                                             53.28
                                             30.76
                                             22.95
                                               3.97
                                     235

-------
   TABLE 3.3.13  ZINC BIOASSAY, EXPOSURE vs. ACCUMULATION (Nehring 1976)
          Mayfly
                               Stonefly
Exposure
(mg/1)
9, .20
4.32
2.29
1.04
0.60
0.00
Accumulation
(yg/g)
2,361
2,381
2,187
2,029
1,794
1,116
Exposure
(mg/1)
13.6
5.54
2.83
1.61
0.77
0.00
Accumulation
(ug/g)
561.2
497.1
415.7
507.7
439.4
357.2
   TABLE 3.3.14
BIOASSAY PARAMETERS AND CORRELATION COEFFICIENTS
                 (Nehring, 1976)
Test Metal
    Test Insect
Range of Exposure
 (metal in mg/1)
Correlation
Coefficient
Copper
Copper
Copper
Copper
Copper
Lead
Lead
Silver
Silver
Silver
Silver
Silver
Zinc
Zinc
Stonefly
Stonefly
Stonefly
Mayfly
Mayfly
Stonefly
Mayfly
Stonefly
Stonefly
Stonefly
Mayfly
Mayfly
Stonefly
Mayfly
0.74 - 13.9
5.51 - 18.5
6.47 - 12.2
0.63 - 10.0
0.08 r 1.06
1.08 - 19.2
0.69 - 9.24
0.05 - 0.74
0.004- 0.067
0.006- 0.104
0.06 - 0.75
0.01 - 0.15
0.77 - 13.6
0.60 - 9.20
0.986
0.901
0.994
0.982
0.974
0.991
0.985
0.996
0.909
0.830
0.893
0.666
0.779
0.694
                                   236

-------
      The predictable factor,  termed  "concentration  factor",  is  determined
 by  dividing the average level of  exposure  into  the  average level  of metal
 accumulation in the insect.   The  concentration  factor  is very effective in
 estimating the average level  of exposure to  lead, copper, and silver
 (Table  3.3.15) (Nehring,  1976).   In  19  of  28 instances, the  concentration
 factor  estimated the actual level of exposure with  an  accuracy  of 80% or
 better.   In 10 of 28 instances, the  concentration factor estimated the  actual
 level of exposure with an accuracy of 90%  or greater.  Thus  aquatic insects
 as  tested do concentrate heavy metals by some predictable factor.

      TABLE 3.3.. 15  EFFECTIVENESS  OF  CONCENTRATION FACTORS
      IN ESTIMATION OF AVERAGE LEVELS OF EXPOSURE TO LEAD, COPPER
      AND SILVER (Nehring,  1976)
            Percent  Accuracy

              50   -  59%
              60   -  69%
              70   -  79%
              80   -  89%
              90   -  99%
         Frequency

           1/28
           3/28
           5/28
           9/28
          10/28
      In  summary, aquatic  insects  fulfill the three prerequisites mentioned on
pages 231 and 233, and appear to be excellent biological monitors of heavy
metal pollution.  They are more tolerant of metal than fish, they accumulate
metal in relative proportion to the metal concentration in the water and
they concentrate the metal by som6 predictable factor.

      •   Benthos

         o   In a study  (Hansen et  al., 1974a) the American oyster, brown
shrimp and  grass shrimp were found to be about equally sensitive to
Aroclor  1016 (Table  3.3.16).
              TABLE  3.3.16  AROCLOR 1016  (Hansen et al.,  1974a)

           Test Organism             Scientific Name
           Oyster


           Brown Shrimp

           Grass Shrimp
Crassostrea
virginica

Panaeus aztecus

Palaemonetes sp.
10.2


10.5

12.5
                                     237

-------
     In addition to its sensitivity,  the American  Oyster  possesses  a wide
geographic range extending  from Price Edward,  Canada,  along the Atlantic
Coast to the Gulf Coast of  Texas.   It is now  feasible  to  spawn adult oysters,
rear the larvae, and maintain  the  spat and  juvenile  oysters under controlled
laboratory conditions.

          o  The midge  (Chironomus species) was  found  to  be the most
sensitive test organism to  certain metals  (Table 3.3.17)  (Rehwoldt, et al.,
1973):

          Mercury++(24-hr LC50 =0.06 mg/1)

          Copper-H-(24-hr and 96-hr LC50 -  0.65 and 0.03 mg/1)

          Nickel++(24-hr LC50  =10.2  mg/1;  96-hr LC50  =8.6 mg/1)

     In the same study, the scud  (Gammarus species )  was the most sensitive
organism to zinc-H-, cadmium-H-  and  chromium-H-  in  both 24 hour and 96 hour
acute toxicity  study, and to mercury-H- in  96  hour.

          o  Green Algae (Dunaliella  tertiolecta Butcher),  found in marine
and  estuarine waters, has shown the most linear  response  for every  parameter
examined  (McLachlan, 1960).  An additional  advantage of green algae is that
it requires no  outside  sources of  vitamins  (Provasoli, 1963).   Dunaliella
tertiolecta has been shown  to  be a highly  versatile  and consistent  bioassay
organism for nutrient assessment  in marine, estuarine, and  some freshwater
situations.  It  will respond to concentration  at  least  as  low as 2.5 mg
phosphorus  (P)/l; 10 mg ammonia  (N)/l and  50  mg  nitrate (N)/l in defined
media (Specht and Miller, 1973).   Green algae was  also found to be  one of
the  most sensitive species  to  herbicides  (Table  3.3.18) (Hollister  and
Walsh, 1973).

     The following are  the  average EC50 values (ppb) from Table 3.3.18 for
four herbicides and four families  of  marine unicellular algae.
Family
Chlorophyceae
Bac illar iophy ceae
Chrysophyceae
Phodophyceae
Number of Species
Tested
, 6
8
3
1
Neburon
EC50
23
77
24
24
Diuron
EC50
22
67
13
24
Atrazine
EC50
104
265
92
79
Ametryne
EC50
31
65
11
35
The  family  of  Chrysophyceae as a whole was  generally the most sensitive.   In
addition  to Dunaliella  tertiolecta,  Skeletonema  costaturn is an ecologically
important phytoplankton that  is common to a wide geographic range of neritic
waters  and  Thalassiosira pseudonana  is sensitive to  heavy metals and has  an
8  hour  generation time  which  offers  great practical  value in the establish-
ment of toxicological responses.   Both Skeletonema costatum and Thalassiosira
pseudonana  have been recommended by  EPA  (US.  EPA,  1976).
                                     238

-------
           TABLE 3.3.17  THE TOXICITY  (LC50, mg/1) OF SOME HEAVY METAL  IONS  TOWARD BENTHIC ORGANISMS
                                                   (Rehwoldt et al.,  1973)
      Test             Cu  	Zn  	Ni	Cd	Hg  	Cr
    Organisms       24hr  96hr    24hr  96hr   i.  24 hr" 96hr <   .  24hr  96hr   .    24ht   96hr     24hr  96hr

    Bristle Worm    2.3   0.09    21.2  18.4      16.2   14.1      4.6   1.7        1.9    1.0      12.1  9.3

    Scud            1.2   0.91    10.2*  8.1*     15.2   13.0      0.14* 0.07*      0.09   0.01*    6,4* 3.2*
     (amphipoda)

    Caddis Fly .    12.1   6.2     62.6  58.1      48.4   30.2      5.1   3.4        5.6    1.2      58     50
N»            '
U>
"*   Damsel Fly     10.2   4.6     32    26.2      26.4   21.2     11.0   8.1        3.2    1.2      46     43.1
     (zygoptera)

    Midge           0.65* 0.03*   21.5  18.2      10.2*   8.6*     5.1   1.2       0.06* 0.02     16.5   11.0
     (Diptera)
               * i                            *                                                       * '
    Snail (egg)     4.5   9.3     28.1  20.2      26.0   11.4      5.1   3.8       6.3   2.1      15^2   12.4
     (Gastropoda)

    Snail (adult)   1.5   0.9     16.8  14.0      21.2   14.3     10.1  - 8.4       1.1   0.08     10*2   8.4
                                                                                                   »

           *Most sensitive

-------
     TABLE  3.3.IS EC50  (ppb) OF NEBURON, DIURON, ATRAZINE, AND AMETRYNE ON
          OXYGEN EVALUATION BY MARINE UNICELLULAR ALGAE.  STANDARD
          ERRORS (SE) WERE DERIVED  BY UNWEIGHTED PROBIT ANALYSIS
                       (Hollister  and Walsh,  1973)
K.imlly S|u>cI«'N
(.hloropliyrcav
Chl.iinydomon.-in «|>.
Duna 1 !(.• 11 n tcrt loli-cl .1
I'latymon.iH «p.
Clilort-l la sp.
Ni'iu'hlorlM sp.
Chlorocncrun sp.
K.H 1 1 larltipliyccar
Thai OHM IPS Ira I'l uvl.it 1 1 Is
N.ivlruln InHcrta
Agnjiorn cxlgua
Achnanttu-H hrcvlpt-H
Stniirom-lH a.I"nh
-------
     TABLE 3.3.19  THE GROWTH SENSITIVITY OF ALGAE TO COPPER
                          (Erickson et al., 1970)

Organism                             ug Cu/1
                               50   100   500   100   1500   2000   3000
Porphyridium cruentum      +
Monochryais lutheri        4-
Nannochloris oculata       +
Amphldinium carteri        +
Skeletonema costaturn       +
Ohisthodiscus luteus       4-
Chaetoceros sp.            4-
Nitzschia closterium       4-
Platymonas subcordiformis  4-
Cyclotella nana            4*
Dunaliella tertiolecta     4-
Isochrysis galbana         4-
4- - Visible growth after 14 days

     Naricula seminulum« another species of diatom, was found to be the most
sensitive to nitrilotriacetic acid (NTA) (Table 3.3.20) (Sturm and Payne,
1973).   An additional advantage of diatoms is that the use of unialgal diatom
cultures for laboratory bioassay analysis has been an accepted ASTM (American
Society for Testings and Materials) method for several years  (Patrick, 1964).

     TABLE 3.3.20 THE COMPARATIVE STATIC, ACUTE TOXICITY OF NTA TO
           BLUEGILLS, SNAILS, AND DIATOMS EXPRESSED AS mg/1
                      (Sturm and Payne, 1973)
Test Organisms
Bluegill
Snails
Diatoms
Bluegill
Snails
Diatoms
Scientific Name 96 hr
Lepomis macrochirus
Physa leterastropha
Naricula seminulum
Lepomis macrochirus
Physa leterastropha
Naricula seminulum
TL50 ma/1
252
373
185
487
522
477
Water _a
•fl^r/l $SL\
60
60
60
170
170
170
ardness
P°9L






     In addition to the diatom, the following species have been successfully
utilized in bioassay and have been proposed by EPA in the algal assay Bottle
test (Payne, 1975).

          o Selenastrum capricornutum
          o Microcystis aeruginosa
          o Anabaena flos-aquae
                                     241

-------
     Of  the  three  species  selected,  Selenastrum capricornutum is  the easiest
 to culture and  to  use  in testing.   Its  growth  rate  is approximately twice
 that of  the  two blue-green Microcystis  aeruginosa and Anabaena flos-aquae.
 Its growth responses normally  are more  clearly nutrient dependent and test
 results,  therefore, are  easier to interpret.

     • Protozoans

     Protozoa,  algae,  and  bacteria  form the broad bases of the aquatic  food
 chain.   Ciliates are among the most numerous organisms of the estuarine
 benthos  (Borror, 1963),  and  may be  most important as nutrient regenerators
 (Johannes, 1965).  Also, some  ciliates, including Tetrahymena pyriformis,
 can concentrate certain  pesticides  and  PCB's  (Cooley et al., 1972; Gregory
 et al.,  1969).  Tetrahymena  pyriformis  has been used as test organism (Rand
 et al.,  1975) for  the  following reasons:

          o  it  occurs  in freshwater and salt marshes
          o  it  has world-wide  distribution
          o  it  is  readily  grown in  axenic culture
          o  its physiology has been studied extensively

     Tetrahymena pyriformis  strain  W and HSM.  has been used successfully in
 many bioassays  (Elliott  et al.,  1973; Corliss, 1970).

     The  sensitivity of  T. pyriformis strain W to insecticide is shown  in
 Table 3.3.21  (  Cooley, 1973).

     TABLE 3.3.21  SENSITIVITY  OF T.  PYRIFORMIS, STRAIN W, TO INSECTICIDES
                             (Cooley, 1973)

 Toxicant        Growth Rate         96-hr, population   Accumulation (X  in-
	reduction	density reduction   itial concentration)

 Mirex           33% at 0.9 ug/1     12%  at 0.9  ug/1           193 X
 Aroclor 1248    18.9% at 1 mg/1     9.6% at 1 mg/1             48 X
 Aroclor 1254    8% at 1  ug/1        10%  at 1 yg/1              60 X
 Aroclor 1260    19.1 to  25% at      13.6 to 22.4%              79 X
                   1 mg/1            at 1 mg/1


     These data indicate that a  significant reduction in population growth
 and 96-hr population density occurred at low toxicant concentrations.
 T.pyriformis. strain HSM, has been chosen as test species because:

          o it  is a large, mobile cell, easy to observe and
            count under relatively low  power of magnification
          o it has been in culture for  30 years without known
            genetic change
          o its cell is easily grown and has a generation time
            of  3 hrs at room temperature,
                                      242

-------
      Table 3.3.24 shows  the  lethal  concentrations  of certain heavy metals for
 Tetrahymena and several  species  of  fish.

      These data suggest  that with the  exception of lead nitrate,
 T_.  pyriformis  is a more  sensitive indicator  of  water pollution due to heavy
 metal contamination  than fish.   In  addition, T_. pyriformis  is, in turn, part
 of  the zooplankton which serves  as  food for  organisms higher in the food chain.
 Therefore, toxic damage  to T_. pyriformis  should give an indication that
 harmful changes are  likely to occur in those organisms  which are  higher in
 the food chain (Carter and Cameron, 1973).

      • Microorganisms

      Keil et al.  (1972)  described a commercial PCB formulation at a
concentration of 0.1 yg/ml which stimulated the growth of Escherichia coli.
Little information on the interactions of  PCB's with heterotrophic
 microorganisms is available  (Kell et al., 1972).   Bourquin  et al.   (1975),
 in  the study of inhibition of growth of estuarlne  bacteria  by PCB, came to
 the realization that most of the sensitive bacteria were gram-positive
 (Table 3.3.23).

      In addition,  Trudgill et al.   (1971) performed a test  on the comparative
 effects of organochlorine on bacterial growth ( Table 3.3.24). The gram-
 positive bacteria were found to  be  more sensitive  than  gram-negative
 bacteria and,  particularly,  the  Bacillus  species was the most sensitive,
 judged by the  range  of inhibition of growth  by  insecticides.

      •  Species Recommended for use  in  Aquatic Bioassay  Tests

      Some tolerant and sensitive species were recommended for use  in aquatic
 bioassay tests by Arthur Scheier (Academy of Natural Sciences of
 Philadelphia).

      Among the fish  suggested were:

           o the sensitive brook  trout  - Salyelinus  fontinalis
           o the more tolerant free-swimming  bluegill -  Lepomis
             macrochirus
           o the tolerant scavenger  channel catfish  - Ictalurus
             punctatus
                                      243

-------
      TABLE  3.3.22 COMPARISON OF  LETHAL  CONCENTRATIONS OF POLLUTANTS ON
           TETRAHYMENA AND OTHER AQUATIC  ORGANISMS  (McKee and Wolf,  1963)



Compound
Mercuric chloride
Mercuric chloride
ZincsuUatc
Lead nitrate
Lead nitrate
Cobalt sulfate
Cadmium sulfate


Water
condition
Unknown

Distilled
"Sofrg
"Hard"!
Unknown
DistiDcd



_
Organism
Minnows

Minnows
Fathead minnows
Fathead minnows
Stickleback
Minnows



Time
42 min

3J3h
%h
96 h
Unknown
3h


Concentration
(mgl-1)
10

400
3.n
100J
10
1042
Teiraliymfam data from
present study
Water Concentration
condition (mg|-')J
SoftT 3.12
HerdS l.*5
Dotilkd 5.77
Soft 37.75
Hard 250
Distilled 4.08
Distilled O.S4
• McKce and WOLF (1963).                                         .-
t Distilled water containing 20 mg l~' calcium carbonate.
j Tolerance limtt median (concentration which kills 50 per cent of the organisms in 96 b).
{ Distilled water containing 400 mg i ~' calcium carbonate.
| Calcium carbonate concentration not specified.
      TABLE  3.3.23 INHIBITION OF  GROWTH OF  ESTUARINE BACTERIA IN NUTRIENT
            SEAWATER MEDIUM  BY PCB'S  (  Bourquin et al.,  1975)
atttBT
Colcura
Bo.
3
21
35
39
53
54
7
9
31
60
86
100
8
11
42
44
93
43
5
13
28
32
41
67
69
Graa
Reaction &
Morphology
+ ROD
- ROD
- ROD
- COCCOZD
- ROD
+ ROD
+ ROD
+ ROD
. goo
+ ROD
• ROD
- ROD
4- ROD
-ROD
+ COCCUS
* coccus
+ ROD
* COCCUS
-COCCOID
— ROD
-ROD
+ ROD
- COCCOZD
-ROD
•ROD
Genoa
Unknown
Unknown
Flcvobactarluai ap.
Unknown
Unknown
Baeillua ap.
Bacillus *p.
RarH Ina >n .
Unknown
Bacillua «p.
Flavobaccarlua ap.
Paaudoaonaa ap.
CoryiMbaccarlua) ap.
Achroanbaccar ap.
Hlerococcua ap.
Mlcrocoeeua ap.
Unknown
Micxococcua ap.
Sarratia ap.
Aearoaobaccar ap.
AchroMobactar ap.
CorynabactarluB ap.
Unknown
Aebroaobactar ap.
Unknown
AroelorR U42(ag)
0.1 0.25 0.5
•f* +* 4**
•f* +* ++*
•H- ** -M.
++• ++* *4*
•«• 4+* -H^
•^^^ +++ ^^^
+ * +
•*• •»-»• +**
•f + •*+*
+ •»••»•
•f + +
•»• ++• +*
X •»• +*
Z * -M-
Z + -H-
Z + +
Z * +
_ +• +*
_ _ +*
_ ~ +4-
~ ~ 4*
~ * •*•
~ _ +*
~ * •+•
_ _ +*
AxoelorE 1016 (m«)
0.1 0.25 0.5
+* +* +**
+* +* +**
•f •*** 4*4-
+*• *** +**
^^+ ^^^ ^4*^
•f +4+ *++•
Z + **
+• ++ +4+
+• *• ++*•
44- +4+4-
Z ••• +
4> 4* 44-
Z 4-44-
Z + 4*
Z +4+
Z *
_ Z *
_ * 4*
_ _ 44-
"" 4*
~ 44-
I Z *
__ _ 4*
"~ ~ 4-
"" ~ 4+
   Dagraa of aanaiclrlcy:  444>(18-20
       -(not •cnaiclva).
am son*), -H-(16-L8 a*),  -K14-16 am),
                                             244

-------
      TABLE 3.3.24  THE EFFECTS  OF ORGANOCHLORINE INSECTICIDES
           ON BACTERIAL GROWTH (Trudgill et al, 1971)
tficro-organism
Bacillus •egatartuB
B. suRtllla
Streptosqrces aatlbioticus
goeardia sp. B
Corynebacterltsk sp. TI
B. cereus
Soeardia sp. A
Sicrobaeterlisi flavu*
Micrococcus lysodeUttlcus
Stapbylococcus albus
Saxclna 1 t»m * "U*
Streptococcus Csecalis
Arthrobacter sisjplex
Pseudoeooaa iodlnu*
Aeh nTohmet '"'fi.


j?7 **^*^^
•ur^faelM*
aureozacieas
aeoaa.ogeas
cioorescens

pS3rta"
lasecticlde
? 2
>^ «^
. i i 1 5 5
c-a-ook*cs»*
«bb«-a-M^-ox
^OOUMWWOu
C •• -< O>e*O'9»4 si>
5555S<^^A§
Cram-poslclvc
----_-.+• + .*.
....... (+) 4.+
------ + + **
- .
----- + *. + +.*
Crsst-aegeclve

* * *
* * •*• *
-«• •*••*• +

I } •*• * +•
* * +••*••*• +
* * * *••»••*•+••»•
* + + + *•*• + + +*
•*• + * + *(•*•)**• * +
+. Growth not Inhibited; (+), growth slightly inhibited; -, growth severely or completely lahlhlted.

     Among the Invertebrates were:

          o  the mayfly -  Isonychia bicolor
          o  the waterflea - Daphnia pulex
          o  two snails;
               - the sensitive gilled snail - Amnicola limosa
               - the tolerant  pulmonate snail - Physa heterostropha
                         f                              ™^

     Algal species suggested by Scheier are:  the diatoms - Nitzschia
closterium and Navicula seminulum.   Mount (1968) lists some twenty fish  :
species which have merit as bioassay test organisms  (Table 3.3.25) and
recently U.S. EPA  (1975a)  has  listed recommended species for general bioassay
use (Table 3.3.26).
                                     245

-------
TABLE 3.3.25  FISH SPECIES RECOMMENDED FOR USE IN AQUATIC BIOASSAY
                             TESTS (Mount, 1968)
           Common Name

           Threadfin shad

           Brook.trout

           Rainbow trout

           Northern pike

           Emerald shiner

           Fathead minnow

           White sucker

           Channel catfish

           White.bass  . •    ...

           Blueglll

           Largemouth bass

           Yellow perch

           Limited Distribution

           Coho salmon

           Lake trout

           Lake herring

           Mountain whitefish

           American smelt

           Smallmouth bass

           Walleye
Genus and Species

Dorosoma petenense

Salvelinus fontinalis

Salmo gairdneri

Esox lucius

Notropis atherinoides

Pimephales promelas

Catoatomus commersoni

Ictalurus punctatus

Morone chrysops

Lepomis macrochirus

Micropterus salmonides

Perca flavescens



Oncprhynchus kisutch

Salvelinus namaycush

Coregonus artedii

Prosopium williamsoni

Osmerus mordax

Micropterus dolomieui

Stlzostedion vitreum
                                  246

-------
      TABLE 3.3.26  RECOMMENDED SPECIES AND TEST TEMPERATURES
                                (U.S.  EPA,  1975)

                                                   Recommended test
 Recommended species	temperature (°C)

 Freshwater
    Vertebrates
      Coho salmon, Oncorhynchus klsutch                  12
      Rainbow trout, Salmo gairdneri                     12
      Brook trout, Salvelinus fontinalis                 12

      Goldfish, Carassius auratus                        22
      Fathead minnow,  Pimephales pro.melas                22
      Channel catfish, Ictalurus punctatus                22
      Bluegill, Lepomis macrochirus                      22

    Invertebrates
      Daphnids, Daphnia magna or I), pulex                17
      Amphipods, Gammarus lacustris, G. fasciatus,       17
              or _G. pseudolimnaeus                       17
      Crayfish, Orconectea species, Cambarus species     22
        Procambarus species,  or Pacifastacus leniusculus 22
      Stoneflies, Pteronarcys species                    12
      Mayflies, Baetis species or Ephemerella species    17
      Mayflies, Hexagenia limbata or H. bilinata         22
      Midges, Chironomus species                         22

 Marine and estuarine
    Vertebrates
      Sheepshead minnow, Cyprinodon variegatus           22
      Mummichog, Fundulus heteroclitus                   22
      Longnose killif ish., Fundulus similis                22
      Silverside, Menidia species                        22
      Threespine stickleback, Casterosteus  aculeatus     22
      Pinfish, Lagodon rhomboides                        22
      Spot, Leiostomus xanthurus                         12
      Shiner perch, Cymatogaster aggregata                12
      Pacific staghorn sculpin, Leptocottus armatus      12
      Sanddab, Citharichthys  stigmaeus                   12
      Flounder, Paralichthys  dentatus, £. lethostigma    22
      English sole, Parophrys vetulus                    12

    Invertebrates
      Shrimp, Panaeus setiferus, P_. duorarun P_. aztecus  22
      Grass shrimp, Palaemonetes species                 22
      Shrimp, Crangon species                            22
      Oceanic shrimp,  Pandalus jordani                   12
      Blue crab, Callinectes  sapidus                     22
	Dungeness crab,  Cancer  magister                    12

                                      247

-------
     •  Source and Size of Test Organisms

     Bioassay organisms are obtained from one of two sources:  natural
sources such as lakes or streams, or from commercial suppliers.  Organisms
obtained from natural sources are generally preferred because they
represent the condition of naturally occurring organisms, especially if the
organisms are from the water body under study.

     However, because their previous exposure to various chemicals is not
readily known, performance of bioassay analyses on these organisms
may, on occasion, lead to erroneous results.  Another disadvantage is that
because these organisms must be captured, the source of supply is not always
assured.  Specimens obtained from commercial suppliers have the advantage
that they are usually from sources where the history of exposure is known.
A disadvantage with the supply house organisms is that they often come
from sources quite different from the water being assayed; even the same
species from different sources may have quite different susceptibility to
test materials.  Additionally, some of these organisms have been inbred,
resulting in various strains that are ideal for test accuracy and
reproducibility (Lenon, 1967), but data obtained may be difficult to apply to
natural populations of the same species.

     Organisms captured by electroshocking should not be used.  All organisms
in a test should be from the same source and be as healthy and uniform in
size and age as possible.  Whenever trout are to be used, certified disease-
free fish (free of infectious pancreatic necrosis, furunculosis, kidney
disease, and whirling disease) should be obtained.  Freshwater amphipods,
daphnids, and midge larvae should-.be reared, in.the. testing facility from
laboratory cultures.  Daphnids from cuJLtures^ in which ephippla are being
produced should not be used (U.S.  EPA,  1975a).

     The size of the test organism is a major consideration.  The organism
should not be so small that it is difficult to observe and contain in the
test cell, especially if the tests incorporate large continuous flow
apparatus with a continuous discharge of test water.  This notion is
changing with increased emphasis on diatoms, protozoans, and small
invertebrates as bioassay organisms.  Test species, on the other hand,
should not be so large as to limit their activity, body functions, and
handling advantages in the test units (Rand et al., 1975; Sprague, 1971).

          o  Fish
     Very young (not yet actively feeding), spawning or recently spent fish
should not be used.   The use of fish that weigh between 0.5 and 5.0 g each
is usually desirable.   Embryos and newly-hatched fish are sometimes more
sensitive than older ones and can be tested if appropriate precautions are
taken.  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
(U.S. EPA, 1975a).
                                     248

-------
           o  Invertebrates

     Immature organisms should be used whenever possible.  Daphnids should be
in the first instar; amphipods, stoneflies and mayflies in an early instar;
and midges in the second and third instar (U.S. EPA, I975a).

           o  Amphibians

     Young larvae should be used whenever possible (U.S. EPA, 1975a).

           o  Shrimps

     Larval stages should be used.

           o  Mollusks

     For mussel and oyster, adults or Juveniles should be used.

           o  Lobster

     Adults or juveniles should be used (Rand et al., 1975).

     • Care and Handling

     It is of utmost importance for bioassay studies that the test animals
be kept in excellent condition before the test.  Never allow abrupt changes
in environmental conditions.  In general, aquatic organisms should not be
subjected to more than a 3°C change in water temperature in any 12-hour
period.  During transport to the laboratory, do not crowd the organisms,
supply plenty of oxygen and maintain a favorable temperature (U.S. EPA,
1975a).

     The dissolved oxygen concentration must be maintained between 60% to
100% of the saturation concentration; gentle aeration may be used if
necessary (U.S. EPA, 1975a).  Provide adequate flow-through water so
that the dissolved oxygen, pH, carbon dioxide, salinity, hardness, and other
characteristics are favorable.  Generally use a flow-through rate
equivalent from 6 to 16 tank volumes per day (Rand et al., 1975).

     Test organisms should be fed at least once a day and the tank scrubbed
at least twice a week.  Remove within 24 hours all uneaten food that collects
on the bottom or in corners.  Recommended diets and feeding schedules are
given in Table 3.3.27  (Lenon, 1967).

     Shield the tank with curtains or some other means  to protect the
organisms from nearby movements and noise.  Provide photoperlods and light
intensities favorable  to the organisms.  In long-term studies for those
species that require annual light cycle photoperiods, simulate the natural
seasonal daylight and  darkness period with appropriate  twilight periods.
Make adjustments in photoperiod on the first and  fifteenth  of every test
month.
                                     249

-------
      TABLE  3.3.27 DIETS AND  FEEDING  SCHEDULE  (IN DAYS PER WEEK*) AT THE
      FISH CONTROL LABORATORY FOR VARIOUS BIOASSAY  SPECIES  (Lenon,  1967)




Species






Rainbow trout (Salmo gairdneri)
Brown trout (Salmo trutta)
Brook trout (Salvelinus fontinalis)
Lake trout (Salvelinus namaycush)
Northern pike (Esox lucius)
Goldfish (Carassius auratus)
Carp (Cyprinus carpio)
Fathead minnow (Pimephales promelas)
White sucker (Catostomus commersoni)
Black bullhead (Ictalurus melas)
Channel catfish (Ictalurus punctatus)
Green sunf ish (Lepomis cyanellus)
Bluegill (Lepomis macrochirus)
Smallmouth bass (Micropterus dolomieui)
Largemouth bass (Micropterus salmoides)
Yellow perch (Perca flavescens)
Walleye (Stizostedion vitreum)
CO

41
iH rH
Cfl i-l
•H 4)
O P.
)H
01 4J

B O
O M
O 4J
7
7
7
7

7
7
7
7
7
5
7






4-1 CO
CO 1-1
•H CO fl
B 4) Q
•H d
B i-4 P
O Q) h
60 P. 41 4)
41 >>
533
1 2
2 5
5
2 5
2 7
2
2
5
1 5
1 5
1 5
5
5 2
5 7
252
252
257






Sfi-
41 S; u
N 0 -H 4)
O -H I-l .C
M h X 4J
Pt« ua co o




2 2(a)



2(b,c)
2(b,c)
2(b)

7
2 2 (a)
7 2 (a)
2
2 2 (a)
    *Large size fish are ndt fed on weekends.
(a)  minnows  (Pimephales promelas).
(b)  soybean meal.
(c)  torula yeast.

For details see Table 3.3.28 Test Photoperiod for Brook Trout, Partial Life
Cycle (Rand et al., 1975).  In short-term tests, standard photoperiod of 14
hour light, 10 hour dark is suggested, but often the usual laboratory light-
ing is adequate.

    Hold field collected animals in quarantine for at least seven days to
observe them for disease, stress, physical damage or mortality.  If more
than 10% of the collected animals die after the second day or they are
heavily parasitized or diseased and the problem cannot be controlled, destroy
the lot and clean and sterilize all containers and equipment used.  At the
end of the quarantine period, transfer the test organisms that appear to be
disease-free to the regular laboratory stock tanks.  The handling should be
done as gently, carefully, and quickly as possible.  Organisms that touch a
dry surface or are dropped or injured during handling must be discarded.
Small dipnets are best for handling small fish.  Smooth glass tubes with
rubber bulbs should be used for transferring smaller organisms such as
                                      250

-------
    TABLE  3.3.28 TEST  (EVANSVILLE, INDIANA) PHOTOPERIOD FOR BROOK TROUT,
                       PARTIAL LIFE CYCLE  (Rand et al, 1975)
Dawn to Dusk Time
6:00-6:1?
6:00-7:00
6:00-7:30
6:00-8:15
6:00-8:45
6:00-9.15
6:00-9:30
6:00-9:45
6:00-9:45
6:00-9:50
6:00-9:00
6 .00-8: JO
6:00-8:00
6:00-7:30
6:00-6:45
6X10-6:15
6:00-5:10
6:00-5:00
6:00-4.45
6:00-4:50
6:00-4:30
6:00-4:45
6:00-5:15
6:00-5:45
Date
Mar. I
15
Apr. 1
15
May 1
15
June 1
15
July 1
15
Aug.- 1
15
Sept. I
15
Oct. 1
15
Nov. 1
15
Dec. I
15
Jan. 1
15
Fd>r 1
15
Day Length (hr & mm)
12:15
13.00
13:30
14:15
14.45
15:15
15:.U>
15:45
15:45
15:30
15:00
14:30
14.00
13:30






Juvenile-adult exposure






12:45 )
..' . ? Spawning and egg incubation
11:00 )
10.4?
10:30
10:30
10:45
11:15
11:45


Alcvin-juvenile exposure


Daphnlds and midge larvae.   Equipment used to handle aquatic organisms
should be sterilized between uses with an lodophor, 200 mg of Hypochlorite/
liter or 30% Formalin plus 1% Benzalkonium chloride (U.S. EPA, 1975a).

    Generally organisms should not be treated for disease during the first
16 hours after they arrive at the facility because they are probably stressed
due to collection or transportation and some may have been treated during
transit.  However, immediate treatment is necessary in some situations, such
as treatment of bluegills for columnaris during hot weather (U.S. EPA 1975a).
To reduce mortality and to avoid introduction of disease into stock tanks,
treat with a wide-spectrum antibiotic immediately after collection or during
transport.  Holding in tetracycline (15mg/l) for 24 to 48 hours can be very
helpful (Rand et al., 1975).

    Table 3.3.29 gives recommended prophylactic and therapeutic treatments
for freshwater fish (U.S. EPA, 1975a).
                                     251

-------
     TABLE 3.3.29 RECOMMENDED PROPHYLACTIC AND THERAPEUTIC TREATMENTS FOR
                                      ,a
                       FRESHWATER FISH  (U.S. EPA, 1975a)
Disease
External
bacteria
Chemical
Benzalkonium chloride
(Hyamine 1622 )
Concentration
(mR/1)
1-2 AIb
Application
30-60 minc
Monogenetic
trematodes,
fungi, and
external  ,
protozoans
Parasitic
copepods
Nitrofurazone (water mix)

Neomycin sulfate

Oxytetracycline hydrochloride
(water soluble)

Formalin plus zinc-free
malachite green oxalate

Formalin

Potassium permanganate

Sodium chloride


DexonR (35% AI)

Trichlorfon
(Masoten )
   3-5 AI

    25

    25 AI


    25
    0.1

  150-250

    2-6

15000-30000
 2000-4000

    20

    0.25 AI
                                                                30-60 min
                                                                30-60 minc
                                                                30-60 minc
   1-2 hours
  30-60 min
  30-60 minc
5-10 min dipe'C
  30-60 min
    f
a    These recommendations do not imply that these treatments have been
cleared or registered for these uses.  These treatments should be used only
on fish intended for research, and researchers are cautioned to test treat-
ments on small lots of fish before making large-scale applications.  Before
a treatment is used, additional information should be obtained from sources
such as:  Davis (1954), Hoffman and Meyer (1974), Reichenbach-Rlinke and
Elkan (1972), Snieszko (1970) and Van Duijn (1973).

b    Active ingredient.

c    Treatment may be accomplished by:

          o Transferring the fish to a static treatment tank and back to
            holding tank
          o Temporarily stopping the flow in a flow-through system, treat-
            ing the fish in a static manner and  then resuming the flow to
            flush out the chemical
          o 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 (1967).
                                      252

-------
d    One  treatment is usually sufficient except for "Ich", which must be
treated daily or every other day until no sign of the protozoans remains.
This may  take 4 to 5 weeks at 5 to 10°C and 11 to 13 days at 15 to 21°C.
A  temperature of 32°C is lethal to "Ich" in one week.

e    Minimum of 24 hours but may be continued indefinitely.

f    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.
       t
     Freshwater invertebrates and amphibians must have been in holding tanks
for at least 10 days and fish for at least 14 days before they are used for
basic tests;  all test organisms must have been in holding tanks for at least
four days before they are used for effluent tests.   They should be held
under stable condition of temperature and water .quality in uncontaminated,
constant-quality water in a flow-through system with a flow rate at least two
water volumes per day.  Water from a well or spring should be used for fresh-
water organisms whenever possible.  Only as a last resort should a dechlori-
natedwater be used.  The cold-water freshwater organisms are best held between
5°C to 15°C, usually well below 15°C.  Hold warm-water organisms at temperature
between 10°C to 25°C depending on the season.  Hold aquatic invertebrates
within the temperature range of the water from which they were obtained unless
they are  being acclimated for special temperatures or other tests.  If possible,
follow the natural variations in temperature.  During long holding periods,
hold most test organisms in the lower range of favorable temperature rather
than at higher temperature because the metabolic rate and the number and
severity  of disease outbreaks are reduced in the cooler water.

     The  acclimation of the test organisms to the test condition begins
from one  to two weeks before they are to be used in bioassays.  There should
be few or no deaths due to parasites or diseases during this period.  Use
only those groups of organisms that are free from parasitic infection and
diseases  and in which the mortality is less than 10% during the laboratory
holding period.  Never allow abrupt changes in environmental conditions;
often it  is helpful to follow the natural seasonal variations in environmental
conditions such as temperature and the seasonal daylight patterns.  There
should be no supersaturation of gases.  If the organisms in the holding tank
are not exposed to the same conditions as those to be used in the bioassays,
gradually acclimate them to temperature and other conditions to which they
will be exposed in the actual bioassays.   Freshwater amphipods, daphnids, and
midge larvae should be acclimated to water quality and temperature by rearing
them in dilution water at the test temperature.   Other organisms can be
acclimated (in a flow-through system with a'flow rate of at least two water
volumes per day for flow-through tests) simultaneously to the dilution
water and test temperature by transferring the appropriate number of similar-
length individuals from a holding tank to an acclimation tank.  They should
be acclimated to the dilution water by gradually changing the water in the
acclimation tank from 100% holding water to 100% dilution water over a period
of 2 or more days for basic tests and for at least 24 hours for effluent
                                     253

-------
tests before they are used for  test.  For basic test, water  that may be con-
taminated by undesirable microorganisms should be passed  through an ultra-
violet sterilizer and the un-ionized ammonia concentration in the acclimation
tanks should be less than 20 yg/1.  They should be acclimated to the test
temperature by changing the water temperature at a rate not  to exceed  3°C
within 72 hours for basic test  and not to exceed 3°C within  24 hours for
effluent tests until the allowable test temperature range is reached.  They
must be maintained for at least 48 hours for basic tests  and 24 hours  for
effluent tests at the allowable test temperature range before tests are
begun with them.  Longer acclimation times are generally  desirable.

     A group of organisms must  not be used for a test if  the individuals
appear to be diseased or otherwise stressed or if more than  3% for basic
tests or 5% for effluent tests  die during the 48 hours immediately prior to
the beginning of the test.  If  a group fails to meet these criteria, all
individuals must be either discarded or treated, held an  additional 10 days
for basic tests or 4 days for effluent tests, and reacclimated if necessary.

     Young amphibian larvae and fish that have been actively feeding for less
than about 20 days, amphipods,  daphnids, and midge larvae must be fed, and
all other Insects may be fed, up to the beginning of the  test.  For basic
tests all other amphibian larvae and fish over 0.5 g each must not be  fed
for 96 hours and all other invertebrates over 0.5 g each  must not be fed for
*>8'hours before the beginning of the test. . For effluefnt-test, all other  •
amphibian larvae, fish, and invertebrate over 0.5 g each  must not be fed for
48 hours before the beginning of the test (U.S. EPA, 1975a).

3.3.1.5  Design of Experiment—

     The precision of a test procedure depends on the following factors
(Rand et al., 1975):

     • The variability of the organisms in their response
     • The number of organisms  exposed to each test concentration
     • The number of replicates being made
     • The test substance to which the organisms are exposed
     • How close the mid-concentration tested happened to be to the
       LC50 and how closely the concentrations of the test substance
       solutions cluster round  the LC50 concentration

     For a given test under similar conditions, increasing the number  of test
organisms increases the precision.  The use of more organisms and replicate
test containers for each test substance concentration is  often desirable to
reduce variability  (U.S. EPA, 1975a).

     The number of organisms to be exposed in each test concentration  is
governed by a number of considerations:
                                      254

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          o the size of the organisms
          o the expected apparent normal mortality
          o the extent of cannibalism
          o the availability of dilution water, toxicant, and test organisms
          o the desired precision of the estimate of the toxlcity of the
            test material.

     Replicates must be true replicates with no water connection between the
test containers.  If replicates are used, random assignment of one test
container for each test concentration in a row followed by random assignment
of a second test container in a second row or an extension of the same row
is recommended rather than total randomization (U.S. EPA, 1975a).

     A representative sample of the test organisms should be impartially
distributed to the test chambers, either by adding one (if there are to be
less than 11 organisms per container) or two ( if there are to be more than
11 organisms per container) test organisms to each chamber, and then adding
one or two more, and repeating the process until each test chamber has the
desired number of test organisms in it.  Alternatively, the organisms can be
assigned either by random assignment of one organism to each test chamber,
random assignment of a second organism to each test chamber, etc., or by
total randomization.  It is often convenient to assign organisms to other
containers and then add them to the test chambers all at once.
                       • *                '                  * •
     Every test requires a control in which the same dilution watei; conditions,
procedures, and organisms are used as in the remainder of the test.  If any
additive is present in any of the test chambers, an additive control is also
required.   This additive control is treated the same as the regular control
except that the highest amount of additive present in any other test chamber
is added to this test chamber.  A test is not acceptable if more than 10%
of the organisms die in any control in a test determining an LC50 or show
the effect in a test determining an EC50.  It is desirable to repeat the
test at a later time to obtain information on the reproducibility of the re-
sults of the test (U.S. EPA, 1975a).

3.3.1.6  Test Methods—

     Toxicity tests with aquatic organisms should be conducted according to
uniform, detailed methods whenever possible to maximize the number of reliable
comparisons that can be made concerning relative toxicity and relative
sensitivity.  Tests shall include control groups to determine if any observed
effects have developed or occurred independent of the test substances.  The
control group shall be maintained in the same manner as the test group (Anon.,
1977).  One or more control treatments should be used to provide a measure
of the acceptability of the test by giving some indication of the health-
iness of the test organisms and the suitability of the dilution water, test
conditions, and handling procedures.  Widespread adoption of uniform methods
will promote the accumulation of comparable data and increase its effective
use (U.S. EPA, 1975a).
                                     255

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     Whenever toxicity tests are conducted with aquatic organisms, the methods
recommended by U.S. EPA  (1975a) should be followed as closely as possible.
Use of these methods for special purposes may require modifications or
specification of additional details, such as choosing one particular species.
Since not all details are covered In these methods, the successful execution
of these methods will require some training or experience In aquatic toxicol-
ogy or aquatic biology or both.  It Is essential to conduct tests so that
they meet specific needs but these methods should cover most situations
(U.S. EPA, 1975a).
    Some novel bloassay  procedures  that  have  been suggested are outlined
below:
    Roberts  (1975)
    Walker et al.,
        (1975)
    Boree  (1975)
    Jensen  (1976)
    Baudouln and
    Scoppa  (1975)
     Canton  et  al,
        (1975)
Byssus formation In mussels was sensitive to
pesticides and PCP's.  Byssogenesis test was
proposed as a rapid and convenient technique
for routine screening of potential marine
pollutants.

Barnacles were suggested as possible indicators
of Zn pollution based on the studies of Zn
accumulation in Balanus, Elimlnus, and Lepas.

A photomlcrographic method was proposed to determine
the degree of response of the protozoan Tetrahymena
pyrlformls to metal levels which was similar to that
of the bluegill sunflsh.

A procedure based on the hatching rate of eggs of the
brine shrimp Artemla Salina revealed the convenience
of a bioassay organism that could be stored dry
in the laboratory.  The method offers an easy way
to get Information of the toxicity of a particular
matter.  The experimental results of the hatching
tests show a characteristic graph typical of many
toxicity tests.

Nucleic acids were used as indicators of blomass in
mixed planktonic populations.  DNA and RNA showed
large variations among different planktonic species,
between zooplankton and phytoplankton and among
seasons.

A tentative method for deriving an EC50 (ecological
limit) was proposed.  The criteria included mortality,
Immobilization, growth, reproduction, hIstopathologic
changes, and enzyme activities.  The procedure was
based on short-and long-term toxicity studies with
d-thexachlorocyclohexane and a variety of organisms
including algae, Crustacea,  and fish.
                                      256

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     The following are specific laboratory procedures that are used to insure
maintenance of sample integrity and treatment.

     •  Receptacles, pipettes, and other instruments used for handling
        specimens must be kept separate from those used for chemicals.

     •  Specimens generated from field investigations are designated by
        the field data sheet number; specimens generated from laboratory
        investigations are given a data sheet number) these numbers are
        then entered in a log book

     •  Fixation (within 24 hours)

     Davidson's fixative has been recommended as follows:

            Formalin                 20 parts
            Glycerin                 10 parts
            Ethyl alcohol 95Z        30 parts
            Glacial acetic acid      10 parts
            Distilled water          30 parts

     Since the nature of the fixing agent has considerable effect upon the
affinity of the structures for various stains, special staining procedures
require the use of different fixatives.  When fixatives other than Davidson's
are used (i.e., Formalin) the specimens are washed in running water or alcohol
to remove the fixative before proceeding with dehydration.

     •  Preservation

     To prevent disintegration or alteration of important constituents
     of fixed tissue, specimens are kept in a solution of one part
     glycerol to nine parts of 70% alcohol.  Since the staining qualities
     of tissues begin to deteriorate after the tissues have remained In
     alcohol for weeks or months, specimens which are eventually to be
     stained and mounted are transferred to glycerol for storage.

     •  Decalciflcation

     Specimens which contain deposits of calcium salts which are too hard
     to be cut with a microtome knife are decalcified with a 3% solution
     of hydrochloric acid in 70% alcohol.  This causes no serious damage
     to tissues.  The tissues are then placed in neutral 70% alcohol.

     •  Dehydration

     To prevent violent diffusion currents which would cause the collapse
     of cavities or the distortion of specimens, a graded series of alcohol
     concentrations is used.
                                      257

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    • Embedding

      The dehydration and embedding steps are carried out using an
      Autotechnicon.  The procedure is as follows:"

            702 alcohol                  1 hour
            952 alcohol                  1 hour
           1002 alcohol                  1 hour
           1002 alcohol                  1 hour
           1:1 benzene:  1002 alcohol    1 hour
           1002 benzene                  1 hour
           1002 benzene                  1 hour
           paraffin                      2 hours
           paraffin                      2 hours

Vacuum infiltration - 20 minutes at 15 psi, or 15 to 20 minutes at 12 psi for
tissues that are relatively delicate.  Each specimen is placed in its own
labelled tissue capsule before being placed in the Autotechnicon.  Once a
day the beakers containing the 1002 alcohol and the 952 alcohol are changed.
Every three to five days the beaker containing the 1:1 benzene alcohol is
changed.  If the instrument sits for a few days, all the solutions are
changed except the paraffins.

    The paraffin in the vacuum infiltrator is changed once a week if used
frequently.  After complete paraffin infiltration, the specimens are placed
in a labelled embedding mold and made into paraffin blocks.  If the
paraffin blocks are not sectioned immediately, they are labelled and stored
in a specimen cabinet.

    Once slides are finished they are stored in labelled slide boxes.

      Staining (trichome type staining)
    1.  1002 xylene - 3 mln (50 to 56°C) (3 changes)
    2.  1002 ethanol - 3 mln (room temp.) (2 changes)
    3.  952 ethanol - 3 min (room temp.)
    4.  502 ethanol - 3 min (room temp.)
    5.  102 ethanol - 3 min (room temp.)
    6.  distilled water - 3 min (room temp.) (2 changes)
    7.  42 ferric ammonium sulfate - 15 min (50 to 56°C)
    8.  tap water - quick rinse to remove any excess
    9.  hematoxylin stain - 15 min (50 to 56°C)
  *10.  distilled water - couple of rinses
   11.  destalning - 42 ferric ammonium sulfate, room temp. - about 1.5 mln
   12.  tap water - 3 to 4 min
   13.  basic ethanol - 30 sec to 1 mln
  *14.  water bath
   15.  acid fuchsin - 4 quick dips
   16.  distilled water - 4 quick dips
   17.  drain on paper towel to remove excess liquid
   18.  12 phosphomolybdic acid - 5 min (room temp.)
                                      258

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    19.  drain for 30 sec
    20.  aniline blue stain - 1.5 min
    21.  drain - 1 to 2 min
    22.  destaining - 1Z acetic acid - 1 min (4 changes)
    23.  dehydration - 1% acetic acid in acetone - 1 min (3 changes)
    24.  100Z xylene - 1 to 2 min
    25.  100% xylene - (keep out of sunlight) - can keep here 24 to 48 hours
    26.  mount - use #1 coverslip (try to flatten out the cover slip as much
         as possible)

     *   The slides can be held at these steps for at least 24 hours.

3.3.1.8  Data Handling—

      Data Collection

    For maintaining a quality bioassay capability, all information about
conduct of the experiment collected should be recorded on either a Bioassay
Biota Log Sheet (Figure 3.3.3) or a Bioassay Water Quality Log Sheet
(Figure 3.3.4) (U.S.EPA, 1975b).  A typical schedule of checks and main-
tenance during studies carried out in tanks could look as follows (U.S. EPA
1975b).

         o  Daily:  check all tanks for signs of disease, abnormal
            organisms behavior and dead organisms.

         o  Hon., Wed., Fri., or every other day:  feed organisms
            and remove unconsumed food within one hour.

         o  Filter cleaning:  high volume pump - once every three weeks
            Dyno flow - once a week.  Filter may need change sooner if
            tank appears cloudy or going bad.

         o  Water exchange:   Monthly (drain half of the water, then add
            distilled water and chemicals).

    Similarly, a typical schedule of checks to be performed with holding tanks
and experimental units could look as follows (U.S. EPA, 1975b):

         o  Holding tanks:  Determine daily air temperature, water
            temperature, and dissolved oxygen.  Determine monthly pH,
            alkalinity, hardness, calcium, conductivity, and salinity.

         o  Experimental units:  Determine every 24 hr dissolved oxygen,
            pH, air temperature, water temperature, and conductivity.
            Determine at end of test hardness, calcium, alkalinity and
            salinity.

Note:  all water samples should be taken at mid-depth.
                                      259

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        WATER TYPE:  SALT   F-WATER
              (CIRCLE ONE)
        TANK TYPE:  HOLDING EXPERIMENTAL
                    (CIRCLE ONE)
DATE


TIME


TANK
SECTION


BIOTA
SPECIES


CHANGE
IN NO


BAL


REASON
FOR
CHANGE


NOTES
BEHAVIOR,
APPEARANCE
CONDITION


MAINTENANCE
FEEDING
TYPE


FILTER
CHANGE


WATER CHANGE
TYPE


AMOUNT


REASON


CSJ

o
                               Figure 3.3.3 Bloassay biota log (U.S. EPA,  1975b)

-------
NJ
DATE

-
TIME


TANK *
SECTION


°C TEMP
AIR


W


nil
pH


rag/ 1 AS CaCOt
TOTAL
ACIDITY


A1.K


HARD


Ca


f\
~o2
og/l


COND
pmho
cm


SAL
Zo


REMARKS


                           Figure 3.3.4 Bloatsay water quality log (U.S. EPA, 1975b)

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SERIES:	
TECHNICIAN:
COMPANY:
	DATE:_
 STARTING  HOUR:
MATERIAL BEING TESTED:_
SOURCE:
SOURCE OF DILUTION WATER:_
TEST SPECIES:
                       TEMP.  RANGE:
NO. INDIVIDUALS PER CONCENTRATION^
                START
DILUTION:
DO
PH
HARDNESS
OTHER

























CONTROL




             24 HOURS
I NO SURVIVING
' % SURVIVAL
DO
, PH
OTHER






























             48 HOURS
1 NO SURVIVING
! % SURVIVAL
! DO
! pH
j OTHER






























              72 HOURS
, NO SURVIVING
i Z SURVIVAL
! DO
: pH
OTHER






























              96 HOURS
:NO SURVIVING
IZ SURVIVAL
'DO
|pH
IOTHER























•






            Figure 3.3.5  Bioassay record sheet  (U.S. EPA, 1975b)
                                    262

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    Itesults of the experiment should be recorded on a form similar to
Figure 3.3.5.  In addition to toxicity data,  the following information
should be recorded:   . ,    «          •

         o  name of method, investigator, and laboratory,
            and date test was conducted
         o  detailed description of the toxicant or
            effluent
         o  source of dilution water
         o  detailed information about the test
            organism
         o  a description of the experimental design and
            test chambers - the depth and volume of solution
            in the test chambers, flow rate,  etc.
         o  definition of the criterion used  to determine
            the effects and a summary of general observations
            on other effects or symptoms
         o  percentage of organisms that died or showed
            the effect in the control treatment
         o  the average and range of the acclimation
            temperature; test temperature
         o  methods used for and the results  of, all chemical
            analyses of water quality and toxicant concentra-
            tions
         o  anything unusual about the test;  any deviation
            from these methods and any other  relevant
            information (U.S. EPA, 1975a).

     Photography may be used to document organisms response, test set-up
and physical appearance of waste concentrations  (U.S. EPA, 1975b).

     •  Biological Response

     The most common toxicity test response with aquatic animals is the
mortality which is counted to obtain information about a median lethal
concentration (LC50).  The data produced by the test generally consist
of the percentages or organisms that are killed by different concen-
trations of a toxicant after specified lengths of exposure.  A statistical
estimation method is then used to obtain the  best estimate of the LC50
from the concentration mortality data for each length of exposure
(Stephan, 1976).

     The precision of a toxicity test is limited to a number of
factors including the normal biological variation among individuals of a
species.  Toxicity studies with a randomly selected species cannot be
expected to give accurate information on the  toxicity of that material to' other
species and life stages or to an entire biota.  A toxicity test with one
species yields an accurate estimate of the toxicity only to others of that
species of similar size, age and physiological condition and in water with
the same characteristics and under similar test  condtions  (Rand ct al. 1975).


                                    263

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     In order to obtain information about the precision of the acute
mortality test, replicate test must be conducted at different
times in one laboratory and/or in different laboratories (Stephen,
1976).

     •  Statistical Estimation Method

     The statistical estimation method should meet the following
criteria:

     o  The method should be a strictly computational
        method
     o  The method should be just as useful whether or
        not the toxicant concentrations are in a
        geometric series and whether or not the complete
        range from 0% to 100% kill is covered
     o  The method should not require exposing the same
        number of organisms to each toxicant concentra-
        tion
     o  The use of adjusted or assumed data should
        not be required for any set  of data

     Based on statistical considerations, the log - probit method
has been highly recommended by Sprague (1969).  It has the advantage
of:

     o  Complete toxicity curves for easy interpolation
        of results
     o  An incipient LC50 instead of one for an arbitrary
        time
     o  A mathematical instead of a subject estimate of
        incipient LC50

     It allows the toxicity of different pollutants to be compared easily
and meaningfully.   Analysis of results by the rapid graphic methods of
Litchfield-Wilcoxon (1949)  is recommended (these improvements are also
suggested by Rand et al.  1965).   To carry out the Litchfield-Wilcoxon
procedure,  actual percentage mortality in each test tank at the selected
time beyond the lethal threshold is plotted on log-probit paper (Figure
3.3.6).   A line is fitted to the points by eye.  Its goodness of fit is
estimated by a rapid chi-square  value.  The incipient LC50 is then read
from the graph.   If desired, the more formal but more time-consuming
mathematical procedures of Finney (1964)  may be used to estimate the
incipient LC50 (Sprague,  1969).
                                  264

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 V


 S
 CO
     i
•§



i
 g

CO
20
   50
 flj

•0

H 80
« 95
G
0)


a! 99
0k
 99.8
                  2           A     6     8   9 10
            Concentration of Fluoride,p.p.m F
                                                        20
40
Figure 3.3.6.  Estimating the median lethal concentration.   In  this case the

incipient LC50 is estimated since the exposure time was  long.   Percentage

response of trout is plotted on the vertical probit scale.   The median lethal

concentration is 8.5 mg/1 and its confidence limits could be estimated as

described in the text.  The 5% response is also shown.   From Herbert and

Shurben (1964).
                                   265

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     The moving average method can be used  to  calculate  the best estimate of
 the  LC50  and  its  95% confidence limits  for all  acute mortality tests, except
 that confidence limits cannot be obtained  if  there  are  no partial kills.

     A modification of the moving average method is  the  moving average angle
 method suggested  by Bennett (1952).   The purpose of using an angle  trans-
 formation with binomial data is to  improve linearity and to stabilize the
 variance, thus allowing equal weight to be given to each transformed
 observation.

     Whenever  any  method is used to  analyze concentration-mortality  data, the
logarithmic transformation should  probably be  used on  the concentration data.
 If the log  transformation is not used,  the formula  LC50 = (A+B)/2 will give
 the  same  result as the moving average method  (A = the highest toxicant
 concentration in  which none of the  test organisms died  and B is the lowest
 concentration in  which all of the organisms died).  The following is the
 recommended scheme for analyzing concentration  mortality data from  acute
 mortality tests with aquatic animals:

          o  With  one or more partial kills, use a moving average
            method and log concentration,
          o  With  no partial kills,  use  either a moving  average
            method or the formula 1/2(A+B)  to obtain an estimate
            of the LC50,  and use A  and  B in place of 95% confidence
            limits if at least five organisms were  exposed to each
            treatment.

     Regardless of what method is used to obtain an  LC50 and confidence limits,
 the  results should always be compared with the  original concentration-
 mortality data to determine if they are reasonable  (Stephan, 1976).

     • Control Mortality

     Control mortality should be virtually  absent.   It should not be greater
 than 10%  and  preferably not more than 5%,  representing  an occasional weak
 organism  in a group.   Make corrections  for higher mortality in controls
 by Abbott's formula (Rand et al., 1975).   According to  Stephan (1976)
 the  use of  Abbott's formula for some sublethal  acute toxicity tests may be
 appropriate if a  percentage of the  test organisms consistently shows the
 effect in the absence of the toxicant.

 3.3.2 Experimental Procedures in Aquatic  Bioassay

     Aquatic bioassay procedures may  be  categorized  as:

     • acute  or chronic bioassay, depending on  whether  effects are  observed
 in the short  or the long term;

     • static or  flow-through bioassay, depending on whether the water in
 the  tank  is still or continuously flowing;
                                      266

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     •  basic or effluent, depending on whether the toxicant is added to
 the  water or whether discharge water already containing the toxicant is
 used.

     These categories are not mutually exclusive.  The static procedure is most
 often used for acute bioassay because of its advantages in short term
 applications.  Similarly, the flow-through procedure is most often used for
 chronic studies because of the advantages it has for long-term tests.  Also,
 by the  nature of  the water  supply,  testing of effluents  lends itself  best  to
the flow-through  procedure.

     Whether an acute or chronic bioassay is used depends on the objectives of
 the  experimenter and on the stage of experimentation.  The acute test may by
 itself satisfy the aim of the experimenter or it may be used as a precursor
 of a chronic test.  There may be a series of intermediate stages such as
 repeated dose and sub-chronic tests, each adding more information and
 building up to the long-term, usually very expensive, chronic bioassay.
 Whether a static or flow-through procedure is used is a matter of choice on
 the  part of the experimenter who will use the kind of experimental set-up
 most suitable to his purpose.  Also, whether basic treatment of the water
 or effluent water is used depends upon the nature of the situation being
 examined.

     In naming an aquatic bioassay protocol,  the essential descriptors are
 "acute" or "chronic".  These terms may or may not be accompanied in the
 name of the test by "static" or "flow-through15 because the instructions for
 performance of the test make the conditions of the test explicit.  The terms
 "basic" or "effluent"  do not often appear in the name but the condition
 which applies is apparent from the context of the test.

     •  Static Bioassay

     In addition to its short-term characteristics, the static bioassay
 procedure offers the following advantages:

         o  it allows for testing of different toxicants in parallel
         o  it allows for testing of several species at the same time
         o  homogeneous water is used
         o  fewer numbers of animals are required
         o  lower cost
         o  more easily reproduced (replicated)
         o  requires minimum space, equipment and maintenance

     The disadvantages of the procedure are:

         o  production of Irregular concentrations if test
            material is volatile
         o  usually gives a lower LC50 reading than flow-through
            bioassay (Martin, 1973)
         o  dissolved oxygen, metabolic products and food wastes
            may create problems.
                                     267

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    •  Flow-through bioassay

    The flow-through bioassay procedure has the following advantages:

       o  It Is useful when the test material is volatile, easily
          precipitated, or when the expression of its effects is
          long coining
       o  can be used for life-time tests
       o  more readily represents natural systems
       o  good for determining response of lethality

    A disadvantage is that it is more complicated and requires close attention
over long periods of time.

3.3.2.1  General Factors in Aquatic Bioassay—

    •  Experimental design

       Usually the design consists of:

       o  One  control and  5 or  6  concentrations  of  toxicant
       o  At least 10, but preferably 20 organisms exposed in
          each treatment and the control groups.  The use of
          more organisms and replicate test chambers for each
          treatment is desirable, but "loading" must be avoided
       o  True replicates with no water connection
       o  Tanks and the test organisms assigned either by
          stratified randomization or total randomization
       o  Randomization of the treatment
       o  A control consisting of the same dilution water,
          conditions, procedures, and organisms as are used in
          the remainder of the test (U.S. EPA, 1975a).

    •  Dissolved oxygen concentration

    Test solutions must not be aerated in the test chambers or in the
toxicant delivery system.  For static tests, the dissolved oxygen
concentration in each test chamber must be between 60% to 100% saturation
during the first 48 hours of the test and must be between 40% and 100%
saturation after 48 hours.  For flow-through tests, the dissolved oxygen
must be between 60% and 100% saturation at all times (U.S. EPA, 1975a).

    •  Test Temperature

    For basic tests, the test temperature must be selected from the series
7°, 12°, 17°, 22°, and 27°C.  The actual test temperature must not deviate
from the selected test temperature by more than 1°C at any time during the
test.  For aquatic invertebrates, the selected test temperature should be
within 5°C of the temperature of the water from which they were obtained.
                                      268

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     For an effluent test, the selected test temperature should be the
 temperature of the receiving water measured just outside the zone of
 influence of the effluent at noon on the day before acclimation begins,
 because the temperature at noon usually approximates the average
 temperature for the day.  The actual test temperature must not deviate from
 the selected test temperature by more than 2°C at any time during the test
 (U.S. EPA, 1975a).

     The suggested test temperature for vertebrates and invertebrates is as
 follows (U.S. EPA, 1976):

             Region*                      Temperature
             I                               20°C
             II** and III                    25°C
             IV, VI and IX                   30°C
             X                               15°C

     • Salinity

     The salinity of the test water should be that of the discharge site if
 effluent water is used or if artificial sea water is prepared.  The salinity
 of any other natural sea water should be greater than or equal to 15%
 (U.S. EPA, 1976).

     • Loading

     The grams of organisms per liter of solution in the test chambers must
 not be so high that it affects the results of the test.  The loading must be
 limited to insure that the concentration of dissolved oxygen and toxicant is
 not decreased below acceptable levels, that the organisms are not stressed
 due to crowding, and that the .concentration of metabolic products does not
 increase above acceptable levels.  For static tests, lower loadings must
 be used if necessary to maintain the concentration of dissolved oxygen above
 60% saturation for the first 48 hours of the test and above 40% saturation
 after 48 hours.  For flow-through tests, lower loadings should be used to
 maintain the concentration of dissolved oxygen in the dilution water above
 60% at the beginning of the test, to keep unionized ammonia below 20 yg/1,
 and to limit to 20% the lowering of toxicant concentration because of uptake
 by the test organisms.  In order to determine the effects of the test
 organisms on the dissolved oxygen concentration during effluent tests, the
 dissolved oxygen concentration should be measured in duplicate test chambers
that do not contain test organisms (U.S. EPA, 1975a).
     *  Temperature should be revised to the highest average monthly
 temperature of oceanic surface waters in each region.

     ** Puerto Rico and Virgin Islands are in Region II but should use
 temperatures suggested for Region IV.
                                      269

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    •  Feeding

    The test organisms must not be fed while in the test chambers
(U.S. EPA, 1975a).

    •  Range-finding

    Generally, groups of five organisms are exposed to three to five
widely spaced toxicant concentrations and a control for 24 to 96 hours using
either the static or flow-through techniques.

    Range-finding tests may often be difficult to conduct for effluents
because the characteristics of the effluent and the receiving water may
vary significantly within short periods of time.  If a range-finding test
is to be conducted with the same grab sample of the effluent with which a
definitive effluent test is to be conducted, the range-finding test can last
8 hours at the most (U.S. EPA, 1975a).

    •  Definitive test

    A definitive test must meet both of the following criteria so that the
LC50 or EC50 can be calculated with reasonable accuracy:

       o  Except for the controls, the concentration of toxicant
          in each treatment must be at least 60% of the next
          higher one for basic  tests and at least 50% of  the next
          higher one for effluent  tests.

       o  One  treatment other  than the control must have  killed or
          affected more than 65% of the organisms. If an  LC or EC
          near  the extremes of  toxicity is to be calculated, such
          as LC10 or EC90, at  least one treatment must have killed
          or affected a percentage of test organisms, other than 0%
          and  100%, near the percentage for which the LC  or EC is to
          be calculated.  This  requirement might be met in a test to
          determine the LC50 or an EC50, but special tests with
          appropriate toxicant  concentrations will often  be necessary
           (U.S. EPA, 1975a).

     •  Control  Test

    A concurrent control test  should be performed along with each test of any
concentration  of  the substance  assayed or with each series of tests of
different concentrations tested simultaneously  (Doudoroff et al., 1951).

     It should be performed in  exactly the same manner as  the other test, but
using the diluent water alone  as the medium in which the  test organisms
(control) are held.  There should be no more than 10% mortality among the
controls during the course of  a test and at least 90% must remain apparently
in good health.  Otherwise, the results cannot be deemed  reliable.
                                      270

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 3.3.2.2  Static Bioassay—

     The static test utilizes a procedure where the test or dilution water is
 initially dosed with the desired concentration of material and the solution
 is adjusted from time to time to maintain the selected concentration.  While
 static tests are relatively easy to operate and maintain,  they do not always
 afford the best procedure because the concentration of the test material may
 vary considerably.   Variations in material concentrations  may be caused by
 several factors, including precipitation of the test substance, chemical
 interactions in the solution, deposition of test material  on the container
 wall, uptake by the test organism, or interactions of test materials and
 excretion of the organisms. In the static test procedure,  it is rather easy
 .to prevent the concentration of test material from exceeding a maximum in the
 test cell but it is usually rather difficult to maintain the desired
concentration.  Because little equipment is usually required, the static
 bioassay is relatively easy to set up and it can be operated in a minimal
 area.  The static test, in which no effluent is discharged, allows the
 accumulation of waste products which may themselves be toxic.  As a result,
 static tests should be short-term tests.  In static tests, it is advisable
 to utilize duplicates and even triplicates to insure test  precision (Martin,
 1973).

     •  Beginning the test

     Static tests are begun either by:

        o  adding toxicant to the test chambers 18 to 24 hours
           after the test organisms are added
        o  or adding test organisms to the test chambers within
           30 minutes after the toxicant is added to the dilution
           water

     The first alternative allows the test organisms to partially acclimate
 to the test chambers and precludes loss of toxicant due to hydrolysis,
 sorption, or evaporation prior to exposure of the test organisms.  The
 second alternative  conserves dissolved oxygen and prevents the exposure of
 test organisms to the toxicant before it is evenly dispersed; this alternative
 must be used when the tests are conducted on aged solutions of a toxicant in
 dilution water (U.S. EPA, 1975a).

     •  Duration

     Test organisms  must be exposed for 96 hours in basic static test, for 48
 to 96 hours in effluent static tests (U.S. EPA, 1975a).

 3.3.2.3  Flow-through Bioassay—

     Flow-through bioassay is more sophisticated than static bioassay and
 frequently involves a considerably greater amount of equipment.  This
                                      271

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methodology is the one currently being utilized by the majority of bioassay
users, as it more closely approximates natural conditions.  In this concept,
the test organisms are held in a unit or cell into which continuous input of
test solution, premixed in a dilution water, is metered.  The operation
requires maintenance of desired concentrations of test material; determin-
ation of residence time and the solution flow rate to the test cell must also
be known.  To accomplish the maintenance of a homogeneous concentration in
the test unit requires the mixing of dosing solution of known concentration
with a standaridized dilution water (Sprague, 1971).

    In the flow-through bioassay, chemical tests should be run at
intervals during the continuous flow test to assure that the test material
concentrations are maintained in the desired range.  Provisions must be made
to feed and maintain the organisms during the test and excess food must be
removed to limit the development of high bacterial populations.  One of the
major problems with the continuous test is that the dosing apparatus is
sometimes difficult to control.  The continuous flow-through test is
particularly applicable where the wastes being tested are easily decomposed
by bacterial action or when they are volatile or unstable and have a high
biochemical oxygen demand (Martin, 1973).

    •  Flow-rate

    The flow-rate must be at least 5 water volumes per 24 hours.  The flow-
rate through the test containers should not vary by more than 10% from any
one test container to any other or from one time to another within a given
test (U.S. EPA, 1976).

    •  Beginning the test

    Flow-through tests are begun either by:

       o  placing the test organisms in the test chambers after the
          test solutions have been flowing through the test chambers
          long enough so that the toxicant concentrations are constant
       o  or activating the toxicant metering device in the toxicant
          delivery system several days after the test organisms were
          placed in test chambers that had dilution water flowing
          through them

The first alternative allows the investigator to study the behavior of the
toxicant and the toxicant delivery system immediately prior to the beginning
of the test, whereas the second alternative allows the test organisms to
partially acclimate to the test chambers before the beginning of the test
(U.S. EPA, 1975a).
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     •  Duration

     A test begins when the test organisms are first exposed to the toxicant.
In the flow-through bioassay, all organisms must be exposed for at least 96
hours.  When basic flow-through tests are conducted with large organisms
(over 0.5 g each), it is usually desirable to determine the shape of the
toxicity curve; i.e., LC50 or EC50 vs. time, throughout an 8-day exposure
(U.S. EPA, 1975a).

3.3.2.4  Acute Bioassay—

     Acute toxicity tests are generally used to determine the level of toxic
agents that produce an  adverse effect on a specified percentage of the test
organisms in a short period of time.  The most common acute toxicity test is
the acute mortality test.  Experimentally, 50% effect is the most
reproducible measure of the toxicity of a toxic agent to a group of test
organisms  (U.S. EPA, 1975a).

     •   Experimental procedure

     There are two procedures in current use:

        o  Approximate mortality times are recorded for most individual
           animals.  The time taken to obtain 50 percent mortality is
           estimated for each test tank.  The series of median lethal
           times is generally used to estimate an approximate threshold
           concentration for lethal effect (TL ).
        o  Mortality is recorded only at 1, 2 and 4 days.  The concentration
           lethal to half the test species at each time period is
           estimated (LC50)

     The first procedure entails more complete observations and hence will
also provide the answers yielded by the second procedure.  However, the
two procedures tend to yield similar results when exposure is for 4 days or
more (Sprague, 1969).

     •   Required volume of test solution

     This would probably depend on the size and shape of the holding tank to
which the test animals were previously accustomed.  Some recommendations
about minimum depths and volumes are given by Doudoroff et al., (1951).

     However, there does not seem to have been any investigation on exactly
what size or shape of tanks are necessary to eliminate stressing the  test
species and affecting test results.  It must be left in large part to the
judgment of the investigator to provide enough water for a reasonable amount
of free activity by the test animals.
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     EPA has proposed  that  for  large  fishes  (over 0.5 g each)  the test
solution should be between  10 and  30  cm deep  (U.S. EPA, 1975a).  This
problem may be avoided in a continuous flow  test.  Alabaster and Abram  (1965)
recommended that  the supply of  new test solution should be sufficient to
maintain dissolved oxygen in the test tank.   This also keeps toxicant and
waste products within  desirable limits.  The  extreme values which they mention
for required amount of replacement solution  are 0.5 and 10 liter per gram
(for fish) per day (Sprague,  1969).

     •   Measuring response  at each concentration

     The reason for using a group  of  test  animals in each test tank instead
of one animal, is that individuals vary in resistance.  Ever since Trevan
(1927), it has been generally recognized that in bioassays, the least and
most resistant individuals  in a group show much greater variability in
response than individuals near  the median  for the group.  A good deal of
accuracy may therefore be gained by measuring some average response rather
than a minimum or maximum response, which  might represent one  animal in ten
or might happen to represent only  one animal in a thousand.

     •   Randomization

     A serious systematic error could result  from placing each successive
batch of 10 captives (for fish) in a  test  tank in order of concentration.
According to Gaddum (1953),  distribution of  animals by a process like
dealing out a pack of  cards (for example six tanks were to receive fish,
the first fish which was caught would be placed in the first tank, the
second into the second tank etc.,  the seventh into the first tank) still has
a tendency to put more easily caught  animals  into certain concentrations.  To
avoid this, Finney (1964) suggests using random numbers.  An improvement
of this has been  used  in research  by  the U.S. Federal Water Pollution
Control Administration and  is hereby  recommended as follows:

     For six tanks, the first six  fish to  be caught from the holding tank are
distributed one to each of  the  test tanks, in random order according to
occurrence of the numerals  1 to 6  in  a table  of random numbers or by drawing
numbered slips of paper; the seventh  to twelfth fish are distributed one to
each of the six tanks  by the same  process;   this is continued  until the tank
is filled.  In addition, test concentrations  should also be assigned to the
tanks by formal randomization to guard against any effect of position.

    •   Duration

     To establish the  time  factor  involved to produce an LC50  in acute
bioassay several  schemes have been used.   Katz (1971) in one experiment
(Table 3.3.3.7) showed that the 96-hour bioassay is unnecessarily long and
does not yield anymore worthwhile  information than does a 24-  or 48-hour test.
                                      274

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     TABLE 3.3.30  TIME FACTOR IN TOXICITY BIOASSAY TESTS3 (Katz, 1971)

waste

ppm .

pH

24 hours
Living
Dead
48
Fish Survival
hours
72 hours
Living Dead Living
Replicate




55.0
44.0
16.5
0.39
7
7
7
7
.68
.69
.83
.89
0
10
10
10
10
0
0
0
0
10
10
10
1: July 21
10
0
0
0
- July
0
10
10
10
Dead
24b
10
0
0
0
96 hours
Living

0
10
10
10
Dead

10
0
0
0
(control)
Replicate




49.0
45.0
43.0
0.13
7
7
7
7
.69
.69
.69
.89
2
4
9
10
8
6
1
0
2
4
9
10
2: July 24
8
6
1
0
- July
2
4
9
10
29C
8
6
1
0

2
4
9
10

8
6
1
0
(control)
a
b
c
Test
TLm:
TLm:
conditions: flowing
49
46
ppm
ppm
waste
waste
water,


1.5


liters/hour














     But according to Sprague (1969),  the most popular exposure period is 4
days or 96 hours (Table 3.3.31).
TABLE 3.3.31  ESTIMATES OF TIME REQUIRED FOR CESSATION OF ACUTE LETHAL ACTION
        IN VARIOUS BIOASSAYS REPORTED IN THE LITERATURE (Sprague,  1969)
Toxicant
Species
Apparent Time of
Lethal Threshold
h=hour, d=day,
w=week
Authors
Cyanide
Cyanide

Ammonia
Ammonia
Ammonia
Fluoride

Chlorine
High pH

Zinc

Copper, zinc

Zinc
Phoxinus
Trout

Trout
4 freshwater fish
Phoxinus
Trout

Trout
Trout

Minnow fry

Salmon

Zebrafish
about 2 d
4 d or more

5 h
less than 4 d
about 2 d
about 7 d

more than 7 d
more than 15 d

1 d or less

1 to 3 d

1 to 6 d, var-
 ious young stages
Wuhrmann, 1952
Herbert and Mer-
 kens, 1952
Lloyd, 1961b
Ball, 1967a
Wuhrmann, 1952
Herbert and Shur-
 ben, 1964
Merkens, 1958
Jordan and Lloyd,
 1964
Pickering and
 Vigor, 1965
Sprague and Ramsay,
 1965
Skidmore, 1965

        (continued)
                                     275

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                          TABLE 3.3.31  (Continued)
Toxicant
Species
Apparent Time of
Lethal Threshold
h=hour, d=day,
w=week
Authors
Copper

Copper, zinc
Heavy metals
Zinc

Zinc
Zinc
Cadmium
Eighteen
 metals
Copper

Thallium
Various (6)
Corrosion
 inhibitors
ABS detergent

ABS detergent

Detergents


LAS detergent

ABS, LAS
 detergents
Phenol

Phenol
Various
 phenolics
Various
 petrochemicals
Trout

Trout
Freshwater fish
Minnow eggs

4 freshwater fish
Bream
Trout
Stickleback
Crayfish

Perca
Tubificid worms
Trout

Bluegill

11 freshwater fish

Trout


5 freshwater fish

Minnow eggs

Trout

4 freshwater fish
Trout

Freshwater fish
2 to 4 d

4 d or less
2 d or less for
 about half of 59
 cases; 4 d or
 longer for other
 half (static tests)
7 d or less

4 to 5 d
7 d or more
7 d
7 d or more in
in each case
Liepolt and Weber,
 1958
Lloyd, 1960, 1961a
Pickering and
 Henderson, 1966a
10 to 15 d (de-
 layed mortality)
more than 14 d
2 d or less
14 d or more

1 d or less
 (static tests)
2 d or less (con-
 tinuous flow)
acute 1 d, sub-
 acute continued
 12 w
more than 4 d
 (continuous flow)
9 d or more
 (continuous flow)
1 d or less
 (saline water)
5 h to 1 d
1 d

62 of 75 cases,
 1 d or less; re-
 mainder 4 d or
 more (static
 tests) 1, 5 d
Pickering and
 Vigor, 1965
Ball, 1967b
Ball, 1967b
Ball, 1967c
Doudoroff and Katz,
 1953, from data
 of Jones, 1938
 and 1939
Hubschman,  1967

Nehring, 1962
Marvan, 1963
Herbert, 1965

Lemke and Mount,
 1963
Thatcher, 1966

Herbert et al.,
 1957

Thatcher and
 Santner, 1966
Pickering, 1966

Brown et al.,
 1967b
Wuhrmann, 1952
Brown et al.,
 1967a
Pickering and
 Henderson, 1966b
                                                                   (continued)
                                     276

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                          TABLE 3.3.31  (Continued)
Toxicant
Species
Apparent Time of
Lethal Threshold
h=hour, d=day
w=week
Authors
DDT (acetone)    Salmon
DDT

Five insecti-
 cides

Chlorinated
 hydrocarbon
 insecticides
Trout

2 Stoneflies


4 freshwater fish
Organophosphate  6 freshwater fish
Various
 pesticides
Freshwater fish
Sewage effluent  Trout

Pulp mill        Salmon
 effluent
Many pollutants  Various inverte-
                  brates, especially
                  Daphnia
                      1, 5 d
acute 1, 5 d;
 subacute 2 w
30 d or more
 (several modes
 of action)
14 cases, 2 d or
 less; 8 cases,
 4 d or more
 (static tests);
 continuous flow
 tests, 20 d or
 more
41 cases, 2 d or
 less; 27 cases,
 4 d or more
 (static tests)
25 cases, 2 d or
 less; 13 cases,
 4 d or more
 (static tests)
1 case, 8 h; 3
 cases, about 3 d
about 12 d

of 82 cases, 1 d
 or less, 26 cases;
 1 to 3 d, 14
 cases; 2 d or more
 13 cases; 4 d or
 more, 29 cases
 (static test)
Alderdice and
 Worthington,
 1959
Abram, 1967

Jensen and Gaufin,
 1966

Henderson et al.,
 1959
                                          Pickering et al.,
                                           1962
Pickering and
 Henderson, 1966c
                                          Lloyd and Jordan,
                                           1963
                                          Alderdice and Brett,
                                           1957
                                          Dowden and Bennet,
                                           1965
     Sprague (1969) realized that of 375 cases, 211 or 56% showed a lethal
threshold in 4 days or less, while in the remaining 164 cases, lethality
occurred beyond the 4th day.  The overall distribution tended to substantiate
                                     277

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that 4 days or 96 hours was a reasonable limit for occurrence of acutely
lethal toxicity of most test substances.  In view of this information, it
would seem prudent to continue tests for 4 days as a rule.  Tests could then
be stopped if mortality had ceased and the toxicity curve showed a threshold.

     •  Methods

     Examples of protocols for acute static bioassay with freshwater fish
and daphnia and marine animals are given in the following pages:


     EXAMPLE 1:  ACUTE STATIC BIOASSAY WITH FRESHWATER FISH AND DAPHNIA

Purpose of Study

     To determine the toxicity of chemicals to freshwater fish and daphnia.

Design of Experiment

     •  Test Animals

          Fathead.Minnow  Pimephales promelus
          Daphnia pulex (first instar stage)

     •  A series of test  containers each with a different, but constant,
        concentration of  toxicant will be used.

     •  At least 10 but preferably 20 organinisms should be used in each
        container for each treatment.

     •  For the minnow, the 96-hour median lethal concentration (96 hr-
        LC50) and for Daphnids,  the 48-hr median effective concentration
        will be used.

     •  A series of controls will be used in which the water conditions,
        animal species and size  will be the same as those used for each
        treatment group.

     •  The timing of the test and the collection of samples will be
        based on an understanding of the short and long-term operations
        and schedules of  the discharge if possible.

Conduct of Experiment

     •  Select the test organisms.
        QUALITY CONTROL -- Species must be readily available, hearty, and
easy, convenient, and economical to maintain.

        QUALITY CONTROL — All minnows should be from the same year class,
and weigh between 0.5 and 1.0 grams; 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.
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      •  Select dilution water.
         QUALITY CONTROL —  A healthy test organism must survive in the
dilution water for the duration of acclimation and testing without showing
signs of stress, i.e., discoloration or unusual  behavior.

      •  QUALITY CONTROL —  The test organism must survive and  reproduce
satisfactorily in the dilution water.  A water in  which  Daphnids, who are
more sensitive to many toxicants than most other freshwater aquatic animals,
will survive and reproduce should be an acceptable dilution water for most
tests with freshwater animals.

      •  At least two grab samples of effluent should be collected.   The
samples, whether liquid waste or sludge, should be  stirred to a uniform
consistency.
         QUALITY CONTROL — Conduct separate tests on each grab  sample;
more tests may be desirable if there are known sources of variability such as
process changes.

         QUALITY CONTROL -- The sample of the effluent must not  be aerated
or altered in any way except that it may be filtered through a sieve or
screen with 2mm or larger holes.

         QUALITY CONTROL — Samples must be covered at all times, violent
agitation must be avoided.

      •  Prepare stock solution or dilution of waste.
         QUALITY CONTROL — Add the same volume at all test levels.

         QUALITY CONTROL — The stability of the test substance  in the
stock solution should be determined.

      •  Place the test organisms in the test containers.
         QUALITY CONTROL -- Stratified randomization or  total randomization
of the treatment is recommended.

         QUALITY CONTROL -- True replicates with no water connection should
be used.

         QUALITY CONTROL — The use of more animals and  replication of
treatment is desirable.
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Observations and Results
     •  The final result should be expressed as concentration tolerated by
the median or "average" test animal.  A test is not acceptable if more than
10% of the organisms in any control die in a test determining LC50 or show
effect in a test determining EC50.
     •  At a minimum, the number of dead or affected animals must be
observed and recorded at 24-hour intervals.  More observations, however, are
desirable.
Termination
     •  At the end of test period, the bioassays are terminated and the
LC50 or EC50 values are determined.
Records
     •  Any deviation from these methods should be recorded as well as the
following specific information:
        The chemical characteristics of the the dilution water.
        Test organisms.
        Definition of the criterion used to determine the effect;
        abnormal behavior.
        Percentage of organisms that died or showed the effect in the
        control treatment.
        Duration.
        Statistical methods employed to interpret test results.
Report
     •  In addition to the final report, interim reports may be made
available to the sponsor if required.  The frequency of such reports will be
determined prior to study initiation.

            EXAMPLE 2.  ACUTE STATIC BIOASSAY WITH MARINE ANIMALS
Purpose of Study
     •  Toxic Effect
Design of Experiment
     •  Test animals:  juvenile sheepshead minnows (Cyprinodon varieqatus);
                                     280

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adult grass shrimp (Palaemonetes pujio or £._ vulgaris)

     •  20 control animals and 20 test animals must be exposed to each
concentration of test material.

     •  A control and 6 concentrations of effluent in a geometric series
will be used.

     •  Concentration of test effluent that yields LC50 or EC50 values will
be determined.

     •  The animals will be observed for 96 hours.

Conduct of the Experiment

     •  Select the test animals.
        QUALITY CONTROL --  The animals should be healthy and as uniform in
size as possible.

        QUALITY CONTROL —  During holding, acclimation and testing, the
animals must not be disturbed unnecessarily.  When they must be handled, it
must be as gently, carefully, and quickly as possible.

     •  Grab samples of effluent, whether liquid waste or sludge, should be
stirred to a uniform consistency.
        QUALITY CONTROL —  Effluent samples may be filtered through a sieve
or screen with 2mm or larger holes.  The collection of samples should be
based, on an understanding of the short- and long-term operations and
schedules of the discharges if possible.

     •  Check the salinity of undiluted effluent and add an appropriate
amount of salts (Table 3.3.3) to yield a salinity of 10 parts per thousand
as determined by a refractometer.

     •  Two range-finding tests should be performed:  one with aeration and
one without.  To aerate, introduce clean air into the test effluent at the
rate of 100 + 15 bubbles per minute.  Use effluent concentrations of 0.01,
0.1, 10 and TOO percent.  If more than 50 percent of the animals die at 0.01
percent, conduct a new range-finding test at lower concentrations, such as
0.001 and 0.0001 percent.
        QUALITY CONTROL —  The stability of the effluent sample in the
stock solution should be determined.

        QUALITY CONTROL —  Conduct a control test 1n 100% dilution water
at the same time.  The pH of the test media and control must be taken before
and after the test.
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     •  Determine the definitive test concentration from the results of the
range-finding test.  The concentration in each treatment must be at least
50 percent that of the next higher one.  One treatment must kill more than
65% of the test animals and one treatment must kill less than 35%.
        QUALITY CONTROL —  Stratified randomization or total randomization
of the treatment is recommended.

        QUALITY CONTROL —  True replicate with no water connection should
be used.

        QUALITY CONTROL —  The use of more test animals and replication of
treatment is desirable.

        QUALITY CONTROL —  A separate test should be conducted on  at least
two grab samples and more tests may be desirable if there are known sources
of variability such as process changes.

Observations and Results

   • Observe the animals frequently throughout the 96 hours and record the
number of dead or affected animals for each 24-hour period.  The final
results will be expressed as concentration tolerated by the median  or
"average" animal.  A test is not acceptable if more than 10% of the control
animals die.

Termination

   • At the end of the test period, the bioassays are terminated and the LC50
or EC50 values are determined.

Reocrds

   • Records will be maintained on:

        detailed description of the material tested

        test animals

        abnormalities such as erratic swimming, loss of reflex, discoloration,
        behavioral changes, excessive mucous production, hyperventilation,
        opaque eyes, curved spines, hemorrhaging, molting and cannibalism

        percent of control animals that die or were affected in each test
        container during the test

        duration

        statistical method used for interpreting the result
                                      282

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Reports

     In addition to the final report, interim reports may be available to
the sponsor.  The frequency of such reports will be determined prior to
study initiation.

3.3.2.5 Chronic Bioassay—

     Chronic bioassays are of primary value in determining "safe" levels of
toxicants.  All such tests involve exposures through the reproductive period
of the life cycle and subsequent exposures of the eggs and young (Eaton, 1970).
The use of the chronic test allows the test operator to better determine the
most sensitive species or life stages to be assayed and on which organisms
to base toxic limits (Martin, 1973).

     Test procedures considered adequate are available for bluegill, fathead
minnow, brook trout and Daphnia magna and procedures are being developed for
several additional fish and invertebrate species.  Various short-term tests
have also been developed for use in conjunction with chronic tests
(Eaton, 1970).  A rather complete discussion of the chronic bioassay is
presented by Sprague (1971).

     The chronic tests differ from the acute tests in that they are an attempt
to measure concentration harmful or safe to the system in a direct manner
without using a lethal end point.  The chronic test, as with the acute test,
requires similar test operations.  Usually continuous-flow test procedures
are used and test dosages are maintained at levels below lethal concentration
and the test is usually carried well beyond the conventional time period for
the acute and/or static test (Martin, 1973).

     Only this kind of exposure demonstrates the "safe" toxicant concen-
trations at which most life processes are protected.  Usually the safe
toxicant concentrations as determined by chronic bioassays are 10 to 100
times lower, and sometimes as much as 200 to 500 times lower than concen-
trations determined by acute bioassay using 50% mortality as an end point
(Eaton, 1973).

     • Experimental Procedure

     Acute flow-through bioassays should be conducted prior to initiation of
any chronic test. It is desirable for these tests to be on at least two
different age classes (e.g., fry, juveniles or adults).

     Concentrations selected for chronic toxicity experiments should be
based on results of acute flow-through bioassays.  Concentrations should be
selected so that at least one will adversely affect some life stage of the
test animal and one will not affect any stage.
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     Chronic bioassay usually includes exposure of animals to five or six
toxicant concentrations along with a control; consecutive concentrations
usually differ from one another by a factor of 2 or 3.  Fish tests often
start with 40 to 50 individuals per tank, and numbers are reduced at
intervals for closer examination for toxicant effects and to adjust sex
ratio so that only 6 to 20 remain at the time of spawning (Eaton, 1970).
Fish chronic exposures routinely take about 10 months to a year to complete
whereas Daphnia magna are exposed for only 3 weeks, as they go through an
entire life cycle in that time (Eaton, 1973).  Use true duplicates for each
level of toxic agent with no water connections between duplicate tanks
(U.S. EPA, 1973).  For Daphnia magna, true quadruplicates should be used
(Biesinger, 1975).

       o  Water source

     Freshwater:  should be from a well or spring if at all possible, or
alternatively from a surface water source.  Only as a last resort should
water from a chlorinated municipal water supply be used.

     Saltwater:  should be natural sea water with salinity greater than or
equal to 15%.

     Any proposed source must be analyzed for possible pollutants such as
pesticides, PCB's and heavy metals.  Special determinations should be made
for those toxicants being investigated  (U.S. EPA, 1976).

       o  Dosing apparatus

     A number of apparatuses would be acceptable for this bioassay including
those of Mount and Brungs, 1967; Hansen et al., 1971; Hansen et al., 1974b;
or Schimmel et al., 1974  (U.S. EPA, 1976).  The diluter should be checked
daily, either directly or through measurement of toxicant concentrations.
An automatically triggered emergency aeration and alarm system must be
installed to alert staff in case of diluter, temperature control or water
supply failure (U.S. EPA, 1973).

       o  Toxicant mixing chamber

     A container to promote mixing of toxicant should be used between
diluter and tanks for each concentration.  Separate delivery tubes should
run from this container to each duplicate tank.  The whole system should
be checked at least once every month to see that the intended amount of
water is going to each duplicate tank or chamber (U.S. EPA, 1973).

       o  Spawning chamber

     The spawning chamber should be small enough to be placed in an
aquarium, but large enough to permit the female to avoid the aggressiveness
of the male, and should be designed 30 eggs would sink through mesh bottom
and fall on a surface for collection (Hansen and Parrish, 1976).
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       o  Embryo and fry chamber

     These chambers should be constructed to allow for adequate exchange of
water and to insure that the proper quantity of material Is entering the
chambers.  Care must be taken that each embryo and fry chamber receives an
equal amount of the toxicant solution (U.S. EPA, 1976).

     Exposure chamber, spawning chamber, hatching container, growth chamber
and other equipment are varied to meet the needs of the different organisms
used in the test (Rand et al., 1975).

       o  Photoperiod

     Simulate the natural seasonal daylight and darkness periods with
appropriate twilight periods.  Make adjustments in photoperiods on the first
and fifteenth of every test month (Rand et al., 1975).  It may be desirable
to control lights by a timing switch (Drummond and Dawson, 1970).

       o  Cleaning

     All aquaria should be cleaned whenever material builds up.  Aquaria
should be brushed down and siphoned to remove accumulated material a
minimum of 2 times weekly (U.S. EPA, 1973).  Care should be exercised in
cleaning to prevent loss, or damage to the fry, juveniles, or adults (U.S.
EPA, 1976).

       o  Disturbances

     All test chambers should be shielded from excessive outside distur-
bances.  Tanks should be shielded from all outside light sources that would
interfere with the photoperiod (U.S. EPA, 1976).

       o  Test Animals

     There are several criteria to be considered when choosing test
organisms for a chronic bioassay:

          The test organisms should be able to reproduce readily in
          close confinement, producing large numbers of eggs;

          fertility as well as survival to adulthood should be high;

          the organisms should mature rapidly, yet be small enough at
          adult size to maintain large, statistically valid numbers of
          test organisms in the bioassay;

          the test organisms should be relatively sensitive to toxic
          pollutants (Schlmmel & Hansen, 1974).
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     The test organisms should be obtained from the same source, either
from wild population or suitable culture laboratory (U.S. EPA, 1976).  To
obtain a sufficient number of eggs to begin a chronic exposure, two methods
may be employed:

          natural spawning from laboratory stocks;

          artificial inducement by injection of human gonadotrophic
          hormone and fertilization with sperm excised from males
          (Schimmel et al., 1974).

     The former may be preferable.

       o  Food

     Each batch of food should be checked for pesticides (DDT, Dieldrin,
Endrin, etc.) and the kinds and amounts should be recorded (U.S. EPA, 1976).

       o  Disease

     Disease outbreaks should be handled according to their nature with
each aquarium being treated similarly even though disease is not evident
in all aquaria.  All treatments should be kept to the minimum and
recorded as to type, amount, and frequency (U.S. EPA, 1976).

     As mature adults begin courtship, separate pairs should be placed in
individual spawning chambers in the aquaria.  Pairs should be left in the
chambers until a sufficient number of eggs have been collected to insure
statistical comparisons of fecundity and fertility,  and survival counts of
embryo and fry can be made.  All eggs should be removed at a fixed time of
each day so that the adults are not overly disturbed and that disruption of
activity will not occur.  Daily records of spawning and egg numbers must be
kept.  Each pair should be observed daily for a minimum of 2 weeks.
Impartially, 50 fertile eggs should be collected and incubated.  If no
spawning occurs at the highest concentration, eggs should be transferred
from control spawns and incubated in the highest concentration to gain
additional information.  Survival of embryos, time required to hatch,
hatching success, and survival of fry will be determined and recorded.
Additional groups of 50 eggs from contaminated aquaria should be placed in
control aquaria to determine if they contain chemicals toxic to embryo or
fry.

     Daily records on embryos and fry should be kept of mortalities and
development of abnormalities.  Termination of the chronic test is
considered as the time when no spawning activity has occurred over a 2
week interval (U.S. EPA, 1976).
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       Data that must be reported for each tank of  a chronic  test  are:

            number and individual total length of normal  and  deformed  test
            animals at 30 and 60 days;  total  length,  weight and number  of
            either sex,  both normal  and deformed, at end  of test;

            mortality during the test;

            number of spawns and eggs;

            hatchability;

            fry survival, growth and deformities (U.S. EPA, 1973).

         o  Concentration of toxicant

       A minimum of 5 concentrations of toxicant and a control, all duplicated,
  should be utilized in all chronic  tests.  Concentrations selected for chronic
  toxicity experiments should be based  on results of acute flow-through
  bioassays.  Concentrations should  be  selected so  that at least one will
  adversely affect some life stage of the test animal and one will not  affect
  any stage (U.S. EPA, 1976).

       Concentrations of the toxicant should  not vary by  more than + 10 to 15%
  from the selected test concentration  because of uptake  by the test organisms,
  absorption,  precipitation and  other causes  (Rand  et al., 1975).

       Analyses should be made of the material itself, of the water during this
  test and of  the test organisms (adult)  at the conclusion of the  test.  At a
  minimum, water from each aquarium  at  the beginning and  end  of the test, and
  test animals from each aquarium (10 or more test  animals each) at the end of
  the test, should be analyzed.   It is highly  desirable to chemically analyze
  additional samples of water and of test animals including,  at each life
  stage, muscle tissue and gametes (U.S.  EPA,  1976).

         o  Preparing a stock solution

       If a toxicant cannot be introduced into the  test water as is, a  stock
  solution should be prepared by dissolving the toxicant  in water  or in an
  organic solvent.  Acetone has  been the most widely used solvent, but
  dimethylformamide  (DMF) and triethylene glycol may be  preferred in many
  cases.  The  use of solvents, surfactants, or other additives should be
  avoided whenever possible.  If an  additive  is necessary, reagent grade or
  better should be used.  The amount of an additive should be kept to a
  minimum, but the calculated concentration of a solvent  to which  any  test
  organisms are exposed must never exceed one-thousandth of the 96-hour
  LC50 for test species under the test  conditions and must never exceed 0.1
  gram per liter of water.  The  calculated concentration  of surfactant  or
  other additive to which any test organisms  are exposed  must never exceed
  one-twentieth of the concentration of the toxicant and  must never exceed 0.1
gram per liter of water.  If any additive is  used,  two sets of controls must
  be  used, one  exposed  to no additives  and  one  exposed  to  the

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highest level of additives to which any other organisms in the test are
exposed (U.S. EPA, 1973).

       o  Measurements of other variables

     Temperature must be recorded continuously.  Dissolved oxygen must be
measured in the tank daily, at least 5 days per week on an alternating
basis, so that each tank is analyzed weekly for pH, alkalinity, hardness,
acidity and conductance, or more often if necessary, to show the
variability in the test water.  At a minimum, the test water must be
analyzed at the beginning and near the middle of the test for calcium,
magnesium, sodium, potassium, chloride, sulfate, total solids, and total
dissolved solids.  Methods described in "Methods for Chemical Analysis
of Watar and Wastes"  (U.S. EPA, 1974) should be used for those measurements.
At a minimum, accuracy should be measured using the method of known additions
for all analytical methods for toxicants.

     If available, reference samples should be analyzed periodically for
each analytical method (U.S. EPA, 1973).

     •  Methods

        An example of a protocol for chronic flow-through bioassay with
fish and aquatic invertebrates is given in the following pages.

       EXAMPLE:  CHRONIC FLOW-THROUGH BIOASSAY WITH FISH AND AQUATIC
                                  INVERTEBRATES

Purpose of the Study

     •  To  determine the quantity of chemical that can be tolerated by fish
and aquatic invertebrates.

Design of Experiment

     •  Start with 40 to 50 animals per tank.  Use at least two different
age classes*

     •  Expose animals in duplicate to five or six toxicant concentrations.

     •  Use a series of controls in which all test conditions will be
        similar to those of the experimental groups, except the toxicant
        will be absent from the test medium.

     •  Observe for 96 hours LC50.
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Conduct of the Experiment

     •  Select test species.
        QUALITY CONTROL -- The chosen species should be able to reproduce
readily in close confinement, producing a large number of eggs.  Fertility
as well as survival to adulthood should be high.

     •  Fish tests should start with 40 to 50 individuals per tank and
number should be reduced at intervals for closer examination for toxicant
effects and to adjust sex ratios so that only 6 to 20 remain at the time
of spawning.

     •  A chronic test should be used which includes exposure of animals
in duplicate to 5 or 6 toxicant concentrations along with a control.
Consecutive concentrations usually differ from one another by a factor of
2 or 3.
        QUALITY CONTROL — Stratified randomization or total randomization
of the treatment is recommended.

        QUALITY CONTROL -- True duplicate with no water connection between
aquaria should be used.

        QUALITY CONTROL — The control should consist of the same water
conditions and animals of the same species as are used in the remainder of
the test.  If any additive is present in any of the test chambers, an
additive control is also required.

        QUALITY CONTROL — An acute flow-through bioassay should be
conducted prior to initiation of any chronic test.   It is desirable for
these tests to be conducted with at least two different age classes.

     •  Use a proportional diluter for all long-term exposures.
        QUALITY CONTROL — The calibration of the toxicant delivery system
should be checked daily before, during and after the test, either directly
or through measurement of toxicant concentration.

        QUALITY CONTROL -- If duplicate test containers are used, separate
delivery tubes can be run from the mixing chambers  to each duplicate.

        QUALITY CONTROL — Check at least once every month to see that the
intended amounts of water are going to each duplicate tank or chamber.

        QUALITY CONTROL -- A container to promote mixing of toxicant bearing
water should be used between diluter and tank for each concentration.

Observations and Results

     •  Observe for mortalities and development of abnormalities.

     •  Obtain water quality criteria by multiplying the 96-hour LC50 of the
most sensitive species tested by an arbitrary application factor.


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Termination

     Termination of the test is considered at the time when no spawning
activity has occurred over a 2-week interval.

Records

     Records will be maintained on:

     •  Detailed description of test material
     •  Test animals
     •  Percent of control animals that died or were affected in each
        test container during the test
     •  Daily records of spawning, egg numbers, fertility
     •  Mortalities and development of abnormalities of embryos and fry
     •  Number of spawns and eggs
     •  Hatchability
     •  Fry survival, growth, and deformities
     •  Duration
     •  Statistical methods used to interpret test results

3.3.2.6  Algal Bioassay—

     The algal bioassay test is intended to identify algal growth-limiting
nutrients, to biologically determine their availability, and to quantify
the biological responses to changes in concentration.

     These measurements are made in a uniform manner by inoculating test
water with a selected algal test culture and determining algal growth at
appropriate intervals.

     •  Species selection

     In choosing species for bioassays, the following criteria are useful
guides:

       o  Whenever possible, indigenous species representing a diversity
          of phylogenetic types from the major seasonal succession should
          be studied.
       o  The more sensitive species should be used.
       o  Conditions of greatest vulnerabilities should be identified for
          the species selected.
       o  Both test species and culture conditions should permit growth
          rates of 0.5 to 1.0 doublings per day under nonstress  conditions
          (U.S. EPA, 1976).

     • Culture conditions

     The culture conditions for the test species generally should reflect
their natural conditions.
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       o  Marine algae

     For temperate species, a temperature of 20+2°C and a light intensity
of 2500 to 5000 lux on a 14-hour light and 10-hour dark cycle (14:10 cycle)
are desirable.

     For cold water species, a temperature of 8+2°C and 2500 to 5000 lux
light intensity on 10:14 cycle is recommended (U.S. EPA, 1976).

       o  Freshwater algae

     A temperature of 24+2°c and "cool white" fluorescent lamps .Riving at
least 250 foot-candles (ftc) (2152 lux), preferably 400 ftc (4304 lux) are
recommended (U.S. EPA, 1977).

     • Selection of test water

       o  Freshwater

     Samples for the test may be:

       surface samples from lakes and rivers,
       wastewaters,
       substances of concern that may ultimately reach surface waters,
       any sample to which nutrients or other substances are added or
       from which they are removed.

       o  Marine water

     Sampling schedules should be arranged to take into account  the tidal
fluctuations, sampling preferably at high water, or at both high water and
the following low water.

     Transport samples to the laboratory at ice temperature.  Temporary
storage in the laboratory should occur under similar conditions.   Each
sample must be tested in triplicate (U.S. EPA, 1977).

     •  Concentration of spike

     The volume of the spike should be as small as possible.  The concen-
tration of spikes will vary and must be matched to the waters being tested.
Two considerations should be taken into account when selecting the
concentrations of the spikes:

       o  The concentration should be kept small to minimize alterations
of the sample, but at the same time it should be sufficiently large to
yield a potentially measurable response.

       o  The concentration of spikes should be related to the fertility of
the sample.  To assess the effect of nutrient additions, they must be com-
pared to an unspiked control of the test water.
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     In addition to spikes for the purpose of determining stimulatory or
inhibitory effects on algal growth in test waters, it is sometimes
necessary to check for the possibility that the test water contains some
toxic materials which could influence results.  To check for toxic
materials, the test waters may be spiked with the elements in complete
synthetic medium.  If no increase in growth occurs, the presence of toxic
materials is suspected (U.S. EPA, 1974b).

     •  Untreated controls

     Control algal cultures must be grown in untreated medium (devoid of
toxicant) at the time bioassays on liquid waste are being done (U.S. EPA,
1976).

     •  Test methods

     Examples of protocols for unicelluar marine algal assay and fresh-
water algal bottle test are given in the following pages.

                     EXAMPLE-  UNICELLULAR MARINE ALGAL ASSAY

Purpose of Study

     To determine biological response to changes in toxicant concentration.

Design of Experiment

     •  Select indigenous algal species or Skeletonema costatum.

     •  Use at least one control and five test concentration groups.  The
five concentrations must be in a geometric series and include concentrations
that inhibit growth by approximately 65 and 35 percent.

     •  All tests should be performed in triplicate.

     •  Measure biomass once daily.

Conduct of Experiment

     •  Maintain algal  stock cultures in artificial seawater medium of 10
parts per thousand salinity prepared from glass-distilled or deionized water.
     QUALITY CONTROL -- Select the more sensitive algal species  and the
conditions of greatest vulnerabilities.

     QUALITY CONTROL -- Test species and culture conditions should permit
growth rates of 0.5 to 1.0 doublings/day under nonstress conditions.

     QUALITY CONTROL -- Stock cultures must be manipulated according to
standard microbiological  techniques to insure a minimum of contamination
by bacteria.
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     •  Perform a toxicant concentration range finding test.
        QUALITY CONTROL -- Perform in duplicate covering concentrations of
4 orders of magnitude.

        QUALITY CONTROL — If growth stimulation occurs, use 5 concentra-
tions in a geometric series between a concentration without effect and 100
percent waste.

     •  When a range has been identified, dilutions of toxicant solutions
should be prepared in distilled water or suitable solvent.
        QUALITY CONTROL — Stock solutions or dilutions of a waste should
be prepared to assure that the same volume is added at all test levels.
This addition should not exceed 1 ml  per 50 ml of test medium with
waste water.

     •  Control
        QUALITY CONTROL — Algal cultures must be grown in untreated
medium at the time bioassays on liquid waste or sludge are being done.

Observations and Results

     •  Determine absorbance of the culture every day between days 3 and 12.

     •  Plot the average absorbance for each day using semi logarithmic
paper and examine the shape of the curve.
        QUALITY CONTROL — Be careful in interpretation of data; some
toxicants inhibit growth in the early stages of a test.

     •  Estimate final biomass on the 12th day by weighing an aliquot of
each culture.
        QUALITY CONTROL — Use a vacuum less than 0.5 atmospheres to prevent
cell breakage.

Termination

     At the end of the 12-day test period, terminate the bioassays and
determine the EC50.

Records

     Record the following test data:

     •  EC50 at 12 days and other days of importance to be decided by the
shape of the growth  curve.   ,

     •  The specific growth rate between days 3 and 12 and any other period
depending upon the shape of the growth curve.
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                 EXAMPLE 1:   FRESHWATER ALGAL BOTTLE TEST

Purpose of the Study

     •  to identify algal growth-limiting nutrients;
     •  to determine biologically the availability of growth-limiting
        nutrients;
     •  to quantify the biological  response to changes in concentrations
        of growth-limiting nutrients.

Design of Experiment

     •  Test algae:

          Selenastrum capricornutum Printz
          Microcystis aeruginosa Kutz. emend Elenkin
          (Anacystis~cyanea) Drouet and Daily
          Anabaena flos-aquae (Lyngb.) De Brebisson
          Diatom - cylotella sp.
                 - Nitzschia sp.

     •  The starting concentrations in the test water should be as
follows:

          S_. capricornutum 103 cells/ml
          ML aeruginosa and A. flos-aquae 50 x 103 cells/ml

     •  Measure biomass at least once daily

Conduct of the Experiment

     •  Select test species.
        QUALITY CONTROL -- Test species should be representative cross
sections of types of algae found in waters of differing nutritional  status.

     •  Collect water samples.
        QUALITY CONTROL -- Collect water samples in nonmetallic  and auto-
el avable storage containers.  Leave a minimum of airspace in transport
container; keep in dark and at ice temperature.

        QUALITY CONTROL -- Do not reuse  containers if toxic or nutrient
contamination is suspected.

        QUALITY CONTROL -- Remove indigenous algae by membrane  filtration
(0.45 p at 0.5 atmosphere or less)  or autoclaving.  Water can also be
prefiltered through glass fiber filter.

        QUALITY CONTROL — Duration of storage should be minimized.

     •  Select spikes of nitrogen,  phosphorous, iron, sewage effluents, etc.
        QUALITY CONTROL -- Volume of the spikes should be as small as
possible.

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      QUALITY CONTROL —  The concentration of the spike should be related
to the fertility of the sample and should be kept small  to minimize the
alteration of the sample.

   •  The effect of nutrient additions must be compared  to an unspiked
control of test water.
      QUALITY CONTROL —  Check for the possibility that the test water
contains some toxic material which could influence results.

   •  Test each sample in triplicate.
      QUALITY CONTROL -- For statistical purposes divide each into three
aliquots before filtration and thereafter treat as separate samples.

Observations and Results

   The fundamental  measure used in the bottle test to describe algal  growth
is the amount of suspended solids (dry weight) produced; this is determined
gravimetrically.  Several different biomass indicators should be used when-
ever possible because biomass indicators may respond differently to any
given nutrient-limiting condition.

Record

   The following data should be recorded:

   •  the EC50 at 12 days and other days of importance to be decided  upon by
the shape of the growth curve;
   •  the specific  growth rate between 3 and 12 days and any other period that
should be reported  depending upon the  shape of the growth curve.

Report

   In addition to the final report, interim reports may  be made available to
the sponsor if required.  The frequency of such reports  will  be determined
prior to study initiation.

   •  Results

   Growth responses should be statistically analyzed and significant levels
of differences reported.  For most purposes a 95 percent significance
level can be considered statistically  significant.  The  EC50 can be
estimated by interpolation by plotting the data on semi logarithmic
coordinate paper with concentrations on the logarithmic  axis and percentage
growth in relation  to the control on the arithmetic axis.  Draw a straight
line between two points on either side of the 50 percent growth value.  The
concentration at which  the line crosses the 50 percent growth line is the
EC50 value (U.S. EPA, 1977).

3.3.2.7  Community  Studies —

   Two examples of  community studies follow:
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      o  Purpose of study

      To determine the effects of various types of alterations such as pred-
ator  pressure, variability of the environment, and competition between
species on species living together in a community.

      •  Experimental design

         o  Periphyton community

      To study the effect of germanium dioxide on a community, an
experiment setup  using periphyton was designed by Dickman (1969).
Periphyton was chosen as representative of the community because (Rand et al.,
1975):
         o  they  are  a very, important food source for most forms of aquatic
            life that feed_upon plants;
         o  they carry out the process of photosynthesis which is so
            important in the generation of oxygen needed by all organisms
            in order to carry out the metabolic processes;
         o  because of the large number of species, one will find many
            species present in almost all natural conditions;
         o  because as a group, they consist of many species that have
            populations composed of varying numbers of specimens;
         o  they are an excellent group to treat statistically in analyzing
            their reaction to varying ecological conditions.

      •  Conduct of Experiment

      The basic procedure is to expose a set of slides with a suspected
toxicant to the water column of a lake or stream where it would be possible
for the periphyton to colonize it.  A second set of slides identical to the
first in every respect but lacking the suspected toxicant is suspended
nearby for comparison.  The species composition of the periphyton colonizing
the two types of slides (treated and control) can then be compared at weekly
intervals by harvesting some of the slides and allowing the remainder to
continue to incubate.  Significant differences in the species composition
between the control and the treated slides can then be attributed to the
presence of the substance which was impregnated on the slides.

      In this study a chemical with a known toxic effect was chosen in order
to test the proposed technique (in theroy, however, this technique should
be applicable to any water-soluble substance).   Germanium dioxide was chosen
because its mode of action has already been demonstrated.  In concentrations
above 1.5 mg per liter the germanium dioxide suppresses silicon uptake and
 hence fission in diatoms.

      •  Periphyton sampling

      The location on the slide at which a particular alga settles and grows
is controlled largely by chance.  Many of the algal species which appear to be
rare in the water column may soon come to dominate the slides on which they
settle.  Five replicates should probably be a minimum under such circumstances.

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One rime-saving device is to record the data directly onto IBM forms so that
they are ready for immediate punching.  This also reduces the probability of
error in copying the data from one form to another.

     •  Data analysis

        The data from the enumeration of each slide were punched onto IBM
computer cards.  A program was written which:
        - listed species counted per slide in the order of their relative
          abundance,
        - calculated the diversity indices,
        - calculated the mean abundance (density) and standard deviation
          of each species from the replicate slides,
        - compared the above means for the treated and control slides at
          each time interval by means of Student's t-test.

     •  Disadvantages of this method

     The major disadvantage in applying this technique to general use is that
the concentration of the toxicant at the gel-water interphase is neither con-
trollable nor known.  The concentration of the toxicant to which the periphyton
colonizing the slide are exposed will be a complex function of the following
factors:
        - the rate of water renewal at the gel-water interphase,
        - the solubility of the compound being tested,
        - the viscous flow characteristics and permeability of the gel.
Some of these factors are controllable.  The acrylamide polymer gels have the
advantage that they are not biodegradable as is agar.

     •  Advantages

     The major advantage of this technique is its wide potential applicability.
Whenever a pollutant is suspected, it could be impregnated in a gel and ex-
posed to the periphyton in the same or similar area as that into which the
potential toxicant would be released.

     This method can be applied to marine as well as freshwater environments,
flowing as well as stagnant waters.  It can be employed at any time of the
year and at nearly any possible location.   In any case, such an approach
obviates the necessity of extrapolating from over-simplified laboratory simu-
lations or modeled environments.  Furthermore, the necessary equipment is
minimal and inexpensive.  The results are easily quantifiable as a record of
the effects of that particular compound (Dickman, 1969).

     •  Planktonlc larvae community

     A test procedure using a planktonic larvae community was designed by
Hansen (1974) as follows:

        - a planktonic larvae community is exposed to a test substance for


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              a relatively long period of time (usually 4 months);
            - 10 aquaria and 10 replicates for each treatment are used (treat-
              ment includes control and contaminated apparatus);
            - at the end of 4 months, the effects of contaminants on develop-
              ment of the community are determined by comparing the number,
              species and diversities of animals (Hansen, 1974).

         o  Planktonic larvae are selected because:

            - plankton have long been used as indicators of water quality;
            - some species flourish in highly eutrophic water while others are
              very sensitive to organic and/or chemical wastes;
            - they have short life cycles; planktons respond quickly to en-
              vironmental changes, and hence the standing crop and species
              composition indicate the quality of water mass in which they are
              found.  As a group, they consist of many species that have popu-
              lations composed of varying numbers of specimens.  They are an
              excellent group to treat statistically in analyzing their re-
              actions to varying ecological conditions (Rand et al., 1975).

      •  Test Substance

      Polyethylene glycol 200 is recommended as solvent for most pesticides
 because this compound at 0.68-mg-per-liter, 2-ml-per-day concentrations, did
 not affect development of two species of crabs, and concentrations up to 1%
(v/v) were not lethal to grass shrimp or sheepshead minnows in 96-hour static
 tests.  The toxicity of 5 ug/liter of Aroclor 1254 to brown shrimp and pin-
 fish was not increased by increasing the concentration of solvent up to 100
 times (0.1 to 10.0 mg per liter).  The same amount of solvent should be used
 in the control apparatus (Hansen, 1974).

      For long-term studies, the concentrations of test substances shall be
 determined at the start of the study and samples shall be collected and ana-
 lyzed periodically to verify concentrations (Hansen, 1974).

        o  Flow rate

      The flow rate through each aquarium should be maintained at 200 ml per
 minute (Hansen, 1974).

        o  Concentration

      The range 0.1, 1.0, 10.0 yg per liter of the toxicant seems adequate
 (Hansen, 1974).

      •  Termination

      At the end of test period (4 months) the study is terminated and the
 index of species diversity as well as the percent occurrence of various
 species is determined (Hansen, 1974).
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     •  Observations and results

     Water is analyzed twice monthly and sediment is analyzed at the end of  a
4-month period.

     Modifications of the Shannon-Weaver method are used to assess effects of
pollution on the natural community
                                     s
                             H  =  - I Pi log pi
                                    i=l

where  p±  =  proportion of the ith species in the collection
        s  =  number of species.

Pooled data from each toxicant concentration and control are compared statis-
tically using the x2 test for independent samples.  Data from each of the 10
aquaria receiving one treatment are compared with data from 10 aquaria receiv-
ing a different treatment using the Mann-Whitney "U" test.  Differences are
considered real at alpha = 0.01.


3.3.2.8  Food-Chain Accumulation—

     •  Food-Chain Model

     The buildup of certain substances, such as heavy metals, pesticides, etc.,
in the ecological food chain has been the subject of considerable study in
recent years.  Ecologists have attempted to analyze the flow of such material
into various sectors of the ecosystem.   To better understand the movement and
transfer of toxicants throughout an estuarine trophic level, several food
chain models or systems have been designed.

     The model food chain is, in essence, a simple means to estimate, under
controlled conditions, the movement of  an organic synthetic chemical (i.e., a
"foreign" molecule or xenobiotic) in certain representative trophic levels of
a natural aquatic ecosystem.

     A food-chain model should be inexpensive,  simple to maintain,  reproducible,
ecologically relevant and able to produce clearly definable data (Johnson and
Schoettger, 1975).

     Thoman et al.  (1974) have described a food-chain model of cadmium in
western Lake Erie which is a mathematical model of the transfer of toxicants
in the food chain.   The purpose of the  model is to:

        - examine the structure of the  buildup  of potentially toxic substances
          in the food chain;
        - determine what  data would be  required for a verification of the
          model;
        - determine the utility and applicability of linear food-chain model
          in broadscale ecosystem planning;
        - demonstrate the interfacing of nonlinear and linear modeling frame-
          works .

                                     299

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The model has proved useful in large-scale planning applications provided
that additional data have been collected on the various trophic levels.

     In a study of pesticide biodegradability, Metcalf et al. (1975) have
proposed a laboratory model ecosystem with a terrestrial-aquatic interface
and a seven-element food chain.  The seven elements are:

        Algae  (Oedoeonium cardiacum)
        Snail  (Physa)
        Plankton
        Water flea (Daphnia manga)
        Mosquito pupae  (Culex pupae)
        Mosquito larvae  (Culex larvae)
        Mosquitofish (Gambusia affinis)

This food-chain model has been found very useful in estimating the potential
environmental effects of DDT and other pesticides, particularly in regard to
ecological magnification and biodegradability.

     •  Experimental Design

     Generally the test procedure consists of:

       - a series of test containers each with a different, but constant,
         concentration of toxicant;
       - at least one control and three concentration groups;
       - the number of animals per exposure ranging at least from 45 to 60;
       - control consisting of the same water conditions, and animals of the
         same species and size which are used for the treatment groups;
       - all tests performed in triplicate (Hamelink, 1976).

The use of 1UC compounds is recommended (Johnson and Schoettger, 1975).

     •  Test Animals

     All test animals should be healthy and as uniform in size and age as
possible.  Test animals should be acclimated to laboratory  test conditions for
at least 10 days.  Mortality of animals should not exceed 1% of the stock in
the 48 hours immediately preceeding the test (U.S. EPA, 1975a).

     Frequent disturbance and unnecessary handling should be particularly
avoided because the environment of the animals has an immediate and profound
influence on their respiration and metabolism.

     The number of animals per exposure level is relatively large compared to
most other toxicity tests.  As a general rule, around 45 to 60 animals per
tank is considered minimal.  This quantity is necessary because three or more
animals have to be sampled each period in order to accomodate the amount of
individual variance encountered (Branson et al., 1975; Macek et al., 1975).
About 12 to 15 sampling periods are usually required to establish the dynamics
of both uptake and depuration (Hamelink, 1976).
                                     300

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     A representative sample of test animals should be impartially distributed
 to the test containers by adding one or two test animals to each container,
 and then adding one or two more to each test container, and repeating the
 process until each test container has the desired number of animals in  it.
 Alternatively, the animals can be assigned either by total randomization or
 by stratified randomization (U.S. EPA, 1975a).

     For fish, only small fish must be used.

     •  Test Substance

     The test sutstance should be technical grade.  If a carrier or vehicle
 is used to dissolve or dilute the test substance, it should be chosen to
 possess as many of the following characteristics as possible:

     - it should not interfere with absorption, distribution, metabolism
       or retention of the test substance;
     - it should not alter the chemical properties of the test substance
       and not enhance, reduce or alter the toxic characteristics of the
       test substance;
     - it should not affect the food and water consumption of the test
       organism;
     - at the level used in the study, it should not produce physiological
       effects or have local or systemic toxicity (Anon., 1977).

     If a solvent is used, two sets of controls, one with and one without sol-
 vent, should be used.   The concentration of the toxicant  under investigation
 should be relevant to the potential use of the information for registration or
 environmental impact statement reviews.   The concentration of the toxicant
 used for a food-chain study should be selected on the basis of acute toxicity,
 recommended use rates, or information on probable concentrations likely to
 occur in aquatic ecosystems.   Acute toxicity data or LC50 (lethal concentra-
 tion) values probably represent the best information at present on which to
 base the selection of concentrations.   Select the LC50 that represents the
 least tolerant member of the food-chain  model.   Concentrations of the toxicant
 used should not exceed the LC50.   Concentrations between  l/10th and l/1000th
 of the LC50, depending on the  slope of the toxicity curve,  should be used.
However,  other nonlethal concentrations  are preferable if they can be esti-
irtated from an anticipated use  rate from a concentration projected or measured
 in aquatic ecosystems  (Johnson and Schoettger,  1975).

     •  Water Quality

     Water should be uncontaminated and  of constant quality and should meet the
 following qualifications:

     - suspended solids <20 mg per liter;
     - TOC or COD <10  mg per liter;
     - unionized ammonia <20 ug per  liter;
     - residual chlorine <3 Ug per liter;
     - total organophosphorous pesticides  <50 ng per liter;
     - total organochlorine pesticides plus PCB's <50 ng per liter.

                                     301

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     Water is considered to be constant in quality if the monthly ranges of
the hardness, alkalinity, specific conductance TOC, or COD, and salinity are
less than 10% of the respective averages and if the range of pH is less than
0.4 unit.  Alternative  freshwater should be obtained from an uncontaminated
well or spring if possible; only as a last resort should dechlorinated water
be used.  If dechlorinated water is used, it must be shown that either first
instar Daphnids can survive in it unfed for 48 hours or that residual chlorine
measured below 3 mg per liter at the beginning of the test (U.S. EPA, 1975a).

     •  Test Duration

     Test duration is determined by the time required to reach equilibrium.
For a great majority of the pesticides studied by Macek et al. (1975), equil-
ibrium was observed in  a relatively short period of time (less than 3 weeks).
However, in order to assess metabolism of the chemicals by fish and to be
confident steady-state  conditions have been reached, exposure periods ranging
from 28 to 45 days are  often employed.

     •  Size of Exposure Tank, Flow Rate and Turnover Time

     Size of exposure tank and turnover time are determined by the total weight
of the animals in each  exposure level.  Fish appear to require a minumum of
1 liter of water per gram per day (Branson et al., 1975; Macek et al., 1975;
Reinert et al., 1974).  There is a general tendency to increase the water
turnover frequency as the average weight of the fish increases.  This arises
simply because it is generally easier to increase the flow rates than tank
size (Hamelink, 1976).

     The flow rate through the test chambers should not vary by more  than 10%
from any one test chamber to any other or from one timfe to another within the
test.

     •  Sampling

     All samples should be taken in replicates of 3 to 5 and expressed as mean
values + standard error; however, the chemical nature of the toxicant may
necessitate a larger sample size.  Data should not be utilized when mortality
within the experimental group exceeds that in the control by 5% (Johnson and
Schoettger, 1975).

     •  Chemical Analysis

     Chemical analysis  used in measuring uptake and degradation requires sen-
sitivity sufficient to  detect and quantify nanogram amounts.  Radiolabeled
compounds and radiometric assays consisting of liquid scintillation spectrometry
and autoradiography or  thin-layer chromatograms are recommended.  The radio-
active material, preferably ll*C-labeled, should occupy the most stable portion
of the molecule.  Efficiency of the radiometric system should be based on
comparison with a spiked control (Johnson and Schoettger, 1975).
                                      302

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     •  Analysis of Results

     Report all samples in terms of degradability of toxicant, percentage of
degradation, and chromatographic identification of degradation products.  Ex-
press all data as the mean + standard error.

     •  Calculation

        o  Plateau method
                                                         •
     Calculate the mean and standard deviation of the concentration of chemi-
cals in the water.  A range of less than ±20% of the mean is desired.  Al-
ternatively, a time-weighted average can be determined by integration.
Divide the concentration of chemical observed in the animals by the average
concentration in the water.  These values constitute the observed bioconcen-
tration factor.  Plot the observed bioconcentration factor versus time.  If a
plateau is observed, report the bioconcentration factor (BF) at or about the
plateau region.

        o  Kinetic methods

     Plot the concentration observed in the animals versus time during ex-
posure.  Determine the slope for that initial period which can be observed to
be linear to fit the uptake equation

                    C  =  a + Kit                                   Eq. 3.3.1

where   C  =  chemical concentration in animal (mg/liter)
        a  =  y intercept
       KX  =  uptake rate (mg/g/h)
        t  =  time in hours

     Plot the concentration observed in the animal during depuration on semi-
log paper versus time.  If a straight line is apparent, determine the depura-
tion equation

                    In C  =  a - K2t                                Eq. 3.3.2

where (C, a, and t are defined as above)
       K£  =  clearance rate

     When these two rates are equal, the equilibrium concentration is

                    0  =  K! - K2Ce                                 Eq. 3.3.3

where  Ce  =  concentration of chemical in animals at equilibrium

     By solving Eq. 3.3.3 for the Ce and dividing by the average concentration
of chemical observed in the water (W),  the projected BF at equilibrium is
derived.

                    T?  '  «.
                                     303

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3.3.2.9  Metabolic Bioassay—

     Recurring pollution of natural waters from the manufacture and use of
pesticides has accentuated the need for suitable monitoring methods.  The de-
termination of fish brain acetylcholinesterase (AchE) activity has been used
for monitoring purposes.  Gibson et al. (1969) exposed fish to organophosphate
pesticides and showed  that the mortality and recovery from organophosphorus
poisoning are not necessarily related to the degree of AchE inhibition.  Test
specimens experiencing over 90% inhibition may fail to develop pronounced
symptoms or organophosphorus poisoning and recover completely when removed to
freshwater.  They found great inhibition of AchE without death and death with
little inhibition and  therefore questioned the usefulness of AchE activity in
the fish brains for monitoring.  The confusing relationship between mortality
and the degree of AchE inhibition jeopardized logical interpretation of data,
i.e., the degree of AchE inhibition is not always related to the amount of
toxicant present or to the length of exposure.  Also, the cholinesterases are
inhibited by more substances than any other group of enzymes.

     However, recent laboratory and field studies have indicated that brain
AchE inhibition in fishes is related to organophosphate insecticide poisoning.
A specific level of brain AchE inhibition was shown to be related to deaths
that occurred in a test population of sheepshead minnows (Cyprinodon variegatus)
exposed to organophosphate insecticides in water under controlled static
conditions in the laboratory (Coppage, 1972).  Similar findings were made for
AchE inhibitions in brains of cod (Gadus callarias) exposed in seawater in
the laboratory to Paraoxon, a metabolite of the organophosphate insecticide
parathion (Alsen et al., 1973).

     Also, several field studies have shown that AchE inhibition in fish
brain is correlated with water pollution or spraying with organophosphate
pesticides in both fresh and estuarine water (Williams and Sova, 1966;
Holland et al., 1967;  Mayer and Walsh, 1970; Carter, 1971; Macek, et al.,
1972).

     A field study of  three species of estuarine fishes showed that brain
AchE inhibition was correlated with mosquito control operations with the
organophosphate Malathion (Coppage and Duke, 1971).

     Several methods have been  used for the assay of cholinesterase.  Most
methods are based on the determination of the rate of disappearance of
acetylcholine or the rate of formation of acetic or butyric acid from the
hydrolysis of acetylcholine, acetyl-B-methylcholine or butyrylcholine
(Witter,  1963).

     There are two prerequisites for a satisfactory procedure:

     - The rate of the reaction measured must be proportional to the amount
       of enzyme present.   In other words,  a straight line relationship must
       exist between enzyme concentration and enzyme activity.

     - Enzyme measured under conditions of  the assay must be a cholinesterase.
       Usually this is demonstrated by showing that low concentration of the

                                     304

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       specific inhibitor eserine inhibits the hydrolysis of the substrate
       (acetylcholine).

     Coppage (1971), in the study of the characteristics of brain AchE of
sheephead minnow (Cyprinodon variegatus), has proposed a method for in vivo
inhibition, utilizing the pH stat which overcomes many of the limitations
and sources of error of other AchE assay methods.

     In this test, 5 to 10 brains of adult fishes (40-70 mm total length)
were pooled, weighed wet, homogenized in distilled water and diluted with
distilled water to the desired tissue concentration.  Acetylcholine iodide
(Ach), acetyl-B-methylcholine iodide (Mech) and butyrylcholine iodide were
used as ester substrates.

     In  vitro inhibitors were:  guthion, phorate, diazinon and eserine
sulfate.

     For enzyme assay instrument, a Sargent recording of pH stat was used.
Indicating the ability to meet prerequisites, the Figure 3.3.7 shows that the
rate of hydrolysis of acetylcholine increased linearly with increasing amount
of enzyme (brain homogenaj:e).  In addition, eserine completely inhibited
hydrolysis of Ach at 1x10 **M concentration and inhibited hydrolysis by 81.5
percent at 1x10 6M, indicating hydrolysis is primarily caused by AchE
(acetylcholine).

     The inhibition values (Table 3.3.32) indicate that the presence of
organophosphate pesticides can be detected by the pH stat brain AchE assay,
but it is obvious that in vitro inhibition is not closely related to the
toxicity of the compounds.  Guthion is approximately 30 times as toxic as
parathion but causes only about twice the inhibition.

     This poor correlation between in vitro inhibition and in vivo toxicity
can be explained by the fact that toxicity depends on in vivo AchE inhibition.
Therefore, only in vivo inhibition could be a meaningful indicator of toxicity.

     •  Coppage's Proposed Techniques

     Data from this study indicate that the following procedure is suitable
for measuring normal and in vivo inhibited brain AchE with the automated pH
stat:  pool 5 to 10 brains from fish of similar size, weigh wet, homogenize
in distilled water, and dilute with distilled water until tissue concentra-
tion is 5 mg per ml; mix 2 ml of diluted brain homogenate with 2 ml of 0.03M
acetylcholine iodide in distilled water; titrate the liberated acetic acid
with carbonate-free 0.01N NaOH; carry out the reaction at pH 7 and 22°C while
passing nitrogen over the liquid to prevent absorption of atmospheric carbon
dioxide.   Calculate the micromoles of substrate hydrolyzed per unit of time
from the number of micromoles of NaOH required to neutralize the liberated
acetic acid per unit of time, and express AchE activity as micromoles of Ach
hydrolyzed per hour per mg brain tissue.

     For interpretation of in vivo inhibition, bioassay tests of fish in  the
laboratory should be made to determine the relationship of AchE inhibition

                                     305

-------
                 60
              3
              O
              0)
              N
              O
              t-i
              J3
              O
              m
              0)
              o
              u
              •H
              S
                 50
                 40
30
20
                 10
                             10        20        30
                        Homogenate concentration
                        (mg. wet weight/vessel)
                                          40
Figure 3.3.7  Hydrolysis of acetylcholine  (15mM) by  sheepshead minnow brain
              homogenate as a  function of  homogenate concentration.
 TABLE 3.3.32  IN VITRO ORGANOPHOSPHATE PESTICIDE  INHIBITION OF  SHEEPSHEAD
                  MINNOW BRAIN AchE COMPARED TO TOXICITY
Pesticide
Guthion
Phorate
Parathion
Diazinon
Percent inhibition at
1x10 **M concentration
59.3
31.5
27.8
100.0
48-hour LD50 (yg per
liter of aquarium water)
3.5
9.0
100.0
100.0
                                    306

-------
to pesticide concentration, length of exposure and death.

     The assay method derived from studies in this work, when applied in
tests comparing in vivo brain AchE inhibition and toxicity in sheepshead
minnow, yields AchE activity measurements that correlate well with exposure
and observed toxicity.

     It is likely that a similar characterization and assay method would lead
to improved correlation between brain AchE inhibition and observed toxicity
in other fish.

     The confusing relationship between mortality and degree of in vivo AchE
inhibition reported by Gibson et al.  (1964) is not evident in this test with
the pH stat.

     •  Advantages of the method utilizing pH stat.

        - This method overcomes many  of the limitations and sources of error
          of other AchE assay methods.
        - It does not utilize buffers.
        - It is rapid and simple to operate.
        - Rate curves are obtained by continuous recording of hydrolysis;
          also, pH, temperature, and  enzyme and substrate concentration can
          be adjusted and maintained  to permit studies of kinetics and
          optimum conditions.
        - It is not subjected to errors from color interference inherent in
          spectrophotometric methods.
        - It is not necessary to use  substrates foreign to the enzyme,  and
          small errors in substrate concentration would not significantly
          alter results as would be the case where residual Ach is measured
          (Coppage, 1971).
                                     307

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Abram, F. S. H.  1973.  Apparatus for control of poison concentration in
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Alabaster, J. S., and F. S. H. Abram.  1965.  Development and use of a direct
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     accurate continuous-flow tests.  Water  Res.  6:  1597-1599.

Gregg, J. H., and A. G. Heath.  1975.  A method for intermittent chlorine
     dosing in continuous-flow toxicity tests.  Bull. Environ. Contam.
     Toxicol.  12(5):  588.

Gregory, W. W., et al.  1969.  Accumulation of parathion and DDT by  some
     algae and protozoa.  J. Protozool.  16:  69-71.

Hamelink, J.  1976.  Current bioconcentration test methods and theory.  In:
     Aquatic Toxicology and Hazard Evaluation.  Proceedings of a  Symposium
     Sponsored by the ASTM Committee E-35 on Pesticides.  F. L. Mayer  and
     J. L. Hamelink  (eds.).  Memphis, Tenn.  Oct.  25-26, 1976.

Hansen, D. J., et al.  1971.  Chronic toxicity, uptake and  retention of

                                     311

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     Aroclor  1254  in  two  estuarine  fishes.   Bull.  Environ.  Contain.  Toxicol.
     6:  113-119.

Hansen, D. J.  1974.  Effect  on  composition  of  developing estuarine animal
     communities in the laboratory.  Mar.  Sci.  18:   19-33.

Hansen, D. J., P.  R.  Parrish, and J. Forester.   1974a.  Aroclor  1016:  Tox-
     icity to and  uptake  by estuarine animals.   Environ. Res.  7:   363-373.

Hansen, D. J., et  al.  1974b.  Aroclor  1254  in  eggs  of  sheepshead  minnows.
     Effects of fertilization success and  survival of embryo and fry.  Proc.
     28th Ann. Conf.  Southeast.  Ass. Game  Fish.  Comm. (in press).

Hansen, D. J., and P. R.  Parrish.   1976.   Suitability of sheepshead minnows
     (Cyprinodon variegatus)  for life cycle  toxicity tests.  In:   Aquatic
     Toxicology and Hazard Evaluation.  Proceedings  of  a Symposium Sponsored
     by the ASTM Committee E-35  on  Pesticides.   F. L. Mayer and J.  L. Hame-
     link (eds.).  Memphis, Tenn.   Oct. 25-26,  1976.

Harris, E. K.  1959.  Confidence limits for  the LD50 using  the moving average-
     angle method.  Biometrics 15:  424.

Henderson, C., et  al.  1959.  Relative  toxicity of hydrocarbon insecticides
     to four species  of fish.  Trans. Amer.  Fish. Soc.  88:  23-32.

Herbert, D.  V. M., and J. C. Merkens.   1952.  Toxicity  of potassium cyanide
     to trout.  J. Exp. Biol. 29:   632-649.

Herbert, D.  V. M., et al.  1957.  Toxicity of synthetic detergents  to rainbow
     trout.   Water Waste  Treat.  6:  394-397.

Herbert, D.  V. M., and D. S.  Shurben.   1964.  The toxicity  of fluoride to
     rainbow trout.  Water Waste Treat.  10:  141-142.

Herbert, D.  V. M.  1965.  Pollution and Fisheries.   Ecol. Ind. Soc.  5th Symp.
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Hoffman, G.  L., and F. P. Meyer.  1974.  Parasites of freshwater fishes.
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Holland, H.  T., et al.  1967.  Use of fish-brain acetylcholinesterase to
     monitor pollution by organophosphorus pesticides.  Bull. Environ.
     Contam.  Toxicol.  2:  156-162.

Hollister, T. A., and G. Walsh.  1973.  Differential responses by marine
     phytoplankton to herbicides:  Oxygen evolution.  Bull. Environ. Contam.
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Hubschman, J. H.   1967.  Effects of copper on the crayfish.  I. Acute tox-
     icity.   Crustaceana  12:  33-42.


                                    312

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Jensen, K.  1975.  Hatching rate as bioassay.  Proposal for a standard
     technique.  Bull. Environ. Contarn. Toxicol.  14(5):  562.

Jensen, L. D., and A. R. Gaufin.  1966.  Acute and long-term effects of
     organic insecticides on two species of stonefly naiads.  J. Water Pollut.
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Johannes, R. E.  1965.  Influence of marine protozoa on nutrient generation.
     Limnol. Oceanogr.  10:  434-442.

Johnson, B. T., and R. A. Schoettger.  1975.  A biological model for esti-
     mating the uptake, transfer and degradation of xenobiotics in an aquatic
     food chain.  Fed. Regist. 40(12):  26906-26909.

Jones, J. R. E.  1938.  The relative toxicity of salts of lead, zinc and
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Jones, J. R. E.  1939.  The relation between the electrolytic solution
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Jordan, D. H. M., and R. Lloyd.  1964.  The resistance of rainbow trout
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Katz, M.  1971.  Toxicity bioassay technique using aquatic organisms.  In:
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Keil, J. E., et al.  1972.  DDT and polychlorinated biphenyl (Aroclor 1242)
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LaRoche, G. R., R. Eisler, and C. M. Tarzwell.   1970.  Bioassay procedures
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Lemke, A. E., and D. L. Mount.  1963.  Some effect of alkyl benzene sulfonate
     on the bluegill.  Trans. Amer. Fish. Soc.  92:  372-378.

Lenon, R. E.  1967.  Selected strains of fish as bioassay animals.  Progr.
     Fish-Cult.  29(3):  129-132.

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     pp. 659-666.

                                     313

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 Litchfield, J.  T.,  and  F.  Wilcoxon.   1953.   The  reliability  of graphic esti-
      mates of relative  potency from dose-percent effect  curves.   J.  Pharmacol.
      Exp. Ther. 108:  18-25.

 Lloyd,  R.  1960.  The toxicity of  zinc sulfate to rainbow  trout.  Ann. Appl.
      Biol. 48:  84-94.

 Lloyd,  R.  1961a.   The  toxicity of  mixtures  of zinc and  copper sulfates  to
      rainbow  trout.   Ann.  Appl.  Biol.  49:  535-538.

 Lloyd,  R.  196lb.   The  toxicity of  ammonia to  rainbow trout.   Water  Waste
      Treat.   8:   278-279.

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      several  sewage  effluents  to rainbow  trout.  J. Inst.  Sew.  Purif., Pt. 2:
      167-173.

 Lowe, J.  I.   1964.   Chronic  exposure  of spot to sublethal  concentrations of
      toxaphene  in seawater.  Trans. Amer. Fish. Soc.  92:  396-399.

 Macek,  K. J., et al   1972.   Toxicity  of the  insecticide  dursban to fish and
      aquatic  invertebrates  in  ponds.   Trans. Amer.  Fish. Soc.  101:   420-427.

 Macek,  K. J., et al.  1975.  Structure-activity correlations in studies of
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      Canada N9A GT3-  Chapter 7,  March  1975,  pp.  119-142.

Marking, L. L., and V. K. Dawson.  1973.  Toxicity  of qulnaldine sulfate to
      fish.  Invest. Fish. Control No.  48.  U.S. Fish  Wildl. Serv., Washington,
      D.C.

Martin, D. M.   1973.  Freshwater Laboratory Bioassays.  A Tool  in Environ-  •
     mental Decisions No. 3.  Contributions from the  Department of Limnology,
     Academy of Natural Sciences of Philadelphia, Pa.   p. 26.

Marvan, P.  1963.   Zur Methodik der Auswertung von Toxizltatstesten.   Sbornik
     Vysoke'skoly Chemlcko-technologlcke.   V. Praze.  Technologic Vody 7(1):
      271-313.

Mayer, F. L.,  and D.  F.  Walsh.    1970.  Multiple exposures of bluegill and
      aquatic invertebrate to abate.  U.S.  Bur. Sport Fish. Wildl. Res. Publ.
      106:  16.

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McKee, J. E., and H. W. Wolf  (eda.).  1963.  Water Quality Criteria,  2nd
     Edition, 548 pp. State Water Quality Control Board, Pub. 3-A, Sacra-
     mento, Calif.

McLachlan, J.  1960.  The Culture of Dunaliella Tertiolecta Butcher.  A
     euryhaline organism.  Can. J. Microbiol.  6:  367-379.

Merkens, J. C.  1958.  Studies on the toxicity of chlorine and chloramine to
     the rainbow trout.  Water Waste Treat.  7:  150-151.

Metcalf, R.,  et al.   1975.  Laboratory model ecosystem studies of the
     degradation and fate of radiolabeled tri-, tetra- and pentachlorobiphenyl
     compared with DDE.  Arch. Environ. Contam. Toxicol. 3:  151-165.

Mount, D. I., and W. A. Brungs.  1967.  A simplified dosing apparatus for
     fish toxicological studies.  Water Res.  1(1):  21-29.

Mount, D. I.  1968.  Test animals for water quality.   Amer. Fish. Soc.
     Newsletter  12(54):  3-6.
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Nehrlng, D.   1962.  Untersuchunger uber fjle toxikologische Wirkung von
     Thalliumionen auf Fische und Fischnahrtiere.  Z. Fisch 11:   557-561.

Nehrlng, R. B.  1976.  Aquatic insects as biological  monitors of heavy metal
     pollution.   Bull. Environ. Contam. Toxicol.   15(2):  147-154.

Ozburn, G. W., and D. Smith Alasdalr.  1976.  A mechanical toxicant injector
     for flow-through toxicity tests.  Lakehead University, Thunder Bay,
     Ontario, Canada.

Patrick, R.  1964.  Tentative method of test for evaluating inhibitory
     toxicity of industrial waste waters.  American Society for Testing and
     Materials.   ASTM Designation D.2037.

Payne, A. G.  1975.  Response of the three test algae of the algal assay
     procedure:   bottle test.  Water Res.  9:  437-445.

Pickering, Q. H.,  et al.  1962.  The toxicity of organic phosphorus insecti-
     cides to different species of warm water fishes.  Trans. Amer. Fish.
     Soc. 19:  175-184.

Pickering, Q. H.,  and W. N. Vigor.   1965.  The acute  toxicity of zinc to
     eggs and fry  of the fathead minnow.   Progr.  Fish-Cult.  27:  153-157.

Pickering, Q. H.  1966.  Acute toxicity of alkyl benzene sulfonate and linear
     alkylate sulfonate to the eggs of the fathead minnow.  Int. J. Air Water
     Pollut.  10:   385-391.

Pickering, Q. H.,  and C. Henderson.   1966a.  The acute toxicity of some heavy
     metals to different species of warm water fishes.  Int. J. Air Water
     Pollut.  10:   453-463.
                                     315

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Pickering, Q. H., and C. Henderson.  1966b.  Acute toxicity of some important
     petrochemicals to fish.  J. Water Pollut. Contr. Fed. 38:  1419.

Pickering, Q. H., and C. Henderson.  1966c.  The acute toxicity of some
     pesticides  to fish.  Ohio J. Sci.  66:  508-513.

Provasoli, L.  1963.  Organic regulation of phytoplankton fertility.  In:  The
     Sea, Vol. II.  M. N. Hill, L. Lasko, C. Shaw, and E. Wirhowski (eds.).
     John Wiley  and Sons, New York, N.Y.  pp.  165-219.

Rand, M. C., A.  E. Greenberg, and M. J. Taras.  1975.  Standard Methods for
     the Examination of Water and Wastewater.  14th Edition.  American
     Public Health Association, American Water Works Association, and Water
     Pollution Control Federation, Washington, D.C.

Rehwoldt, R.,  1973.  The acute toxicity of some heavy metal ions toward
     benthic organisms.  Bull. Environ. Contain. Toxicol.  10(5):  291.

Reichenbach-Klinke, H., and E. Elkan.  1972.  The principal diseases of lower
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Reinert, R. E.,  et al.  1974.  Proceedings 17th Conference Great Lakes
     Research, published by the International Association for Great Lakes
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Roberts, D.  1975.  The effects of pesticides on Byssus  formation in the
     common mussel,  Mytilus Edulis.  Environ. Pollut.   8:  241.

Sanders, H. 0.   1970.  Toxicities of some herbicides to  six species of
     freshwater  crustaceans.  J. Water Pollut. Contr. Fed.  42(8):  1544.

Scheier, A.  1973.  Personal communication.  Limnology Department, Academy of
     Natural Sciences of Philadelphia.  Philadelphia, Pa.

Schimmel, S. C., et al.  1974.  Effects of Aroclor 1254  on laboratory-reared
     embryo and  fry of sheepshead minnow.  Trans. Amer.  Fish. Soc.  103:
     582-586.

Schimmel, S. C., and D. J. Hansen.  1974.  Sheepshead minnow (Cyprinodon
     variegatus):  An estuarine fish suitable for chronic (entire life-
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     Comm.

Shepard, M. P.   1955.  Resistance and tolerance of young speckled trout
     (Salvelinus Fontinalis) to oxygen lack, with special reference to low
     oxygen acclimation.  J. Fish. Res. Board Can.  12:  387-446.

Shumway, D. L.,  and J. R. Palensky.  1973.  Impairment of the flavor of fish
     by water pollutants.  U.S. Environmental Protection Agency, EPA-R--73-
     010, Washington, D.C.

Skidmore, J. F.  1965.  Resistance to zinc sulfate of the zebrafish at

                                     316

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     p. 526.

 Specht, D. T., and C. Miller.  1973.  Development of a  Standard Marine Algal
     Assay Procedure for Nutrient Assessment.   In:  Proceedings of  Seminar
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 Sprague, J. B.,  and B. A.  Ramsay.  1965.  Lethal levels of mixed copper-zinc
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 Sprague, J. B. 1969.  Measurement of pollutant  toxicity to fish.  I.  Bioassay
     methods for acute toxicity.  Water Res. 3:  793-821.

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     Sublethal effects and'safe concentration.  Water Res.  5:  245-266.

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     Toxicology  and Hazard Evaluation.  Proceedings of a Symposium  sponsored
     by the ASTM Committee E-35 on Pesticides.  F. L. Mayer and J.  L. Hame-
     link  (eds.).  Memphis, Tenn.  Oct. 25-26, 1976.

 Sturm, R. N., and A. G. Payne.  1973.  Environmental testing of trisodium
     nitrilotriacetate (NTA).  Bioassays for aquatic safety and algae stimu-
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     Glass (ed.).  Ann Arbor Sci.  Publ., Inc., Ann Arbor, Mich.  pp.  403-424.

 Tattersfield, F., and H. M. Morris.  1924.  An apparatus for testing  the toxic
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     Entomol. Res. 14:  223-233.

 Thatcher, T. 0.   1966.  The comparative lethal toxicity of a mixture  of hard
     ABS detergent products to 11 species of fishes.  Int.  J. Air Water
     Pollut.  10:  585-590.

Thatcher,  T. 0.,  and J.  F.  Santner.   1966.  Acute toxicity of LAS to various
     fish species.  Proc.   21st Ind.  Waste Conf. Purdue Univ. Engin.  Exten.
     Ser.  No. 121:  996-1002.

 Thoman, R., et al.  1974.   A food chain model of cadmium in Western Lake
     Erie.  Water Res.  8:   841-849.

Trevan, J. W.  1927.  The  error of determination of toxicity.  Proc.  Roy.
     Soc.  B. 101:  483-514.

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Trudgill, P. W., et al.  1971.  Effect of organochlorine insecticides on
     bacterial growth, respiration and viability.  J. Gen. Microbiol.  69:
     1-3.

U.S. EPA.  1973.  Biological Field and Laboratory Methods for Measuring the
     Quality of Surface Waters and Effluents.  C. I. Weber (ed.).  U.S. Environ-
     mental Protection Agency, EPA-670/4-73-001, Cincinnati, Ohio.

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     Environmental Protection Agency, EPA-625/6-74-003, Cincinnati, Ohio.

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     mental Protection Agency, PB-239709/9BA, Corvallis, Ore.

U.S. EPA.  1975a.  Methods for Acute Toxicity Tests with Fish, Macroinverte-
     brates and Amphibians.  U.S. Environmental Protection Agency, EPA-660/3-
     75-009, Corvallis, Ore-

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     Park, N.C.

Van Duijn, C., Jr.  1973.  Diseases of Fishes.   3rd Ed.  Charles C. Thomas,
     Publ.  Springfield, 111.  p. 309.

Vigers, G. A., and A. W. Maynard.  1977.  The residual oxygen bioassay.  A
     rapid procedure to predict effluent toxicity to rainbow trout.  Water
     Res. 11:  343-346.

Walker, G., et al.,  1975.  Barnacles:  Possible indicators of zinc pollution.
     Mar. Biol.  30:  57; Selected Water. Res. Abs. W75-11611.

Warnick, S. L., and H. L. Bell.  1969.  The acute toxicity of some heavy metals
     to different species of aquatic insects.  J. Water Pollut. Contr. Fed.
     41(2):  280.

Weil, C. S.  1952.  Tables for convenient calculation of median effective
     dose (LD50 or EC50) and instruction in their use.  Biometrics 8:  249-
     263.

Williams, A. K., and R. C. Sova.  1966.  Acetylcholinesterase levels in
     brains of fishes from polluted waters.  Bull. Environ. Contarn. Toxicol.
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     Health    6:  537.
                                      318

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Wuhrmann, K.  1952.  Sur quelques principles de la toxicologie du poisson
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     suitable for oyster larvae development.  Amer. Zool.  9:  1141.

Zillioux, E. J., et al.  1973.  Using artemia to assay oil dispersant
     toxicities.  J. Water Pollut. Contr.  Fed.  45:  2389-2396.
                                     319

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3.4 MICROBIOLOGIC ASSAY

     The elements of a program to insure validity and integrity of experi-
mental results in microbiologic work are essentially the same as those for
other areas of biology:  personnel, supervision, sampling, procurement and
acceptance specifications, instrument checks and calibration, experimental
design, standard test methods, controls (positive and negative), statisti-
cal analysis of data, and proficiency testing.

     Competent, dedicated, industrious personnel are essential to the success
of any program.  Although there is no substitute for competence, continuing
education, workshops, and on-the-job training can do much to raise the level
of performance (Russell et al., 1969; Prier, 1973; Bartlett et al., 1968;
Lott, 1973).  The dedicated worker can be counted on for the extra time and
effort that oft-times spells the difference between success and failure in a
project.  Indolent employees, on the other hand, may resort to short cuts and
improvisions that can lead to erroneous results.  Automated systems, where
applicable, can eliminate, to a large degree, human errors due to such
factors as eye fatigue.  Automation has been a great boon in analytical
chemistry and hematology.  However, much remains to be accomplished in this
area of microbiology technology (Heden and Illeni, 1974; Kuzel and Kavanagh,
1971; Kavanagh, 1974; Rippere and Arret, 1972).

     Supervision must be professional and thorough.  This important aspect
of the program cannot be delegated to technical personnel or relegated to the
status of a casual walk-through inspection from time to time by a busy admin-
istrator with many other duties constantly demanding attention.  The proper
supervision of a successful quality control program for a large laboratory is
a major administrative task.  The supervisor must make certain that all
elements of the program are in operation at all times.  A perfunctory exam-
ination of logbooks and test results will not insure this; the supervisor
must be in the operation.

     The other elements of the quality control program vary according to the
nature of the project and will be discussed specifically in the following
sections.

3.4.1  Microorganisms - Diagnostic Environmental Microbiology

     Quality control in microbiology received a great impetus with the pas-
sage of the Federal Clinical Laboratories Improvement Act (CLIA) of 1967 which
established minimum standards for clinical laboratories engaged in interstate
commerce in the U.S.A. (PHS, 1968).  A wealth of information and experience
is now available for organizations launching programs in this area (Russell
et al., 1969; Prier et al., 1973; Vera, 1971; Halstead et al., 1971; Glasser
et al., 1971).

3.4.1.1  Sampling—

     Environmental samples for the isolation and identification of patho-
genic microorganisms must be  representative, of sufficient size, and
properly preserved so that viability of the agents isolated is preserved.

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     Water sampling methods for sanitary bacteriology have been developed
and refined over a period of many years and are described in detail in Stan-
dard Methods for Examination of Water and Wastewater, 14th Edition, 1975
(Rand et al., 1975).  Samples for judging water quality according to the 1975
USEPA Drinking Water Standards should  be collected in sterile bottles that
have been properly cleaned and rinsed with distilled water.  A dechlorina-
ting agent should be added unless the sample is collected in broth for direct
plating.  Sodium thiosulfate is usually added for dechlorination prior to
sterilization in an amount sufficient to'yield a final concentration of
100 mg/1 of sample.  Water samples high in copper or zinc or wastewater
samples high in heavy metals should be collected in bottles which also con-
tain a chelating agent such as ethylenediaminetetraacetic acid in an amount
to give a final concentration of 372 mg/1.  Individual samples should be
taken at representative stations over the complete distribution system.  The
minimum number of samples to be collected each month is determined by the
size of the population dependent upon the supply.  Distribution system taps
should be opened for 2 to 3 minutes, or long enough to empty the service line,
before collecting the sample.

     Other samples for bacteriologic examination of water should be repre-
sentative and collected in a manner that precludes contamination.   Well water
should be hand-pumped for about 5 minutes before collecting the sample.  The
critical factor in collecting samples from a stream, lake, reservoir, spring,
or shallow well is that the sample be representative of the body of water
sampled.  Samples from a stream may be taken at one-quarter, one-half,  and
three-quarters the width at various sites.  The sample bottle should be held
near the base and plunged neck downward to the desired depth and then turned
slightly upward with mouth toward the current.   Flow patterns and other hydro-
logic factors in streams as well as the tendency of motile organisms to
gather where light, temperature, oxygen, nutrients, and/or flow are favorable,
present difficulties in collecting a representative sample.   The use of a
standard Kemmerer Sampler for collecting multiple discrete samples at various
depths and the continuous automatic-type sampler for collecting samples pro-
portional to the flow pattern of the stream should merit consideration
(Bicking, 1976).  Samples collected from a boat should always be taken from
the upstream side of the craft.   Samples from moderate depths may be taken
by attaching a weight to the base of the sample bottle.  Deep sampling
devices such as the ZoBell J-Z Sampler may be used for collecting samples
at various depths from a lake or reservoir.  The device consists of a 350-ml
bottle with glass and rubber tubing equipped with a cable and a messenger.
The messenger is released when the bottle is at the desired depth and breaks
the glass tubing at a point weakened by a file mark and the sample is sucked
in under a partial vacuum created at the time of assembly.  Although impounded
waters do not present as many hydrologic problems with respect to sampling,
stratification and other factors make multiple sampling imperative to be truly
representative.  Bottom sediment samples may be collected with a Von Donsul
and Geldreich sampler consisting of a stainless steel frame and a sterile
plastic bag equipped with a nylon cord which closes the bag when the sampler
penetrates the sediment.

     Water samples should be tested as soon as possible after collection to
insure valid  results.  Samples that cannot be analyzed within one hour after

                                     321

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collection should be refrigerated at a temperature below 10°C.  The maximum
time between sampling and transportation of refrigerated samples to the lab-
oratory should be no longer than 6 hours.  Samples handled in this manner
should be refrigerated on receipt at the laboratory and processed within 2
hours.

     The major groups of pathogenic microorganisms that may be present in
surface and groundwaters in the U.S.A. are Salmonella. Shigella, pathogenic
Escherichia coli, Leptospira. and enteric viruses.  Vibrio cholerae should
also be considered in view of present-day widespread world travel.  Standard
methods of sampling for these groups of microorganisms in water have not been
developed at this time.  In general, however, some method of concentrating
the sample must be employed since these organisms are present in much smaller
numbers than the coliforms which are the index of pollution in sanitary
bacteriology.  Three techniques are recommended in Standard Methods (Rand
et al.t 1975).

          •  Swabs are prepared from a 216-cm length of 23-cm wide cheese-
     cloth folded five times at 36-cm intervals.  This provides a rectangle
     23 cm wide on the folds by 36 cm long on the open edges, and six layers
     thick.  Cut this lengthwise to within 10 cm of the head into 4.5-cm
     wide strips or streamers (four cuts making five streamers).  Tightly
     wrap the uncut end with 16-gauge wire.  For sampling, the swab is placed
     slightly below the surface of the stream for 3 to 5 days and traps
     microorganisms and other particulates.  Water expressed from the swab,
     and pieces of the swab itself, are placed in enrichment media for
     analysis.  Gauze pads of the same thickness may be substituted for the
     cheesecloth swabs.

          •  Diatomaceous earth ("Cellite", etc.) packed over an absorbent
     pad in a membrane filter holder may be used for concentrating micro-
     organisms.  At least two liters of sample should be drawn through the
     filter mass by vacuum.  Representative samples of the filter "plug" are
     then sampled for analysis.

          •  Commercial membrane filters, 0.45-ym pore diameter, are satis-
     factory for concentrating pathogenic microorganisms in samples with low
     turbidity.  Several liters of sample should be used.

     Human enteric viruses excreted with the feces into domestic sewage con-
stitute a special problem in water management.  Viral particles in the center
of clumps, covered by debris, or otherwise protected, may escape inactivation
and eventually find their way to fully virulent form into a community water
supply.  Although there are only six viruses known to be shed in large
numbers from the human intestinal tract - poliovirus, echovirus, coxsackie
virus, reovirus, adenovirus, and infectious hepatitus virus - each occurs in
varying numbers of different antigenic types so that today well over 100
different human enteric viral serotypes are recognized.  Outbreaks of water-
borne viral disease continue to be reported both here and abroad and there is
considerable concern about larger and more serious outbreaks in the future
(Craun et al., 1976).  Fortunately, viruses are unable to multiply outside
living cells and, unlike bacteria, do not increase in numbers in the water

                                     322

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supply.  This creates a special problem  in water  virology,  however, since
large volumes of sample (400-1900 liters) must be processed through filters
or adsorbents to insure isolation of sufficient infectious  units for viral
identification.  Great progress has been made in  this  area  during the past
decade, largely through the efforts of Metcalf  (1961),  Oliver (1967), BergetaL
(1971), Jakubowski et al.  (1974), Hill et al. (1976),  Wallis and Melnick
(1967), and Wallis et al.  (1972).  A tentative microporus filter technique
for enteric virus concentration in finished waters has  been included in the
latest (14th) edition of Standard Methods (Figure 3.4.1.).
         PRESSURE
         RELIEF
         VALVE
FLOW
METER
       FLUID
       PROPORTIONER

PRESSURE /
GAUGE  /    MIXING
       //S3\  O AMBER
                       CHEMICAL
                       ADDITIVE
                       CONTAINERS
                        HOSE
                        OUTLET
    Figure 3.4.1.   Diagrammatic view of the virus-concentrator apparatus.
     Ancillary component parts are shown mounted on a two-wheeled  dolly
     constructed of angle iron.  Note:  Use stainless steel fittings  for
                    all connections.  (Rand et al., 1975)

     The main features of the virus-concentrator apparatus are:

          •  Virus adsorbent

             8-ym + 1.2-um stack of 293-mm cellulose nitrate membrane filters
                                     or
             5-um + 1-pm stack of 267-mm epoxy-fiberglass-asbestos discs
                                     or
             3 epoxy-fiberglass tubes, 8 ym, 24.5 x 63.5 mm in parallel

          •  Sample treatment (continuous)

             pH 3.5

             Sodium thiosulfate (1:100 final cone.)
                                     323

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          •  Sample size

             400 to 2000 liters (to detect 1 to 2 infectious units/400 1)

          •  Flow rate

             4 to 10 liters per minute

          •  Elution

             0.05M glycine buffer, pH 11.5

          •  Reconstitution

             Adjust pH to 3.5.  Add A1C13 to final cone. 0.0005M.  Filter
             through stack of 47-mm AA3Cox M-780 fiberglass filters 5 urn
             and 1 urn.  Elute with glycine buffer, pH 11.5, into buffered
             Hanks balanced salt solution with nutrient broth or 20% fetal
             calf serum, adjust to pH 7.4.

     Wallis et al. (1972) have developed a portable virus concentrator for
isolating viruses from highly turbid tapwater.  Yarn-wound clarifying filters
are used in conjunction with a 293-mm size membrane filter or fiberglass
textile filter.  A commercial unit that concentrates viruses from water and
elutes them as well is now available also (Rand et al., 1975).

     Since subclinical enteric viral infections are quite common during the
summer months the following quality control practices have been advocated by
Akin and Jakubowski (1976) to safeguard against false positive results in
water analysis.

          •  Personnel directly involved in sample collecting and handling
             should routinely have throat and rectal swabs collected.  They
             should be processed if a virus-positive water sample is found

          •  Aseptic technique and a closed system should be used for sample
             collecting and processing

          •  When samples are to be stored prior to testing, they should
             be placed in ultralow temperature freezers that contain no
             other type of virus sample

          •  Samples should be processed in isolation facilities where no
             other type of virus sample is handled

          •  Multiple barriers to air contamination should exist, i.e.,
             separate isolation facility, laminar flow hoods, etc.

          •  All isolates must be confirmed as being viral

     A new instrument for large-volume sampling of water supplies for micro-
organisms was announced recently by the  'Bacterial and Parasitic Diseases

                                    324

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Section, Epidemiology Branch, Field Studies Division, Health 'Effects Research
Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio.  The
portable instrument is adaptable for collection of bacteria, viruses, or
Giardia cysts.

     A flow diagram illustrating instrument operation for collection of
viruses is given in Figure 3.4.2.  Slight modifications in flow are necessary
for collecting bacteria or Giardia cysts.
                                 Water cap
                                     i
                 gas or electrical-powered pump (optional)
                                     I
                        orlon prefilter (optional)
                                      1
 hydrochloric acid   	    proportioner motor    	sodium thiosulfate
    cubitainer          *             I           *         cubitainer
1
                              mixing chamber
                                     1
                          virus-adsorbing filter
                                     I
                                water meter
                                     i
                                 to waste


    Figure 3.4.2  Equipment configuration for virus sample collection.


     Bacteria are collected on five Balston 0.3-um filters, using one filter
for each 75 liters of water.  Giardia cysts are collected on a 25 cm long
yarn-wound orlon pre-filter over a 24-hour period from a domestic water
supply at the maximum flow rate obtainable.  The flow rate is reduced to 4
liters per minute if the water is turbid.  Viruses are concentrated in
essentially the same manner as described in Standard Methods for the Exami-
nation of Water and Wastewater, 14th Edition (Rand et al., 1975).

                                     325

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     The atmosphere contains all main groups of microorganisms including
viruses.  Mold spores are particularly prevalent and include species patho-
genic for man, domestic animals, and plants.  Coccidioidomycosis, histoplas-
mosis, cryptococcosis, aspergillosis,  blastomycosis, and nocardiosis are
typical human pulmonary diseases caused by pathogenic fungi found in the air.
Coccidioidomycosis spores are so prevalent in the air in the San Joaquin
Valley in California that the disease there is referred to as San Joaquin
Valley Fever.  Viruses have not been found in significant numbers in the out-
door air although there is some evidence for the airborne spread of both
smallpox and foot-and-mouth disease  (Davies, 1971; Jacobson and Morris, 1976).
Soil and bodies of water are the main sources of bacteria in the outdoor air
but sewage treatment plants, rendering plants, and facilities where solid
waste is shredded for incineration may also give rise to airborne micro-
organisms.  For example, significant increases in the concentration of
Escherichia coli in the air at distances up to 800 meters (half a mile) from
trickling filter sewage treatment plants, in contrast with the level in the
upwind control air, have been found.  Spray irrigation of land with chlori-
nated sewage effluent also produces aerosols which may be carried long
distances.

     Air sampling for microbiologic analysis is usually conducted with
impactors, impingers, or membrane filters.  Impactors are devices in which
the airstream is directed onto sticky surfaces such as petri dishes with an
agar medium or coated plates or slides where the microbes are trapped
(impacted).  Impingers trap airborne microorganisms as they are blown or
sucked into a nutrient liquid or buffer solution.  Membrane or alginate fil-
ters will filter out and concentrate microorganisms in the airstream in pro-
portion to the pore size (Davies, 1971; Jacobson and Morris, 1976; Giever,
1976).

     The two main types of impactors used in recent years for air sampling
are the Slit Sampler and the Cascade Impactor.  The Slit Sampler (Figure
3.4.3) is a device in which air is sucked or blown through a narrow slit
orifice onto an agar medium in a rotating petri dish where the microorgan-
isms are impacted.  Particulates from a 3 cubic meter air sample are impacted
onto each agar dish when the instrument is operated at a flow rate of 50
liters per minute for one hour (Goddard, 1976).  The Cascade Impactor
(Figure 3.4.4) is an instrument with a series of air jets of decreasing size
in series to achieve a  gradation in size of particles passing or being im-
pacted from one stage to another.  Impactors such as Petri dishes with an
agar nutrient medium are positioned beneath each air jet.  The Andersen
Cascade Sampler (Figure 3.4.5) is regarded by some authorities as the best
device today.for air sampling of bacteria.  With this instrument, air is
drawn or blown through a circular opening and then through a series of six
circular plates with 400 holes each onto the surface of agar media in under-
lying Petri dishes where the entrained particles are impacted.  The plates
have progressively smaller holes so that the largest particles are impacted
on the first dish and the smallest at the sixth stage.  Air is sampled at
the rate of 28.'3 liters per minute and retention is reported to be as great
as 100% for single bacteria cells, although there is some loss on walls and
plates.  Impactors made up of seven or more units followed by a filter are
also available (Figure 3.4.6).  Particulates as small as 0.24 urn can be

                                     326

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                    I!
1
Figure 3.4.3   The  Casella slit sampler  (Davies, 1971)
                          327

-------
                    Intake •
Figure  3.4.4  Sectional elevation of the Cascade Impactor   (Davies, 1971)
                        Siooei
StogeS
                             ]  *!•• i • I I I 1 1 I ft.._^«ji •!_•«* •* , n*
                             I  i-      •    --1 — In
                                                ,  Gasket
                                                i   8T
   Figure  3.4.5  Sectional elevation, Andersen  Sampler  (Davies,  1971)
                                     328

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      Figure 3.4.6  Inertial in-stack cascade impactor (Courtesy of
                Meteorology Research Inc.)  (Giever, 1976)


collected in the last stage of typical modern commercial cascade impactors.

     Impingers have several important advantages over impactors in isolating
microorganisms from air:

          •  Aliquots of the liquid sample can be subcultured in a variety
             of enrichment broths and selective media which increases the
             possibility of isolating all of the various types of microbes
             trapped

          •  Loss of delicate, fastidious, and slow-growing species which
             often occur on crowded Petri dish cultures is avoided

          •  Viruses can easily be separated from bacteria and other types
             of microorganisms for isolation and identification

The Porton impinger (Figure 3.4.7), although simple in design and operation,
is regarded as highly efficient for collecting single bacterial cells as
small as 0.5 tolum in diameter.  A slow flow rate (11 liters per minute)
and foaming are its main deficiencies.   The Multi-stage Liquid Impinger
(Figure 3.4.8), consisting of three chambers separated by sintered glass
discs of graded pore size has obvious advantages over the single-cell im-
pingers.  Discs and walls are continuously wetted by the collecting medium
and foaming is reduced to a minimum even at high flow rates.  This instrument

                                    329

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       Figure 3.4.7  Porton impinger and pre-impinger   (Davies, 1971)
is rated as 80-90% efficient for capture of single cells of Bacillus subtilis
or Escherichia coli at an airflow rate of 55 liters per minute (Davies,
1971).  A Multi-Slit Large-Volume Air Sampler (Figure 3.4.9) has been de-
veloped recently in which air is drawn at a rate of 500 liters per minute
through eight radial slits.  Particulates are impinged onto a film of culture
medium which flows continuously over a rotating disc into an effluent con-
tainer.  The concentrating factor is reported to be as great as 100,000.  The
instrument has a high rate of efficiency and samples very large volumes of
air in a short time.                        •
                                    330

-------
                   17-
                               -•  4
  Figure 3.4.8  Sectional elevations, multi-stage liquid impinger,  at right
   angles to each other in the directions I-II and II-II   (Davies, 1971)

   (1, 2, 3 - chambers or stages;  4 - air inlet tube;   5 - connection for
   airflow from stage 1 to stage 2;  6 - connection for airflow from stage
   2 to stage 3;  7 - nozzle;  8 - annular well;  9, 10 - sintered  glass
   discs held by curved glass rods 11 and 12;   13, 14,  15 - rubber  bungs
   in access holes to chambers;  16 - connector for suction containing
   critical orifice;  17 - hemicylindrical metal shield)
     Standard soil sampling methods for microbiological monitoring are not
highly developed.  Representative samples from agricultural soils may be
collected by the method described by Bicking (1976)  in which corings are
taken to a depth of at least 10 centimeters from the center of random one-
square-meter plots.  The sampling device recommended is a 10 cm "bogey" hole
cutter used on golf courses.  The exact diameter of  the core must be recorded
so that the total surface area sampled can be calculated.  It is recommended
that ten to 20 cores representing at least 200 square centimeters be collect-
ed from each sampling area.  If the soil is covered  with grass or a cover
crop, the sward should be cut and removed prior to sampling.  Samples that
cannot be analyzed immediately must be refrigerated  in closed containers to
prevent drying.  Topographical features should be taken into consideration in
                                     331

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                       .tfl ,1 ^> Air flow
                      —* —* —> Liquid flow
                      "      "" Electricity
 Figure 3.4.9  Diagramatic section, Litton  LVS/10K air  sampler (Davies,  1971)

  (1 - airflow ports;  2 - corona  needles;   3  -  inlet  duct;   4 -  liquid in-
  put tube;  5 - high voltage plate;   6  -  collection plate;   7 -  multi-jaw
  coupling;  8 - high voltage power supply;  9 - blower;   10 - pumps;   11  -
  return reservoir;  12 - blower motor;  13  -  fluid reservoir)
selecting sampling sites in areas subjected  to  spray  irrigation  of  chlorina-
ted sewage effluent or treated with sludge as well as  in areas in the vicinity
of sewage disposal plants, solid waste disposal facilities,  rendering plants,
and landfill areas.

     Raw agricultural commodities grown in areas where the possibility  of
contamination with human enteric pathogens exists should be  monitored by
appropriate microbiologic procedures.  Surface  samples from  crops such  as
potatoes, carrots, radishes, tomatoes, egg plant, etc., should be collected;
internal as well as external samples are necessary in  the case of lettuce,
broccoli, cauliflower, celery, etc.  The sample should be representative of
the entire crop area.  After compositing, representative aliquots should be
preserved for the various tests to be performed.
                                     332

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 3.4.1.2  Procurement and Acceptance Specifications—

     Each item of equipment purchased should be tested in the laboratory
 under working conditions as soon as received to make certain that it meets
 the manufacturer's claims and the laboratory's specifications based on the
 work to be performed.  Incubators, water baths, autoclaves, hot air ovens,
 deionization equipment, safety cabinets, etc., should be thoroughly checked
 for consistent performance before routine use (Ellis, 1976).

     All special reagents such as stains, media supplements, diagnostic
 biologicals, buffers and other chemicals, etc., should meet or exceed CDC's
 current standards (CDC, 1969).  The necessity for continuing reagent testing
 by the user laboratory was brought to light by a recent survey in which 14%
 to 27% of microbiologic reagents examined over a 2-year period gave unsatis-
 factory results.  All new lots of reagents should be tested in parallel with
 reference quality control preparations, if available (CDC), as well as with
 a satisfactory lot in current use, with both positive and negative cultures.
 The proposed 4th edition of CDC's Recommended Specifications for Micro-
 biological Reagents  covers over 2000 different products (Suggs, 1973).
 Each reagent lot should be dated and stored at all times in accordance with
 the manufacturer's recommendations.  Care should be exercised to avoid con-
 tamination.  Performance testing should be repeated each time a new batch of
 reagents is prepared.  Many laboratories go much beyond this.  Gram staining
 solutions, for example, are tested at the beginning of each day with at
 least one gram-positive and one gram-negative organism as a control on per-
 formance.  The Quality Control Supervisor should make certain that unsatis-
 factory substitutions are not made for specific brand products stipulated in
 test procedures.

     Most laboratories today use commercially-prepared dehydrated or "ready
 to use"•culture media for microbiologic work (Power, 1973).   In spite of the
 fact that most lots are subjected to quality control tests by the manufactur-
 er before release, contamination and unsatisfactory performance are still
 reported  (Russell et al., 1969;  Halstead et al., 1971).  In one survey, of
media collected from eight different laboratories, 46% of the chocolate agar
 plates failed to support the growth of Hemophilus influenzae or Hemophilus
 parainfluenzae (Barry and Feeny, 1967).   Another laboratory discarded 5% of
 all media lots purchased over a period of time because of contamination,
 poor performance, etc.   In another survey, however, 900 lots of 46 different
media from two commercial suppliers tested over an 8-month period by a uni-
versity medical school and a general hospital, revealed only four lots as
 unsatisfactory on the basis of performance,  contamination, or physical
 properties (Power, 1973).   Nevertheless, each new lot of media should be
 sterility-tested and tested for performance upon receipt and each time a new
batch is prepared (Blazevic et al.,  1976).

     Quality control supervisors should make certain that performance fail-
ures with dehydrated commercial media are not due to error in final pre-
paration or to improper storage.   Errors in weighing, amount of water, pH
measurement,  and in addition of supplements, incomplete mixing, overheating
during sterilization, and  use of unclean glassware (residue of chemicals  or
detergent) must be avoided.   Water should meet U.S. Pharmacopeia XIX require-

                                     333

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ments (U.S. Pharmacopeia, 1976).  Aluminum foil for capping glassware for
sterilization should be oil-free.  The use of soft glass Pasteur-type dis-
posable pipettes which release alkali and organic contaminants may also be
responsible for errors in certain types of performance tests (Ellis, 1976).
Loss of moisture from media during storage can cause performance failures.
For example, it was found that unwrapped, poured petri dishes lost 7%
moisture per week at 4°C, cellophane-wrapped dishes lost 2% per week, while
those wrapped in polystyrene and stored in polystyrene containers lost only
0.5% per week under the same conditions (Power, 1973).

     Performance tests on culture media and reagents must be conducted with
standard control cultures with documented satisfactory performance by a
reliable source.  Such cultures are available from the American Type Culture
Collection, Rockville, Maryland.  Laboratories with the proper equipment and
trained personnel may prepare a large number of lyophilized stocks from the
official control cultures which, if properly stored, will retain viability
with unchanged characteristics over a long period of time (Morton, 1973).
A list of control cultures for use in performance tests on a variety of
standard bacteriologic media and reagents together with a description of the
correct reaction in each case has been compiled by Bartlett (1973) and is
presented, with permission, in Appendix C.  It is recommended that each batch
of culture media, yeast extract, and peptone be checked by gas-liquid chroma-
tography for the presence of fatty acids and that those containing excessive
amounts be discarded.  Standard reference toxins, antitoxins, antigens,
antisera, and other diagnostic biologicals may be purchased from one of the
reliable manufacturers listed in Section IX of the CDC Quality Control
Manual for Microbiological Laboratories (Ellis, 1976).  Procedures and
reagents for quality control work in the highly specialized area of fluores-
cent   antibody techniques are given in CDC manuals by Cherry et al. (1960)
and Hebert  et al. (1972).

     The essentials of performance tests with equipment, culture media,
reagents, and other supplies in the areas of mycology, virology, and para-
sitology are, in general, the same as those outlined above for bacteriology.
Specific procedures, cultures, and materials required are outlined in two
American Public Health Association publications - Diagnostic Procedures for
Bacterial, Mycotic, and Parasitic Infections (Bodily et al., 1970) and
Diagnostic Procedures for Viral and Rickettsial Infections (Lennette and
Schmidt, 1969).  Viral and fungal cultures are available from The American
Type Culture Collection; parasitologic specimens may be obtained through the
suppliers listed in the CDC Quality Control Manual for Microbiologic
Laboratories (Ellis, 1976).

     A logbook should be maintained for all quality control tests on all
culture media, reagents, and other supplies.  Name of product, lot number,
manufacturer, date of receipt, and storage data should be logged in for all
products upon receipt.  Data on each performance test should include date,
product, lot number, medium or reagent(s) used, type of test, standard con-
trol culture data, completion date, results (whether satisfactory or unsatis-
factory) together with disposition of the lot or batch.  All personnel, in-
cluding those responsible for preparation of media, reagents, test cultures
should sign the logbook.  The Quality Control Supervisor should make certain

                                    334

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that all performance tests are conducted according to the program schedule
and protocols.

3.4.1.3  Instrument Checks and Calibration—

     A wide variety of precision instruments and other types of mechanical
equipment are necessary for diagnostic microbiology.   New instruments must be
properly calibrated and tested before use; instruments in use must be recali-
brated at stated intervals.  Instrument calibration and a regular check on
all mechanical equipment is an important part of the  quality control program.
Serious errors can result from variation in incubator temperature, anaerobic
jar failures, fluctuations in C02 levels in capneic incubators, etc.  Cold
rooms, walk-in incubators, and freezers should be equipped with Hi-Lo ther-
mometers, recording thermometers, and alarm systems.   A dry-ice box should
be available in the event of a breakdown of mechanical freezers.  Daily checks
are recommended for incubators, water baths, hot blocks, refrigerators,
freezers, anaerobic and carbon dioxide incubators. Autoclaves and centri-
fuges should be tested weekly.  Safety cabinets should be checked for face
velocity each month and filters should be replaced every six months (Russell,
1974).


     Airborne contamination can be one of the main sources of error in diag-
nostic microbiology.  Most laboratories today solve this problem through the
use of laminar flow hoods or cabinets in combination  with High Efficiency
Particulate Air (HEPA) filters.  HEPA filters regularly retain 99.9% or more
of particles as small as 0.3 um in diameter and tiius  remove most bacteria
and some viruses from the airstream.  The general principle of the laminar
flow cabinet is a rapid "piston-like displacement" of all air in the cabinet
by egress of filtered air from a whole wall or ceiling and withdrawal from
the opposite side.  Any particles entering from the outside are quickly swept
away by the air flow before contamination can occur.   In addition to the gen-
eral type of cabinet, other hoods are available for special purposes.  The
Class I partial containment cabinet is designed to give maximum protection to
the operator by drawing all air through front of cabinet across the work area
and exiting it through filters at top of cabinet.  The Class II partial con-
tainment cabinet protects both operator and experimental materials.  The in-
ward flow of air at front of cabinet protects the operator and the recircu-
lation of air through the filters provides clean air  over the work area.  The
Class III or absolute containment cabinet is a sealed unit which completely
shields the work material from the external environment and the operator from
any infectious agents associated with the experimental material.  All manipu-
lations are performed by the operator through sealed-in gloves which extend
into the work area (Coriell, 1973a). A fourth cabinet, the horizontal flow
cabinet, directs airflow from back to front of cabinet across the work area;
it can be used only with noninfectious material and is advantageous for pre-
paring petri dish media, tubing media, reagents, etc.

3.4.1.4  Experimental Design—

     Standard methods are available for all diagnostic work the microbiology
laboratory will be called upon to perform.  Moreover, these procedures must

                                    335

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be followed precisely and completely to obtain valid results.  However, two
aspects of sound experimental design should be emphasized - replicate samples
and controls.

     The need for replicate samples in experimentation has been well estab-
lished and needs no further documentation here.  In the more exact sciences,
such as analytical chemistry, the use of duplicate samples is routine.  In
microbiology, however, replicates within a sample are more common than repli-
cate samples.  For example, in the titration of viruses with cell cultures,
it is common to use 5 tubes at half-log dilutions or 10 tubes at log dilu-
tions over a range of 10°•5 to 107.  It would appear, however, that at least
two samples should be used in every experiment even with samples collected
by the most reliable sampling procedure.

     Blanks, vehicle controls, and other forms of negative controls are
usually included in laboratory procedures today.  Positive controls, however,
are often omitted.  Changes in sensitivity of the test system can lead to
grossly erroneous results if not checked with appropriate positive control
preparations.  The continuing sensitivity of any test system should not be
taken for granted.  Historical controls, if available, should also be taken
into consideration in the assessment of results (Prier et al., 1973; Vera,
1971; Russell, 1974).

3.4.1.5  Standard Methods—

     Standard methods in the field of diagnostic microbiology have been test-
ed and refined over a period of many years.  They are available in the follow-
ing standard reference works:

     Water and Wastewater - Standard Methods for the Examination of
                            Water and Wastewater, 14th Edition, 1975
                            (Rand et al., 1975)

                            Handbook for Evaluating Water Bacterio-
                            logical Laboratories - U.S. Environmental
                            Protection Agency (Geldreich, 1975)

     Bacteria, Fungi, &     Diagnostic Procedures for Bacterial, Myco-
     Parasites              tic, and Parasitic Infections. 1970, APHA
                            (Bodily et al., 1970)

                            Manual of Clinical Microbiology.  2nd edition,
                            1974, ASM (Lennette et al., 1974)

                            Diagnostic Microbiology  (Bailey  and Scott,
                            1970)

                            Sergey's Manual of Determinative  Bacterio-
                            logy. 8th Ed.  (Buchanan and Gibbons, 1974)
                                   336

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     Viruses & Rickett-   - Diagnostic Procedures for Viral and
     siae                   Rickettsial Infections, 4th Edition,
                            1969 (Lennette and Schmidt, 1969)

     Basic techniques may be found in:

                            Methods in Microbiology, Vols. 1-9
                            (Davies, 1971).

                            Methods in Virology  (Maramorosch and
                            Koprowski, 1967)

     Sample protocols for bacterial and viral assays are given in the
following pages.

     A Procedure Manual which contains complete protocols for each diagnostic
test performed by the laboratory as well as procedures for all ancillary work
such as media preparation, reagent testing, etc., is essential.  A "loose-
leaf" type which can be revised and updated easily is ideal.   It is a major
responsibility of the Quality Control Supervisor to make certain that this
manual is complete, up-to-date, and is followed without variation at all
times (Bartlett, 1973; Russell, 1974).

     3.4.1.6  Proficiency Testing—

     Proficiency testing in the area of diagnostic microbiology involves the
use of standards or unknowns for identification by the laboratory staff
(LaMotte, 1973; Prier, 1973; Wilson, 1973).  The ability of a laboratory to
correctly identify samples of this type in a consistent manner over a period
of time is probably the best assurance that the quality control program, for
that particular area at least,  is achieving its objectives.

     Two general types of standards are used in proficiency testing -
external standards and internal standards.  External standards are unknowns
made up and distributed by the quality control section of an organization
such as the College of American Pathologists, American Society of Clinical
Pathologists, or the Center for Disease Control.  Internal standards are un-
knowns prepared by the laboratory for evaluating the performance of their
own personnel.  Both types of standards have their own special advantages
and drawbacks.  External standards are prepared by specialists in the area of
quality control.  However, laboratories tend to work more carefully and to
expend extra effort when such unknowns are announced.  Internal unknowns are
prepared on a much smaller scale and usually have less documentation than
external unknowns.  Internal unknowns, however, can be slipped unannounced
into the regular flow of specimens and thus are tested routinely or without
special attention.  Moreover,  they can be tailored to appraise special
efforts to improve performance  of weak areas of the laboratory.  The prepar-
ation and preservation of satisfactory unknowns in certain areas require
great care and considerable "know how".  For example, considerable difficulty
has been encountered in maintaining viability of small numbers of _E. coli in
simulated drinking water samples for distribution to laboratories.  The prob-
lem was finally solved with the discovery that a formate lactose glutamate

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medium  (without  lactose)  with boric acid added to a final  concentration
of 1.8% would preserve viability  for  7  to  10 days.  The suspension is diluted
3:200 for examination  (Gray and Lowe, 1976).   Many  laboratories use both
external and internal standards in  their proficiency testing program thus
profiting by the advantages of each.

     The number  of unknowns employed  and the frequency of  testing varies
from laboratory  to laboratory.  Some  laboratories use as few as two internal
unknowns per month, others include  several each week.  Internal unknowns are
rotated so  that  all areas of  the  laboratory are checked periodically.  Pro-
ficiency testing of clinical  laboratories  subject to interstate commerce
regulations is under the jurisdiction of the Center for Disease Control.
Private laboratories, however, may  set  their own  schedule  for external stand-
ards  testing.  One large teaching  hospital laboratory receives four sets of
unknowns a year  from each of  two  large  private accreditation organizations -
Survey Program of the College of  American Pathologists and the Check Sample
Program of the Commission on Continuing Education of the American Society of
Clinical Pathologists.  Government  agency  laboratories are eligible also for
participation in the CDC proficiency  testing program.

     Proficiency testing results  with both internal and external unknowns
are discussed with laboratory personnel by the supervisor.  Where areas of
deficiency have been revealed, remedial measures are instituted.  Special
on-the-job training, laboratory courses and workshops are some of the methods
that may be used to increase the  quality of performance (Russell, 1974).

     EPA is now in the process of certifying laboratories performing analyses
of drinking water (Geldreich, 1975).

                         EXAMPLE:   BACTERIAL ASSAY

     Multiple-Tube Fermentation "technique - Coliform  Group:  Standard Total
Coliform Most Probable Number (MPN) Tests

Purpose of Study

     •  Ascertainment of the presence or absence of coliform organisms in
water and estimation of their density in terms of the Most Probable Number
as an aid in establishing the sanitary quality of the water.

     •  The coliform group comprises all of the aerobic and facultative
anaerobic, Gram-negative, non-sporeforming, rod-shaped bacteria that ferment
lactose with gas formation within 48 hours at  35°C.

Design of Experiment

     •  Tests:

        Presumptive Test
           Positive test - An indication of coliform organisms in sample
           Negative test - Absence of coliforms
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         Confirmed  Test
            Positive  test  - Additional evidence for presence of  coliforms
            Negative  test  - Absence of coliforms

         Completed  Test
            Positive  test  - Proof of presence of coliform organisms in sample
            Negative  test  - Absence of coliforms

      •   Diagnostic Media:

         Presumptive  Test
            Lactose broth  or lauryl tryptose broth

         Confirmed  Test
            Lactose broth  or lauryl tryptose broth
            Brilliant green lactose bile broth

         Completed  Test
            Lactose broth  or lauryl tryptose broth
            Brilliant green lactose bile broth
            Endo medium or Eosin methylene blue (EMB)  medium
            Agar slant

      •   Inoculum and Number of Fermentation Tubes

         U.S. Environmental Protection Agency Standards for water:
            five tubes of Presumptive medium, 10 ml  or 100 ml  of water sample
            each

         Other waters presumed to be of drinking-water quality:
            five tubes, at least, in each of at least  three dilutions

Conduct  of  Experiment

      •   Presumptive Test:   Inoculate a  series  of fermentation  tubes  ("primary"
fermentation tubes) with appropriate graduated quantities (multiples  and sub-
multiples of 1  ml) of the water to be tested.   Bottles to contain 100-ml
sample portions should be prewarmed in  a water bath at 35°C;  after adding the
sample, mix thoroughly and aseptically  add a sterile  fermentation vial.   The
concentration of nutritive ingredients  in the  mixture of medium and  added
portion of the sample must conform to the requirements given  in Section  905C,
Media Specification,  Media 2  and 3,  in  reference  at end of this protocol.
The portions of water sample  used for inoculating the lactose  or lauryl
tryptose broth fermentation tubes will  vary in size and number with  the
character of the water under  examination, but  in  general  should be decimal
multiples and submultiples of 1  ml.   These should be  selected  as outlined
under Design of Experiment for types  of  water  indicated.   Incubate the
inoculated fermentation  tubes  at 35  + 0.5°C.
     QUALITY CONTROL  —  The accuracy of  any single test is dependent  upon the
number of tubes used.

     •  Confirmed Test:   Lactose broth  or lauryl  tryptose broth may be used

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for the primary fermentation; however, lauryl tryptose broth is recommended
when experience shows a high proportion of false positive tubes of lactose
broth.
        Use brilliant green lactose bile broth fermentation tubes for the
Confirmed Test.
        Procedure:  Submit all primary fermentation tubes showing any amount
of gas at end of 24 hours of incubation to the confirmed test.  If active
fermentation appears in the primary fermentation tube before expiration of
the 24-hour period of incubation, it is preferable to transfer to the con-
firmatory medium without waiting for the full 24-hour period to elapse.  If
additional primary fermentation tubes show gas production at the end of 48-
hour incubation, these too shall be submitted to the confirmed test.
        Alternative procedure:  Where three or  more multiple portions of a
series of three or more decimal dilutions of a given sample are plated sub-
mit to the Confirmed Test all tubes of the two highest dilutions (smallest
volumes) of the original samples showing gas formation in 24 hours.
        All tubes producing gas in 24 hours that have not been submitted to
the Confirmed Test must be recorded as containing organisms of the coliform
group — that is, as positive - even though all the confirmed tests actually
performed yield negative results.
        Submit to the Confirmed Test all tubes of all dilutions of the
original sample in which gas is produced only at the end of 48 hours.
        If less than three portions of any dilution (volume), or if a series
of less than three decimal dilutions of the original sample is plated sub-
mit all tubes producing gas at 24 and 48 hours to the confirmed test.
        Procedure with brilliant green lactose bile broth:  Either 1) gently
shake or rotate primary fermentation tube showing gas and with a sterile
metal loop, 3 mm in diameter, transfer one loopful of medium to a fermenta-
tion tube containing brilliant green lactose bile broth, or 2) gently shake
or rotate primary fermentation tube showing gas and insert a sterile wood
applicator at least 2.5 cm (1 inch) into the medium.  Promptly remove and
plunge applicator to bottom of fermentation tube containing brilliant green
lactose bile broth.  Remove and discard applicator.
        Incubate the inoculated brilliant green lactose bile broth tube for
48+3  hours at  35 ± 0.5°C.

     •  Completed Test:  The Completed Test is used as the next step follow-
ing the Confirmed Test.  It is applied to the brilliant green lactose bile
broth fermentation tubes showing gas in the Confirmed Test.  The procedure is
as follows.
        Streak one or more Endo or eosin methylene blue plates from each tube
of brilliant green lactose bile broth showing gas, as soon as possible after
the appearance of gas.
        Incubate the plate (inverted, if with glass or plastic cover) at
35 + 0.5°C for 24+2 hours.
        The colonies developing on Endo or eosin methylene blue agar may be
described as typical (nucleated, with or without metallic sheen); atypical
(opaque, unnucleated, mucoid, pink after 24-hour incubation), or negative
(all others).  From each of these plates fish one or more typical well-
isolated coliform colonies or, if no typical colonies are present, fish two
or more colonies considered most likely to consist of organisms of the coli-
form group, transferring each fishing to a lactose broth or a lauryl tryptose

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 broth  fermentation tube and to  a  nutrient  agar  slant.
         The use of a  colony counter  is  recommended  to  provide  optimum magnif-
 ication when colonies are fished  from the  plates  of selective  medium.
     QUALITY CONTROL  -- It is essential  that  the  Endo  or  EMB plates  be  so
 streaked as to insure the presence of some discrete colonies,  separated by
 at least 0.5 cm from  one another.  Careful  attention to the following details
 when streaking plates will  result in a  high proportion of successful isola-
 tions  if coliforms are present:   Use an  inoculating needle slightly  curved
 at the tip; tap and incline the fermentation  tube to avoid picking up mem-
 brane  or scum on the  needle; insert  end  of needle into liquid  in tube to a
 depth  of approximately 5.0 mm;  streak plate by  contacting agar surface  with
 curved section of needle only so  that agar will not be scratched or  torn.
 When transferring colonies, choose well-isolated  colonies separated  by  at
 least  0.5 cm and barely touch surface of colony with a flame-sterilized, air-
 cooled transfer needle, to minimize  danger of transferring a mixed culture.

 Observations and Tests

     •  Presumptive Test:   At end of 24  +  2 hours,  shake  each  tube gently
 and examine it and, if no gas has formed and  been trapped in the inverted
 vial,  repeat this step at the end of 48  +  3 hours.   Record the presence or
 absence of gas formation at each  examination  of the tubes, regardless of the
 amount.
         Interpretation:  Formation within  48  +_  3  hours of gas  in any amount
 in the inner fermentation tubes or vials constitutes a positive Presumptive
 Test.
         The absence of gas  formation at  the end of  48 + 3 hours of incubation
 constitutes a negative test.  An  arbitrary limit  of 48 hours for observation
 doubtless excludes from consideration occasional  members of the coliform
 group  that form gas very slowly and  are  generally of limited sanitary signif-
 icance;  for the purpose of a standard test based  on  the definition of the
 coliform group, exclusion of these occasional slow  gas-forming organisms
 does not compromise the value of  the test.
     QUALITY CONTROL  -- Appearance of an air  bubble  in inner fermentation
 tubes  or vials must not be  confused  with actual gas  production.  If  the gas
 is formed as a result of fermentation, the broth  medium will become  cloudy.
 Active fermentation may be  shown  by  the  continued appearance of small bubbles
 of gas throughout the medium outside the inner  vial  when  fermentation tube
 is gently shaken.

     •  Confirmed Test:  The formation of  gas in  any amount in the inverted
 vial of the brilliant green lactose  bile fermentation tube at  any time  within
 48 + 3 hours constitutes a  positive  Confirmed Test.

     •  Completed Test:  The agar slants and  secondary broth tubes are  in-
 cubated at 35 +_ 0.5°C for 24 + 2 or  48 + 3 hours  if gas is not produced in
 24 hours.  Gram-stained preparations from  those agar slant cultures  corre-
sponding to the secondary lactose  broth tubes  that show gas are examined
 microscopically.
         Interpretation:  The formation of  gas in  the secondary lactose  broth
 tube and the demonstration  of Gram-negative,  non-sporeforming, rod-shaped
 bacteria in the agar  culture may  be  considered  a  satisfactory  Completed Test,

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 demonstrating the presence of a member of the coliform group in the volume
 of sample examined.
         If, after 48+3 hours, gas is produced in the lactose and no spores
 or Gram-positive rods are found on the slant, the test may be considered
 completed and the presence of coliform organisms demonstrated.
      QUALITY CONTROL — A Gram-positive and a Gram-negative culture should be
 used as controls for the Gram-staining process.

 Data Handling and Validation

      •  Record permanently analytical  data in meaningful, exact terms.

      •  Report data in proper form to  an information storage facility for
 future use.

      •  All laboratory personnel must  agree upon precise rules for the use
 of significant figures, rounding off numbers, and arithmetic operations.

      •  Use bound laboratory record books and preprinted report forms.   Lab-
 oratory records should be readily available for inspection and held on  file
 for at least two years.  The multi-copy report forms are highly recommended
 for recording all information from sample collection to calculation of re-
 sults.  One copy of these forms is then forwarded to the appropriate office
 for computer data entry.

      •  A study on analytical quality  control methods for use in validating
 microbiological data by a group of researchers at the Robert S.  Kerr Water
 Research Center at Ada, Oklahoma, demonstrated that precision control  charts
 are a useful  tool for precision but they cannot measure accuracy; that  is,
 the data can be precise and still be inaccurate.   At least duplicate tests
 must be performed for each sample.  The data must be plotted on  an everyday
 basis and problems must be rectified immediately.  Data from the same waters
 under study should be used to construct the control  charts.

 References

      •  Bordner,  R.  H.   1973.  Quality control:   A state-of-the-art.  Proceed-
 ings of the First Microbiology Seminar on Standardiration of Methods.   Office
 of Research and Monitoring, U.S. Environmental  Protection Agency, EPA-R4-73-
 022, Washington,  D.C.,  pp.  170-194.

      •  Rand,  M.  C., A.  E.  Greenberg,  and M.  J.  Taras (eds.).   1975.  stan-
dard  Methods  for  the Examination of Water and Wastewater, 14th Edition.
 American Public Health  Association-American Water Works Association-Water
 Pollution Control  Federation, Washington, D.C.,  pp.  968-975.

                  EXAMPLE:   WATERBORNE  ENTERIC VIRUS  ASSAY

 Purpose of Study

      •  Detection of waterborne enteric viral  pathogens


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Design of Experiment

      •  Entire sample-concentrate must be inoculated into indicator hosts
(see  Section 3.4.1.1)

      •  Indicator hosts
        Cell cultures:  Primary African Green monkey kidney*, and human
embryonic kidney*
        Suckling mice:  less than 24 hours old

      •  Number of mice or cell cultures:
        Suckling mice - at least two litters
        Cell cultures - sufficient for remainder of sample after mouse inocu-
lation

Conduct of  Experiment

      •  Suckling mice:  Mice are inoculated with a portion of the original
sample-concentrate by the intracerebral (0.02 ml) and intraperitoneal (0.05
ml) routes.  Animals are then observed daily for 14 days.
      QUALITY CONTROL — A vertical-flow laminar-air hood is used for all
virus assay procedures.
        Temperature, humidity, and all other environmental conditions in
animal rooms should be maintained at proper level for suckling mice and dams.

      •  Cell cultures:  Remaining sample-concentrate is inoculated onto mono-
layer cultures of two cell types.  Inoculum must not exceed 0.06 ml/cm2 of
cell  surface area.  After a 2-hour adsorption period at 36° + 0.5°C, inoculum
is decanted and maintenance medium is added to cells.  Cultures are then
incubated at 36° +. 0.5°C and observed microscopically, daily for 3 days and
then  periodically for 14 days.  Medium is not changed during observation
period unless required to maintain healthy cells.
      QUALITY CONTROL -- Negative (uninoculated) controls must be included
(both cell  culture types as well as mice).
        Cell cultures must be free from Mycoplasma contaminants and "passen-
ger viruses."
        All tissue culture containers should be permanently labelled.
        Replicate assays should be run for each cell culture type.
        Glassware, pipettes, media, and reagents for cell cultures must be of
tissue culture grade.  Media and reagents should be performance-tested before
use.
        Incubators must be monitored carefully to make certain temperature is
maintained  within prescribed limits.
        Microscopes, incubators and any other precision equipment employed
should be checked frequently and adjusted or recalibrated if necessary.

Observations and Tests

      •  Suckling Mice:  Mice showing no pathology by 14th day of first  passage
 * Other  cultures may  be  used  if  evidence  is  available  to  show that "cells
 have equivalent spectral  sensitivity  for  enteric  virus replication."

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are regarded as  negative  for  Coxsackie  virus group A.
        Mice exhibiting any pathologic  changes  (e.g., flaccid or spastic
paralysis of the extremities)  are  sacrificed and  passaged a second time in
suckling mice  according to procedures and  analyses outlined in Diagnostic
Procedures  for Viral  and  Rickettsial Diseases  (Melnick, J. L., H. A. Wenner,
and L.  Rosen.  1964.   The  Enteroviruses.   In:   Diagnostic Procedures for Viral
and Rickettsial  Diseases, Lennette, E. H.,  and  N.  J.  Schmidt (eds.), 3rd
Edition, American Public  Health  Association, New  York, N.Y., pp. 217-218).

      •  Cell Cultures:  Cell  cultures showing  no  cytopathic effects (CPE) by
the 14th day are frozen and thawed once for re-passage.  Harvest-fluids from
a  single cell  culture type are pooled and  20%  of  the volume is inoculated
onto  a  second  cell  culture monolayer  of the same cell type.  Cell cultures
negative for CPE on 14th  day  of  the second passage are considered negative
for virus.
        All cultures  positive  for  CPE are  confirmed  for presence of virus by
additional  passages.
        Virus  isolates are identified by appropriate serologic procedures
(Diagnostic Procedures for Viral and Rickettsial  Diseases, Lennette, E. H.,
and N.  J. Schmidt (eds.)» 3rd  Edition,  American Public Health Association,
New York, N.Y.,  1964).

Reference

      •  Rand,  M.  C.,  A. E. Greenberg, and  M. J. Taras (eds.).  1975.  stan-
dard  Methods for the  Examination of Water  & Wastewater, 14th Edition.  Ameri-
can Public  Health Association-American  Waterworks Association-Water Pollution
Control Federation, Washington,  D.C., pp.  968-975.

3.4.2  Microorganisms - Mutagenicity Testing

      An important milestone in the history of  mutation research was reached
in 1947, when  for the first time,  a chemical,  mustard gas, was shown to be
capable of  artificially inducing cell mutation  in an experimental test organ-
ism,  Drosophila  melanogaster  (Auerbach  et  al.,  1947).  Since this initial
discovery,  a whole host of chemicals have  been  found to be mutagenic in a
variety of  animals and other  test  organisms.

      In view of  the fact  that  mutation  tests in mammals are time-consuming
and relatively expensive, researchers early turned to simpler forms of life
with  a more rapid generation rate  as a  test system.  Microorganisms (bacteria,
yeasts, mold, protozoa), mammalian  cell cultures, and insects (Drosophila
melanogaster strains  mainly) are widely used today (Anon., 1975).  However,
opinion is  sharply divided regarding the value of mutagenicity testing with
nonmammalian systems.  One school  contends that results of value in predict-
ing the likelihood of mutagenicity  in man  can be obtained only with the in-
tact  animal where  the chemical is  subject  to metabolic and detoxification
mechanisms  (WHO,  1971; Anon.,  1972).  Others take the stand that mutations
result basically  from alterations  in DNA which is essentially the same for
all organisms;  moreover,  metabolic and detoxification mechanisms can be
brought to bear on the test compound in microbial or cell culture assays
through the use of microsomal  systems in vitro  (Ames and Yanofsky,  1971; Ames

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 et al.,  1973a).   Mutageniclty testing takes on added significance with the
 accumulation of  evidence that most mutagens are also carcinogens  and some
 authorities regard mutagenicity testing as  practically  equivalent to carcino-
 genicity testing (Ames and Yanofsky,  1971;  McCann   et al.,  1975).

      Genetic change can be brought about in many different  ways and  no single
 mutagenicity system has been developed to date which will detect  all types of
 mutations.   Major mechanisms involved are:   Gene mutations  and chromosomal
 mutations.   Gene mutations may be point mutations  (base-pair  substitutions,
 frameshift mutations, small deletions or insertions) or small multilocus
 mutations.   Chromosomal aberrations may be  ploidy  changes or  chromosomal
 breaks and/or misreplication and/or misrecombination effects  (U.S. EPA,
 1977).  Consequently, most authorities today recommend  a tier system in
 mutagenicity testing, each tier consisting  of a battery of  tests  in an effort
 to detect all of the kinds of mutations that can be induced by a  chemical:

           Tier 1 - Microbial cultures
           Tier 2 - Mammalian cell cultures  and/or  Drosophila  strains
           Tier 3 - Mammals

 Microbial systems most commonly recommended for Tier 1  are:

           •  Host-mediated assay with Salmonella typhimurium
           •  Ames test - Salmonella tvphimurium in vitro with and without
                          microsomal activation
           •  Mitotic gene conversion test with Saccharomyces  cerevisiae
           •  Repair-deficient E. coli with  activation (Anon., 1975;  U.S.
                          EPA, 1977; Huisingh, 1976; Flamm,  1974;  Zimmermann,
                          1975)

 The bacterial system can be used to detect  gene mutations  (base-pair sub-
 stitution mutation, frameshift mutation, and stimulation of DNA repair).
 Saccharomyces cerevisiae can be used to detect mitotic  recombination and
 mitotic gene conversion as well as forward  and reverse  gene mutations.
 Decisions are made at the end of each tier  test regarding further testing.
. Organizations requiring definitive tests in mammals may use the  tier system
 to establish testing priorities.

 3.4.2.1  Methods—

      •  The Host-mediated Assay

         Although microbial assays have a number of advantages over mammalian
 tests - short generation time, large cell population, short test  period,
 great sensitivity, large range of detectable compounds, simplicity, economy,
 etc.  - the usual in vitro tests do not detect, and may give misleading
 results, with:

         o  Chemicals (promutagens) which must undergo metabolic  transfor-
            mation in the host to become mutagenic  (false negative results)
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        o  Compounds which are detoxified by  the  host  (false  positive results)

The host-mediated assay developed by Legator  and  coworkers  (Gabridge and
Legator, 1969; Legator and Mailing, 1971; Legator,  1976), is  designed to
correct these shortcomings and to bridge the  gap  between  the  in  vitro assay
and definitive mutagenicity  tests in animals.

     In the host-mediated assay, the test organism  is  injected into  an animal
undergoing treatment with a  suspected mutagen.  After  a period of  several
hours, the indicator organisms are withdrawn  from the  peritoneum and plated
on appropriate media to detect any induced  mutants  (Figure  3.4.10).   Since
the chemical is subjected to both metabolic and detoxification systems of  the
host, the possibility of false-negative and false-positive  results is reduced
to a considerable degree.  An in vitro test in  which the  test chemical acts
on the indicator organism directly is included  as a control on the host-
mediated effect.
                                              ItoWMtf
                                               Micr«-*rgaiiciini
                                             AIIOI tor Miami
                                  IntHrt
 Figure 3.4.10  The  technique  of  host-mediated assay involves the comparison
  of the mutagenic power  of  a  chemical  when tested in vitro and when tested
 in the peritoneum of  the rodent  after  oral administration.   It thus gives a
measure of in vivo activation  and detoxication of  the compound (Clayson,  1973)

     An example of a protocol  for the host-mediated assay is given in the
following pages.
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                       EXAMPLE:  HOST-MEDIATED ASSAY

 Purpose of Assay

      •  Mutagenicity testing
                         »
 Design of Experiment

      •  Test Culture:

        Salmonella typhimurium histidine auxotrophs (His~) Strains TA-1537,
          TA-98, TA-100 (Ames)
        Escherichia coli Strains 1212/343/113
        Saccharomyces cerevisiae
        Neurospora crassa

      •  Host:   Swiss albino mice (25 to 30 g) usual host

      •  Dosage  of Test Substance

        Recommended doses:  Maximum tolerated dose (MTD) (highest dose);
 intermediate concentration (intermediate dose); maximum use concentration
 (lowest dose).  If the usage  level is not known, second and third doses can
 be one log and  two logs,  respectively,  lower than the  MTD.

      •  Vehicle:  Corn oil, ethanol, and dimethylsulfoxide are suitable
 vehicles  for substances not soluble in  water

      •  Controls:  Positive,  vehicle, and untreated controls should be in-
 cluded in each  assay.  Positive control compound should be a known pro-mutagen
 of the same chemical .class as the test  compound

 Conduct of Experiment  (S^. typhimurium strains)

      •  Test compound may be  administered to the host  by any route other than
 intraperitoneal.
      QUALITY CONTROL — Test  chemicals  must be collected by a statistically
 sound method to ensure that sample is representative of entire batch or lot.

      •  Tester  (indicator) strains are  injected intraperitoneally to sepa-
rate animals about 4 hours after treatment with test compound in acute tests.
 The inoculum consists of 2 ml of a suspension containing 3 x 108 to 5 x 108   .
 cells per ml at log phase of  growth.
      QUALITY CONTROL — Test  compound should be of highest purity unless
 technical grade, mixture, or  formulation is to be tested.
        Test animals must be  healthy and should be selected randomly for
 various parts of test.

      •  Recovery of Indicator Organism  from Host:  Three hours after adminis-
 tration  of indicator organisms, 1 to 2 ml of sterile  saline is injected
 into  the peritoneal cavity and animals  are sacrificed  for aseptic removal  of
 the peritoneal  exudate.

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     QUALITY CONTROL -- Strict aseptic technique must be used for parenteral
administration of both test chemical and indicator organisms.
        Tester strains must be obtained from a reliable source and monitored
frequently to make certain that no changes in essential test genetic markers
have occurred.

     •  Detection of Mutants:

        1)  Petri dishes with nutrient medium containing only 40 yg/ml histi-
dine (minimal agar) are streaked with 0.2 ml of undiluted exudate for detec-
tion of host-mediated mutants.  Five plates are streaked for each animal.
     QUALITY CONTROL — The streaking technique used must ensure that organ-
isms will develop into discrete colonies which can be counted accurately.
        All culture operations should be carried out in a biological cabinet
equipped with HEPA filters to prevent contamination by ambient airborne
microorganisms.

        2)  To determine total number of surviving bacteria, dilutions of
exudate are streaked in same manner, in triplicate, on tryptone-yeast agar
(complete agar).

        3)  A parallel in vitro test is carried out to ascertain if test
chemical will induce mutation without intervention of a host (mutagen instead
of pro-mutagen).  A  0.1 ml  sample  of an overnight  broth culture of  the tester
strain  is  added  to 2 ml of  cooled  molten  agar  (0.6%) and poured over  a mini-
mal  agar base  plate,and test  chemical spotted  on center of dish.  Positive
and  negative  (vehicle  and untreated) controls  must be  included.

        4)  All  plates are  incubated inverted  at 37°C  for 24 or 48  hours.
      QUALITY CONTROL — Incubators must be monitored during tests to  make
certain that the desired temperature is maintained uniformly  throughout the
appliance.

Observations and Tests

      •  Mutant histidine revertants (His  ) only will grow on the minimal agar
plates  (trace  of histidine) streaked with exudate.  The colonies can  be
counted after  48 hours at 37°C.
      QUALITY CONTROL — Any contaminated  plates must be excluded from the
assay and  the  samples  recultured.

      •  All surviving  organisms  (His" originals and His  mutants) will grow
on the complete  medium, and colonies are  counted after 24 hours at  37°C.
        Mutation frequency  (MF)  is calculated  for  each sample:

           MC_  His mutants/ml	
111   CFU/ml in undiluted exudate
    (CFU = Number of colonies)

MF /MF  - MF of treated sample
nrt/nrc ~ MF of control sample
                                   sample


                                     348

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     •  If mutants  (His  ) are induced in the in vitro test, colonies will
appear in a circle  around the test sample.

     •  Comparison  between direct effect of test substance on indicator organ-
isms (in vitro test) and effect of animal (hostrmediated effect) indicates
whether or not the  mammal can detoxify the compound or metabolize it with the
formation of one or more mutagens.

     •  A negative  result is not conclusive proof that a substance is not a
mutagen or promutagen since not all mutagenic mechanisms are detectable by
the tester strains  now available.  Also, although less likely, the host
employed may not be able to metabolize a given promutagen with the formation
of one or more mutagens.  In spite of this weakness the host-mediated assay
with !>. typhimurium auxotrophs has been widely used with excellent results
and is recommended  by most experts for mutagenicity testing.

Data Collection and Analysis

     •  All results will be recorded on specially designed data sheets.
     QUALITY CONTROL — All data sheets will be dated and signed by opera-
tor(s)  conducting test.   Data will be subjected to appropriate statistical
analysis.

     •  Assays employing other acceptable indicator organisms are conducted
in essentially the  same manner as described above with appropriate minimal
and complete nutrient media and inocula.

     •  Ray et al.  (1973) found that the host-mediated assay using Salmonella
typhimurium tester  strains was considerably more sensitive in detecting
mutagenesis with ethylmethanesulfonate (EMS) in random-bred Swiss CD-I mice
than the dominant lethal or cytogenetic assays.  Relative sensitivities of
three assays as determined by dose-response curves and no-effect dose levels
were as follows:

            Dominant lethal effects not evident until   150 mg/kg used

            Cytogenetic test - no significant breaking
            of somatic cell chromosomes in bone marrow
            cells until                                 120 mg/kg used

            Host-mediated assay - statistically-signifi-
            cant response detected at                    35 mg/kg level
     The second major advance in microbial mutagenicity testing methodology
was the development by Ames and coworkers of a microsomal activation system
which is capable of detoxifying or metabolizing potential mutagens in vitro.
The preparation,  obtained from the livers of rats induced with Aroclor 1254,
can be mixed directly with cofactors,  test chemical, bacteria, and culture
medium in a single petri dish test system (Ames et al., 1973b; Ames et al.,
1975).   In addition to being simple,  rapid, and inexpensive, the system is
also extremely sensitive.  Strong mutagens can be detected at levels as low
as a few nanograms.

                                     349

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     A sample protocol for the Ames Test is on the following pages.

     The complete battery of Ames strains "are able to detect almost all
mutagens", according to the authors.  The only additional test required is an
excision repair test.  It should be borne in mind, however, that in the Ames
tert the tost chemical is subjected to a certain fraction of the liver induced
with a single chemical whereas in the host-mediated assay the complete
metabolic and detoxification  systems of  the intact mammal are brought  to bear
upon the  chemical.   Nevertheless, the Ames test  is highly regarded  and is an
almost unanimous  choice of the experts for Tier  1 testing.

     Other mutagenicity tests in recent  use are  outlined in Table  3.4.1.

3.4.2.2  Media, Reagents  and  other  Supplies—

     All media, reagents, and other materials  should  be tested  for  perform-
ance upon receipt and when each batch is prepared.  Microsomal  activation
preparations together with cofactors must be tested with known  promutagens
before use and at intervals during  storage.  Performance specifications are
given in  the various test methodologies  referred to above.

3.4.2.3   Instrument  Calibration and Checks—

     Incubator temperature should be checked at  the beginning and  end  of each
day.  Centrifuges,  freezers,  refrigerators, autoclaves and all  other equip-
ment should be checked regularly according to  the schedule given in Appendix
C.

3.4.2.4   Experimental Design—

     All  mutagenicity assay systems employed should have a satisfactory per-
formance  documentation in the scientific literature.  The  test  system  must
also be reproducible; identical results  should be obtained by various  labora-
tories with the same compounds.  The system should not give false  negative
tests and few false  positive  results  (de Serres, 1974).  Each test  system
should be calibrated before routine use  with known mutagens and negative
chemicals  (U.S. EPA, 1977).   The spot test technique  as well as the standard
plate method should  be used in each assay.

     Test chemical specimens  should include technical grades and formulations
as  well as purified  preparations.

     Dosage ranges vary somewhat depending upon  the type of assay  system and
on  the  individual investigator.  In the  Ames test,  the maximum  dose is usually
set at  500 yg  (or the highest non-inhibitory level); doses  as low as a  few
nanograms of strong  mutagens  are sufficient to induce mutations.   Ames sys-
tems and  types of mutagenic activity detected  are shown in Table 3.4.2
 (Legator, 1976).

      In the host-mediated assay, at least three  doses are  used  initially in
screening chemicals. Dose levels suggested for  several types of chemicals
are given in Table 3.4.3.

                                    . 350

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                                       TABLE  3.4.1.  OTHER MUTAGENICITY  TESTS
         Assay
                                           Strain Used
Experimenter
Cn
    Mitotic gene conversion
    DNA repair
Dominant or recessive
  lethal test

Host-mediated forward
  mutation

Forward mutation
  (Canavanine resistance)

Host-mediated assay

Chlorate reduction


Forward mutation
                                           Saccharomyces  cerevisiae
                                             Strain D-4
                                           Escherichia coli
                                             P3478 polA~
                                                J.  coli
                                                 W3110
                                               Neurospora  crassa
                                                    crassa
                                               j^.   cerevisiae


                                               jS.   cerevisiae

                                               Escherichia  coli
                                               Salmonella typhimurium

                                               S .  typhimurium
Zliranermann (1973, 1975)
Brusick and Andrews  (1974)
Ong et al. (1977)


Slater et al. (1971)
Webber and de Serres  (1965)
de Serres and Mailing (1971)

Legator and Mailing (1971)
Zeiger and Brusick (1971)


Fahrig (1975)


Ames and Yanofsky (1971)

Ames and Yanofsky (1971)

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                         TABLE  3.4.2   GENE MUTATION  DETECTION SYSTEMS (Legator,  1976)
K>
Gene
Alterations
Types detected:
Mutations forward
Reverse
Mitotic gene con-
S. typhimurium
(histidine
auxotroph)
No
Yes
No
N. crassa
(adenine 3
locus)
Yes
Yes
No
E. coli
343/113
Yes
Yes
No
S.
cerevisiae

Yes
Yes
Yes
Chinese
Hamster
Yes
Yes
No
Murine
Leukemia
Yes
Yes
No
   version and re-
   combination

Ease of detecting
 genetic events

Genetic validity of
 detected change

Growth division in
 host as compared to
 in vitro
                            Excellent
                            Established
                             Similar
Fair
Excellent
Fair
Good
Established   Established
No growth     Similar
 or division
              Established  Question-
                            able
              Slight
               growth; no
               division
             No growth
              or divi-
              sion
   Spontaneous mutation     Similar
    frequency in host as
    compared to in vitro

   Ability to localize      With dif-
    genetic effect in         ficulty
    host
                                          With dif-
                                           ficulty
              Fair
              Fair
             With dif-
              ficulty
Good
             Question-
              able

             Similar
                                                                                                Similar
             With dif-
              ficulty
   Utility
                         Good
Questionable  Good
              Good
             Questionable Good

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              TABLE 3.4.3  DOSE LEVELS FOR HOST-MEDIATED ASSAY
Chemical
Highest
Intermediate
Lowest
Environmental

Drugs
Drugs
LD50

Toxic but
permits
survival of
most animals

LD5
LD50/5-LD50/10

Intermediate
between high
and low doses
Intermediate
between high
and low doses
LD50/50-LD50/100

Close to pharma-
cologic thresh-
old of drug
Use-dose of drug
Drugs
General
General
5.0 g/kg*
MTD*
MTD*
500 mg/kg*
Intermediate
between high
and low doses
MTD/ 10*
50 mg/kg*
Maximum use
level
MTD /I 00*
*  If maximum use level or dose is not known, ten animals per dose level per
indicator organism are usually employed in the host-mediated assay (Green
et al., 1976).  Dose response curves should be obtained in all cases where
mutagenicity has been detected in the screening test.  Toxicity data should
be included in the mutagenicity test report on each compound tested.

                    EXAMPLE:  MICROBIAL ASSAY (AMES TEST)

Purpose of Study

     •  Mutagenicity determination

Design of Experiment

     •  Test culture:

        Salmonella typhimurium
         Strains TA-1535, TA-1537, TA-98 and TA-100

        Escherichia coli              .             _
         Strains WP2 uvrA , W3110/polA , P3478/polA

        Yeast
         Strain D4
                                     353

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                PROPOSED TEST SCHEME FOR A SINGLE CHEMICAL

          Nonactivation                      Activation (Rat Liver)

          Positive Controls (2)              Positive Controls (2)

          Solvent Controls (3)               Solvent Controls (3)

            Test Level 1 (1)                   Test Level 1 (1)
                       2 (1)                              2 (1)
                       3 (1)                              3 (1)
                       4 (1)                              4 (1)
                       5 (1)                              5 (1)

     •  Replications:  Each solvent control set will be performed in dupli-
cate; each positive control and each test material will be performed with one
plate for each of the concentrations.  The assays will be performed under
both nonactivation and activation conditions as outlined above.  The total
sum of plates required for the evaluation is also given.

     •  Chemical Levels:  The proposed chemical range will consist of the
following five concentrations:

        Dose range        ul/plate (liquid)        ug/plate (solid)

            1                   0.001                     1
            2                   0.05                     10
            3                   0.5                     100
            4                   1                       250
            5                   5                       500

Adjustments to either higher or lower concentrations will be made to accom-
modate variations in toxicity.

Conduct of Experiment

     •  Strain Culture:  The strains employed will be grown overnight at 37°C
on a shaker in complete medium.
     QUALITY CONTROL -- Before the use of any test culture, samples of each
culture will be removed and monitored for the appropriate marker per Table 1.
Genetics of tester strains are given in Table 2.

     •  Activation System:  Assays conducted in the presence of a microsome
activation system will be performed as outlined above.  The activation
system will consist of the components listed in Table 3 and will use 9,000 x
£ supernatant from hepatic homogenates from the same species and strain of
animals employed in associated carcinogenesis experiments (either mice or
rats).  The animals used to provide the hepatic tissue will be pretreated with
Aroclor 1254 (Ames et al., Mutation Res. 31_:347, 1975) to induce the micro-
somal enzyme activity.
     QUALITY CONTROL — All samples of 9,000 x £ supernatant will be monitored
for protein content and P-450/P-448 activity.  These latter measurements are

                                     354

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                                TABLE 1. MICROBIAL STOCK MONITORING PROCEDURES
Resistance to: Spot Test
Strain
TA-1535
TA-1537
TA-10Q
TA-98
W?2 uvrA"
W3110
P34/8
D4
Genus
Salmonella
Salmonella
Salmonella
Salmonella
Escherichia
Escherichia
Eschertchia
Saccharomyces
Markers Monitored Ampicillina , Crystal Violetb Reversion0
his G, rfa
his C, rfa
his G, rfa, AmpR +
his D, rfa, AmpR +
try.
polA+
polA"
try NA
MNNG
QM
MNNG
NF
+ MNNG
+ NG
+ MNNG
NA MNNG
"x
aAn ampicillin sensitivity disc is placed on a fresh lawn of cells on complete medium.
 The zone of inhibition is compared with TA-98.

 A single drop of crystal violet solution is placed on a fresh lawn of cells on complete medium.
 The zone of inhibition is compared to a G-46 standard (resistant).

°Spot tests with reference mutagens are made to determine both correct mutant response and any
 contamination.

 MNNG « N-Methyl-N -nitro-N-nitrosoguanidine
 QM   = Quinacrine mustard
 NF   - Nitrofluorene
 NG   = Nitrosoguanidine

-------
                                          TABLE 2. BACTERIA AND YEAST  STRAINS
Gene Additional Mutations References for
Strain Designation
TA-1535
TA-98
TA-100
W3UO
P3478
D4
Affected Repair
his G uvrB
his D uvrB
his G uvrB
polA+
polA~
ade2, try 5
LPS R Factor Use Ln Screehiag
rfa - Ames et al
rfa pKMlOl Ames et al
rfa pKMlOl Ames et al
Slater et
Slater et
— — Zimmermann
.. (1975)
. (1975)
. (1975)
al. (1971)
al. (1971)
(1975)
CO

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                                     TABLE 3. AMES ACTIVATION SYSTEM

1.
2.
3.
4.
5.
6.
7.
Component MW Supplier
TPN 801 ICN
Glucose-6- 282 Sigma
phosphate
dibasic
Sodium 142 Sigma
phosphate
MgCL2 95 Sigma
KCL 74 Sigma
Homogenate
H20
Volume of Stock Added/ml Final' Concentration
Stock Preparation of Final Mix of Component /ml in Mix
40 g/500 ml H20
141 g/500 ml H20
14.2 g/500 ml H20
adjusted to pH 7.4
19.2 g/500 ml H2Q
61 g/500 ml H20
Standard KCL 9,000 x £
supernatant

40 yl
5 yl
500 yl
20 yl
20 yl
100 yl
315 yl
4 ymoles
5 ymoles
100 ymoles
8 ymoles
33 ymoles
Approximately 25 mg of
fresh tissue equivalant

Components 1 and 2 are prepared in sterile distilled water and maintained at -20°C.




Components 3, 4, and 5 are prepared in distilled water, sterilized, and maintained at 4°C.




Components 6 is prepared and stored at -80°C until used.




Components 1-5 combined = core reaction mixture. MW = Molecular weight




Components 1-6 combined = complete S-9 mixture.

-------
 added  to ensure reproducibility from sample to sample.

      •  Control  Compounds:   Unless otherwise specified,  dimethylsulfoxide
 (DMSO) will  be employed as  the solvent for all  test materials.   DMSO  has been
 carefully evaluated in the  assay and has  no mutagenic activity.   The  concen-
 tration of DMSO employed in the solvent control  will be  equal  to  the  amount
 of DMSO added along with the highest concentration  of test material and will
 likely not exceed 50 yl per plate.
     QUALITY CONTROL — Records of the manufacturer and  lot number of DMSO
 employed will  be maintained.   Positive control  compounds are .listed in Table 4,

     •  Test Samples:
     QUALITY CONTROL — Upon receipt of the materials, the identifying desig-
 nations and  physical  descriptions will  be entered into a logbook  and  dated.
 All  details  of weighing and dilutions will  be recorded.

 Methods

     •  Preparation of Tissue Homogenates:   Animals will  be stunned,  decapi-
 tated, and bled.   The liver  will  be excised aseptically  and placed in cold
 KC1.   After  washing with additional  KC1,  the tissue will  be homogenized in
 0.15 M KC1 at a  ratio  of one part tissue  to three parts  KC1.  The homogenate
 will be centrifuged at 9,000 x £ for 10 minutes  in  a refrigerated Sorall
 centrifuge.   The supernatant  will  be collected,  pooled,  and frozen at -80°C.
 Samples will  be  assigned a  lot number and assayed for total  protein and P-448
 content.
     QUALITY  CONTROL — The samples  will  also  be checked  for sterility.

     •   Plate  Assay Method:   Approximately  10P cells (0.1  ml) from an over-
 night  culture  of each  indicator strain  will  be added to  separate test tubes
 containing 2.0 ml  of molten agar supplemented with  biotin  and a trace of
 histidine.   For  nonactivation  tests  the five dose levels of  the test compound
 will be added  to  the contents  of the appropriate tubes and  poured over the
 surfaces  of  selected agar plates.   In activation tests five  dose levels of
 the  test  chemical  will  be added  to the  appropriate  tubes with cells.  Just
 prior  to  pouring,  an aliquot  of  reaction  mixture (0.5 ml  containing the 9,000
 x £  liver  homogenate) will  be  added  to  each of the  activation overlay tubes,
 which will then  be  mixed, and  the  contents  poured over the  surface of a mini-
 mal agar  plate and  allowed  to  solidify.   The plates will  be  incubated for 48
 hours at  37°C, and  scored for  the  number  of colonies growing on each plate.
 Positive and solvent controls  using  both  directly active positive chemicals
 and those that require metabolic activation will be run with each assay.

 Dosing  Procedures

     •   All types of chemicals can be evaluated with this technique.   Solids,
 liquids, volatile liquids, and gases  have all been screened through appropri-
ate modifications of the  procedure.   Gases are tested by placing the plates
and a measured amount of  the gas in an airtight  container of known volume
for a fixed duration exposure.

Data Collection and Analysis

                                     358

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                                     TABLE 4. POSITIVE CONTROL COMPOUNDS
  Assay
    Chemical
  Solvent
Probable
Mutagenic
Specificity
 Nonactivation
 Activation
Methylnitrosoguanidine
    (MNNG)

2-Nitrofluorene (NF)

Quinacrine mustard (QM)

2-Anthramine (ANTH)

2-Acetvlaminofluorene

8-Aminoquinoline (AMQ)

Dimethylnitrosamine
Water or saline


Dimethylsulfoxidea

Water or saline

Dimethylsulfoxidea

Pimethylsulfoxide3

Dimethylsulfoxide3

Saline
   BPSD



   FSb

   FSb

   BPSb

   FSb

   FSb

   BPSb
Previously shown to be nonmutagenlc.

bBPS - Base-pair substitution.
  FS = Frameshift.

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      •  The raw data will be recorded on printed forms containing all relevant
 information concerning the test procedures.
      QUALITY CONTROL —All data sheets will be signed and dated by the
 responsible technician as the information is recorded.  Copies of all raw
 data sheets will be attached to the final report.  The standard deviation
 and standard error should be calculated for the solvent control plates from
 a minimum of 20 independent assays with each tester organism.  Any data that
 fall outside the accepted range will be rejected.  A complete set of evalu-
 ation criteria is to be provided with each final report.

 3.4.2.5  Indicator organisms (tester strains)—

      Considerable research has been conducted during the past two decades to
 develop sensitive and stable tester strains of microorganisms.  The Salmo-
tiella typhimurium histidine auxotrophs are the best characterized and most
 widely used strains, particularly those developed by Ames and coworkers.
 Various Escherichia coli mutants, Saccharomyces cerevisiae strains, and
 Neurospora crassa mutants are also widely used (Table 3.4.4).

      New strains are continually being developed and laboratories should keep
 in touch with leaders in the field for advice on strain selection.  With
 reverse mutation systems, as many different strains as practicable should be
 used since each strain will detect mutagens which are able to induce a par-
 ticular type of mutation only (Legator and Mailing, 1971).  Dosage of tester
 strains should be as great as possible to insure detection of compounds with
 low mutation rates.  In the Ames test with Salmonella typhimurium, approxi-
 mately 5 x 108 cells are usually used.  In the host-mediated assay with Sal-
 monella, Neurospora, or Saccharomyces strains, the animals are injected with
 about 6 x 10a cells.

      All tester strains should be tested for original markers before starting
 a testing program and monitored frequently during use since some markers are
 somewhat unstable and can be lost.  Ames strains should be routinely checked
 for 1) histidine requirement, 2) deep rough (RFA) characteristic, 3) R factor
 (TA 98 and TA 100), UvrB deletion, and 5) spontaneous reversion rate.

      Spontaneous mutation (reversion) rates on control plates without mutagen
 and S-9 mix after 48 hours for Ames strains are reported to be as follows
 (Ames et al., 1975):

                Strain                Spontaneous mutants/plate

                TA 100                          160

                TA 98                            40

                TA 1538                          25

                TA 1535                          20

                TA 1537                           7

 Rates are slightly higher on plates with S-9 mix.  The normal bacterial spon-
 taneous mutant frequency ranges from 1 x 10   to 1 x lO"1^ (Jawetz et al.,
 1972).

                                      360

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            TABLE 3.4.4  SOME MICROBIAL INDICATOR STRAINS
            AVAILABLE FOR MUTAGENICITY ASSAYS (Brusick et al. 1976)
Oreanism
Salmonella typhimurium







Escherichia coli






Saccharomyces cerevisiae







Strain
G-46
TA-1530
TA-1535
TA-1536
TA-1537
TA-1538
TA-100
TA-98
WP
WPouyrA
K12/343/113
CM561
CM, , ,
661
W3110/P3478
S288Ca
S211
S138
D3
D4
D5
D5
S288C/774-6A
Probable (*)
Event Detected
R-BPS
R-BPS
R-BPS
R-FS
R-FS
R-FS
R-BPS
R-FS
R-BPS
R-BPS
FM
R-BPS
R-BPS

ER
FM
R-BPS
R-FS
MR
MGC
MR
MR
ER
*  R - Reverse; EPS - Base-pair substitution nutation; FS - Frameshift
   mutation; FM - Forward mutation; MR - Mitotic recombination; MGC - Mitotic
   gene conversion; ER - Excision repair
                                    361

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3.4.2.6  Controls—

     Positive controls as well as negative controls  (including vehicle con-
trols) must be included in every test.  Moreover, the positive compound
should be chemically similar to the test chemical.   Positive controls used
by one commercial laboratory are shown in Table 3.4.5.  In Salmonella assays,
dimethylnitrosamine is usually given in the  form of  a single 50 mg/kg oral
dose.  Ethylmethanesulfonate  is generally  administered  intramuscularly at  a
level of 350 mg/kg in Saccharomyces assays  (Green et al., 1976).
 TABLE 3.4.5  POSITIVE CONTROLS USED IN NONACTIVATION AND ACTIVATION ASSAYS
                            (Brusick et al., 1976)

                                                                  Probable
Assay          Chemical                     Solvent              Mutagenic
	Specificity
Nonactiva-
 tion
Activation
Ethylmethanesulfonate
 (EMS)

Methylnitrosoguanidine
 (MNNG)

2-Nitrofluorene (NF)

Quinacrine mustard (QM)

2-Anthramine (ANTH)

2-Acetylaminofluorene

8-Aminoquinoline (AMQ)

Dimethylnitrosamine (DMNA)
Water or saline         EPS*


Water or saline         BPS*


Dimethylsulfoxide**     FS*

Water or saline         FS*

Dimethylsulfoxide**     BPS*

Dimethylsulfoxide**     FS*

Dimethylsulfoxide**     FS*

Saline                  BPS*
*  BPS = Base-Pair Substitution
    FS = Frameshift
                      **  Previously shown to be nonmutagenic
3.4.2.7  Proficiency Testing—

     In spite of the fact that positive as well as negative controls are
usually included in mutagenicity tests, proficiency testing of unknowns
should be an integral part of the quality control program in mutagenicity
testing.  The use of unannounced unknowns which can be slipped into the daily
flow of work together with routine testing slips should give the best indi-
cation of the laboratory's proficiency in the area.  Interlaboratory coopera-
tion with new chemicals is advisable also.

3.4.3  Microorganisms - General Toxicity Testing
                                     362

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     Numerous attempts have been made to develop microbial assays to replace
the costly and time-consuming animal tests for general toxicity testing of
chemicals and other substances.  The greatest achievement to date in this
area appears to be the Ciliastasis assay using ciliated protozoa (Woodard,
1976).  Cilia of human respiratory tract epithelium keep the respiratory sys-
tem free from foreign matter by sweeping a blanket of mucus toward the
esophagus at the upper end of the tract.  The cilia of several species of
Paramecium and Tetrahymena have been found to respond to components of cigar-
ette smoke in a manner quite similar to the response of human tracheal cilia.
The Ciliastasis assay using these test species is regarded by some workers as
having predictive value for effects of smoke and other pollutants on the human
respiratory tract (Ballenger, 1960; Wang, 1963; Kensler and Battista, 1963;
Weiss and Weiss, 1964; Wynder and Hoffman, 1964; Weiss, 1965; Kennedy and
Elliott, 1970).

3.4.3.1  Methods--

     Early toxicity studies with protozoa used growth inhibition as the end-
point.  Protozoa are similar to both undifferentiated prokaryotic micro-
organisms such as bacteria and the  more complicated metazoans in many re-
spects and have been regarded as bridging the gap between the two groups.
Although protozoa are unicellular organisms they have highly developed and
specialized organelles for locomotion and reproduction.  Metabolic and nu-
tritional requirements are even similar to those of mammalian cells (Woodard,
1976).  Their growth in pure culture has been compared to the growth of
somatic tissue cells in multicellular organisms (Jacob, 1958).

     Some of the assays that have been in use recently are outlined in Table
3.4.6.

     Although the Ciliastasis assay with Tetrahymena and Paramecium has been
developed mainly in connection with cigarette smoke toxicity studies, the
test is also applicable for air pollutants, stack emissions, and environ-
mental contaminants contained in soil and water.

3.4.3.2  Experimental Design—

     Dalhamn and Rylander in 1969 made a critical study of methodologies
employed in the toxicologic evaluation of tobacco smoke on the respiratory
system and developed the following guidelines for smoke toxicity experiments:

     •  The smoke should be analyzed and the concentration of particulates
        be determined.
     •  Exposure of the test organisms should be comparable to human exposure
        during smoking with respect to the amount of smoke and duration of
        exposure.   One 35-ml puff drawn for 2 seconds once every minute
        was found to be the smoking method most widely used.
     •  Smoke should be tested as a suspension in air rather than as a solu-
        tion or condensate.
          In humans,  an average of approximately 35 ml of smoke is inhaled
        into the lungs along with about 200 to 300 ml of fresh air.  Exposure
        is short in the upper respiratory tract but is considerably greater
        deep down in the lungs.
                                     363

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              TABLE 3.4.6  TETRAHYMENA AND PARAMECIUM ASSAYS
Strain Used
Toxic Substance
Experimenter
Tetrahymena pyrifonnis
  (Strain E)
Tetrahymena j>.
Tetrahymena £.
Paramecium caudatum
Paramecium aurelia
   (Strain 51)
Paramecium a.
Aminoazob enz ene
Monomethylamlnoazobenzene
Dimethylaminoazobenzene
3'-Methyl-4-dlmethyl-
 aminoazobenzene
Methyl red
8-Azaguanine

Cigarette smoke
Nontobacco smoke
  Lettuce
  Poa pratensis

Benzo(a)pyrene
Tobacco smoke
Tobacco leachate
Tobacco ash
Cigarette paper ash

Cigarette smoke
Jacob (1958)
Kennedy and Elliott
  (1970)

Gray and Kennedy
  (1974)
Epstein et al.
  (1963)

Wang (1963)
Weiss and Weiss
  (1964, 1967)
Weiss (1965, 1968)
      •  Experiments  should  be  of  long  duration  and  should  correspond  to a
        lifetime exposure situation  in humans.

      •  In vivo systems  are preferred  although  in vitro assays are acceptable
        for screening  studies.  Animal species  most closely  related to man
        should be employed.

3.4.3.3  Quality Control-

     All lots of culture media and reagents  should  be  tested for performance
upon receipt and at  regular intervals  thereafter.   Careful attention  should
be paid to the manufacturer's  storage  recommendations  and  expiration  date.
All sterility tests  should  be  conducted in replicate inside  a biological
safety cabinet with  adequate environmental air  controls.

     Negative and positive controls should be included in each test.
Negative controls should Include reagents, vehicle, etc., as well as untreated
cultures.
                                     364

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Positive control  cultures  should be  treated with a substance known to be
toxic to the  tester  strain which is  of the same chemical class as the test
chemical.

     All assays should be  replicated whenever possible with different samples
and on different  days with different lots of the tester organism.

     The tester strains employed by  the authors of an assay procedure should
always be used unless alternate strains have been specified as being accept-
able.  The preparation of  large lyophilized pools of the test organism is
advisable to  eliminate variation in  this component of the assay system.

     Laboratories engaged  in a routine large-scale screening operation may
profit from a proficiency  test program to identify shortcomings and problem
areas.  Unknowns  can be slipped into the daily samples from time to time and
results used  as the  basis  for corrective measures.  Cooperative interlabora-
tory tests are also  valuable in this respect.

     All precision equipment employed in this assay such as incubators, bal-
ances, etc.,  should  be cared for as  outlined in the manufacturer's maintenance
warranty requirements.  Instruments  should be inspected regularly and recali-
brated at intervals  and by procedures recommended by the manufacturer.

     A quality control record book should be maintained for recording all
quality control operations.  All entries should be signed by the responsible
personnel involved.

3.4.4  Cell Cultures - Mutagenicity  Testing

     The applicability of mammalian  cells in culture for mutagenicity testing
was discovered independently by Chu  and Mailing (1968) and by Kao and Puck
(1968).   Both groups used  essentially the same general experimental proce-
dure:
Hour
-4
Inoculation of
petri dish with
tester cells
-2

Addition
of mutagen
rinse
medium
/ J*+ "
-------
sensitivity, and radiation sensitivity  (Chu, 1971; Anon., 1975).  The use of
human cells is limited somewhat due to  the fact that they are genetically
stable in vitro for only about 50 passages and have a low plating efficiency
(about 10%).  However, the field of mammalian genetics is relatively young
and methods for overcoming these technical difficulties may be forthcoming
soon.  The use of nondividing primary human lymphocytes  (S stage) has already
circumvented some of these difficulties.  Moreover, there is no good reason
for believing that mutational effects in other mammalian cells are not the
same as those in human cells (Chu, 1971).

3.4.4.1  Cell Identification and Monitoring —

     In general, mammalian cells used for mutagenicity testing should have
the following minimal characteristics:

          •  High sensitivity
          •  High plating efficiency
          •  Stable karyotype
          •  Low spontaneous mutation rate
          •  Response to metabolic activation
          •  Absence of mycoplasma and  virus

     All cell cultures should be subjected to identification tests upon
receipt and at regular intervals during use.  Many cell lines look alike and
can be identified only by specialized tests.  Incorrect identification can
result from mislabelling by the manufacturer or by contamination during prep-
arat-ion or in the user's laboratory.  Cell cultures can be identified by the
cytotoxic antibody test or by the use of the fluorescent-labelled antibody
test; both tests require cell-specific  antiserum which is available commer-
cially.
     In the cytotoxic antibody test, samples of the cell suspension (1 x
to 2 x 105) are incubated with specific diagnostic antisera at 37°C for one
hour.  Complement or guinea pig serum is then added and the mixture incubated
for another 30 minutes.  Complement will be fixed only by cells combined with
their specific antibody; these cells will then be killed.  A small amount
(0.1 ml) of trypan blue  (0.25%) is added to each tube and the cells are ex-
amined in a hemocytometer under the microscope.  Blue cells are dead; living
cells are white.  If more than 50% of the cells are dead, the test is positive
for the antiserum present (Greene and Charney, 1973).

     The fluorescent-labelled antibody test is a very sensitive procedure for
cell identification and  can also detect contaminants at levels as low as
1/10,000 cells.  Cell samples are treated with specific labelled antibodies
covering the range suspected and then examined under the fluorescent micro-
scope.  Cells will combine only with their specific antibody in significant
numbers.  Cell-antibody  combinations show an "apple-green" fluorescence under
the scope since the antibodies used are labelled with fluorescein isothio-
cyanate.  The cells are  identified, as in the preceding test, by the specific
antibody with which they have combined (Stulberg and Simpson, 1973).

     Mammalian cell cultures sometimes undergo genetic changes - loss of

                                     366

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markers, change in number or character of chromosomes, etc.  - and must be
monitored frequently for genetic stability.  Karyotype analysis is also of
value for identification.  Log phase cultures are treated with colcemid or
velban (0.06 pg/ml) for 2 hours at 37°C.  After rinsing, the culture is
treated with trypsin or pronase to separate cells.  The suspension is then
washed and transferred to a hypotonic medium (medium and H20, 1:2).  The
cells are next collected by centrifugation and fixed with methyl alcohol and
glacial acetic acid (3:1).  Thin suspensions on slides are then stained with
acetic-orcein, rinsed, air-dried and mounted for chromosome analysis.  Various
banding techniques have also been developed for genetic analysis (Hsu, 1973).
Preparation of a large number of single-test ampoules of cells for storage
in liquid nitrogen (-196°C) will greatly reduce the amount of monitoring
necessary (U.S. EPA, 1977).  A complete history of each cell line or strain
used in mutagenicity testing as well as all monitoring results (identity,
karyotype, mycoplasma, viruses, etc.) should be recorded in the quality con-
trol record book.

     Cell cultures must also be monitored frequently for the presence of
mycoplasma (PPLO) organisms.  Mycoplasmas are bacteria-like microorganisms
which lack cell walls and hence are very pleomorphic.  The smallest repro-
ductive units  (100-125 nm) are as small as medium-sized viruses and readily
pass through many filters.  Several species are members of the normal flora
of mouth and genitourinary tract.  Many continuous cell lines are parasitized
by these agents which, unfortunately, usually give no evidence of their pres-
ence (Clive et al., 1973; Hayflick, 1973; Barile, 1973).

     The three main sources of mycoplasma contaminants are personnel (mouth
pipetting), serum (bovines), and typsin (swine).  With the advent of mechani-
cal pipettes, contaminants from workers have decreased greatly.  Today more
than 50% of the contamination comes from serum.  Serum should be inactivated
at 56°C for 30 minutes and then filtered twice through a 220 nm filter;
trypsin is usually filtered through a 100 nm filter (Barile, 1973).

     Mycoplasmas can be isolated in Edward-Hayflick broth or in the semi-
solid broth (SSB) medium of Barile (Barile, 1973), although samples as large
as 25-100 ml may be necessary.  The broth cultures are usually streaked over
an agar medium for the formation of colonies with the characteristic "fried
egg" appearance.  The colonies are very small and require 50-100 x magnifi-
cation for detection.  Moreover, various artifacts - pseudocolonies  (Ca and
Mg soaps), air bubbles, clumps of tissue cells, and condensed water - make
detection difficult.

     Monitoring cell cultures and reagents (serum and tryspin) for mycoplasmas
requires personnel with specialized training and laboratories not so staffed
should engage the services of a mycoplasmatologist for this work.

     Elimination of mycoplasmas from contaminated cell cultures is very
difficult.  According to Barile  (1973) the best method consists of a  combi-
nation of a high-titered antiserum (5% final concentration)  and tetracycline
(10 Mg/ml) or kanamycin (100 pg/ml) in the growth medium.  However,  if  the
contaminated cell type is commercially available, it may be  more  economical
from the standpoint of time and money to purchase a new stock from a reliable

                                     367

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manufacturer who  supplies  certified mycoplasma-free  cultures.

      In addition  to  the  "mycoplasma menace",  cell  cultures may  carry "pass-
enger" viruses without any evidence of  their  presence.  Many human lympho-
blast cultures have  been found  to  contain  the Epstein-Barr virus  (Sato et al.,
1972).  Uncertified  chick  embryo cultures  usually  contain avian leukosis
virus and  the SV^Q virus may  be present in several types of monkey kidney
tissue cultures  (Jawetz  et al., 1972).   Since some viruses cause  chromosome
aberrations and other genetic effects,  mutagenicity  test results  with such
cultures may be grossly  erroneous, and  are always  suspect.

      Discovery of mycoplasmas,  viruses, or any other contaminants in tester
strains requires, of course,  complete re-assay of  all mutagenicity test
compounds  back to the last previous certification  that  the cultures were
"clean".

3.4.4.2  Cell Population—

     The number of cells employed  in a  mutagenicity  assay should  be sufficient
to detect  mutation at double  the spontaneous  mutation frequency,  at least,
of the tester strain (U.S.  EPA, 1977).   In general,  sensitivity is directly
proportional to the  number of cells used.   However,  several workers have
found that increasing the  cell  population  often decreases the mutation fre-
quency.  Chu and  Mailing (1968), for example,  found  that increasing the cell
inoculum from 2.5 x  105  to 10 x 10s decreased the  mutation frequency from
11.3 to 0.4/105 survivors,  in assays using Chinese hamster V-79 cells to de-
tect mutation at  the glutamine  (gin) and 8-azaguanine (azg) loci.  Shapiro
et al. (1972) discovered that the  "concentration effect" varied with the cell
type.  Increasing the concentration of  Chinese hamster  cells from 101* through
1(P to 106 decreased the mutation  frequency.

     With  certain human  cell  lines (L-53 and  L-54) increase of  the cell popu-
lation at  the same rate  (10**  to 106) caused an increase in the  mutation
frequency.  In all mutagenicity tests,  the plating population to  be used with
the selective medium which is optimal for  the survival  of mutants must be
determined in preliminary  tests before  assays are  conducted (Shapiro et al.,
1972).  In general,  any  mutagenicity assay system  should be calibrated with
known positive and negative mutagens before testing  is  begun.

3.4.4.3  Dosage of Test  Chemical

     Two of the most widely used cell culture assays  are Unscheduled DNA
Synthesis  (with and without microsomal  activation) and  the host-mediated
assay using mouse lymphoma L5178Y cells  (Anon.  1975;  U.S. EPA,  1977; de Serres,
1974; Legator, 1976).

     Dosage varies considerably in unscheduled  DNA synthesis assays as well
as in the host-mediated  assay.  In both  types of test the determining factor
is mainly  toxicity of the chemical (Lieberman  et al., 1971; Stich and San,
1970; Clarkson and Evans, 1972).  A wide range  of doses should  be used.   The
maximum dose must cause  some  toxic effect  and  should  be sufficiently large
to detect weakly-acting mutagens; at least  four lower doses, appropriately

                                     368

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spaced, should be included (U.S. EPA, 1977).  If toxicity of the compound is
not known, the dosage range may be based upon results of a preliminary tox-
icity test (Brusick et al., 1976).  The tester cultures should be exposed to
action of the chemical (dosing period) for at least one hour (U.S. EPA, 1977).
It is important to test technical grades and formulations as well as purified
specimens in the case of commercial chemicals (U.S. EPA, 1977).  Typical
dosage ranges reported in recent assays by various workers are shown in
Table 3.4.7.

     In the host-mediated assay using mouse lymphoma L5178Y asn~ cells in BDF
male mice, Capizzi et al. (1973) obtained 39 mutants per 106 cells with sulfur
mustard at a dosage 100 mg/kg.  In vitro, the chemical-induced mutation
occurred at a dosage as low as 0.001 yg/ml.

3.4.4.4  Methods—

     •  Unscheduled DNA Synthesis

     The unscheduled DNA synthesis mutagenicity test is based upon the assump-
tion that mutagens damage DNA and that this effect can be detected by the
incorporation of DNA precursors into the DNA of nondividing cells (Stoltz
et al., 1974).  Unrepaired or misrepaired DNA damage will result in gene mu-
tations or other changes which affect gene function (U.S. EPA, 1977).

     A variety of mammalian cell cultures, Including Syrian hamster, Chinese
hamster, normal human cell strains (WI-38, etc.) as well as neoplastic human
cell lines (HeLa, etc.) have been used in unscheduled DNA synthesis assays
(Brusick et al., 1976; Painter and Cleaver, 1969; Stich and San, 1970).  Pri-
mary peripheral human lymphocytes have also been used by some workers (de
Serres, 1974; Lieberman et al., 1971; Clarkson and Evans, 1972).  Standardized
human cell strains from reliable repositories are recommended (U.S. EPA,
1977).
       TABLE 3.4.7
INDUCTION OF UNSCHEDULED DNA SYNTHESIS BY VARIOUS
 COMPOUNDS IN VITRO;  DOSAGE RANGE
Chemical
Dosage Range
    Exposure Cell
    Period   Type
Reference
4-Nitroquinoline    1 * 10"5 to"5
 1-oxide
Nitrogen mustard    10'1 to 10~7 M
 (NH2) Ethylmethane-
 sulfonate (EMS)
 Methylmethanesul-
 fonate (MMS)

Nitrogen mustard    10'** to 10~5 M
 (NH2)
M   1.5 hr.  Human;
             Chinese
             hamster

    1.0 hr.  Human
             lympho-
             cytes
                         1.0 hr.  Human
                                  lympho
                                  cytes
                                             Stich and
                                             San, 1970
                                             Lieberman
                                             et al.,
                                             1971
                         Clarkson
                         and Evans,
                         1972
                                     369

-------
     A protocol  for Unscheduled DNA  Synthesis  (UDS)  in Human WI-38 Cells is
given in the  following  pages.

         EXAMPLE:  UNSCHEDULED DNA SYNTHESIS (UDS)  IN HUMAN WI-38 CELLS

Purpose of Study

     •  Mutagenicity  determination

Design of Experiment

     •  Cell  Cultures:   Normal human diploid WI-38  cells are seeded at
250,000 cells  in 60 mm  tissue culture dishes.  The  cells are grown to con-
fluency in Eagle's Minimum  Essential Medium (MEM) plus 102 fetal calf serum
(PCS).  They  are then kept  in MEM containing 0.5% PCS for 5 days.

     •  Dosage of Test  Substance:  Dosages are determined from preliminary
toxicity curves  established  from treatment with 1.0, 0.1, 0.01, and 0.001%
levels of the  test substances; three dose levels of each substance are select-
ed for mutagenicity testing.

     •  Controls:  Positive  and negative (solvent and untreated) controls are
included in each test.

Conduct of Experiment

     •  Nonactivation Assay:  Nonproliferating cultures, arrested in Gj
phase of the cell cycle by contact inhibition and 0.5% PCS synchronization
medium, are exposed to  three doses of the test substance determined as indi-
cated above.   Tritiated thymidine is added to the cultures along with the
test substance.  In order to prevent any scheduled  DNA synthesis from taking
place, hydroxyurea is added  to the cultures one hour before addition of the
test substance and is included in the medium at each change.  Exposure to
both test substance and radioactive  label is terminated by washing the cells
in Hanks'  Balanced Salt Solution that contains an excess of unlabelled thy-
midine.
     QUALITY CONTROL  --  The entire mutagenicity assay system should be cali-
brated with known positive and negative mutagens and promutagens before
routine testing  is begun.
     The cell cultures  must be free  from mycoplasma contaminants which are
capable of incorporating radioactive thymidine in addition to causing other
effects which  invalidate assays.

Conduct of Experiment

     •  Activation Assay:  The activation assay is identical to the noninacti-
vation assay except that an aliquot of 100,000 g liver microsomal extract
containing the following components is included in the incubation of cells
with the test chemical:
                                     370

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               Component                       Final Cone./ml

               TPN  (sodium salt)                  6 pinoles
               Isocitrlc acid                    35 pmoles
               Trls buffer, pH 7.4               28 pinoles
               MgCl2                              2 pinoles
               Liver extract equivalent
                to 25 mg fresh tissue

     QUALITY CONTROL — Donor animals for the microsomal extract should be
pre-induced with a compound known to be effective for the class of substances
to be tested.

     •  Radioactivity incorporated by cells during exposure to test substance
and radioactive thymidine indicates unscheduled DNA synthesis and hence DNA
repair.
     QUALITY CONTROL — Glassware, pipettes, water, media, and reagents must
be of tissue culture grade.  Media and reagents (including tritiated thymi-
dine) must be performance-tested before use.

     •  Amount of tritiated thymidine incorporated into DNA during repair is
determined by solubilizing the cells and extracting the DNA.

     •  One aliquot of the DNA extracted is processed for determination of
the amount of radioactivity by scintillation counting.
     QUALITY CONTROL — All tissue culture work should be performed in lami-
nar flow cabinets equipped with HEPA filters to safeguard against airborne
microbial contaminants.

     •  A second portion is used to determine spectrophotometrically the
amount of diphenylamine-reactive DNA.
        Results are expressed as radioactivity (DPM) per milligram of DNA.

     In unscheduled DNA Synthesis Assays it is essential that the cell cul-
tures be maintained in the nondividing s-state since incorporation of
tritiated thymidine in scheduled DNA synthesis during mitosis would over-
shadow that involved in repair of the mutagen-damaged DNA (Anon., 1975).  In
assays with peripheral human lymphocytes, hydroxyurea (HU) is used to keep
the occasional cell not in the s-state from replicating (Lieberman et al.,
1971).  Arginine-deprivation has been used by Stich and others to prevent
replicative DNA synthesis in a variety of other cell types (Stich and San,
1970; Stich et al., 1971).   It is essential that the laboratory assess the
system employed before starting mutagenicity testing to make certain that
replicative DNA synthesis does not occur in the test system.

     Incorporation of tritiated thymidine (3H-TdR) by nondividing cells is
usually used as the indicator of DNA repair of mutagen-induced damage (Anon.,
1975; U.S. EPA, 1977; Lieberman et al., 1971; Stich and San, 1970).  The
labelled reagent may be added with the test compound in the continuous method
or pulsed for an appropriate period only.  Incorporation of 3H-TdR is measur-
ed by autoradiography or by scintillation counting using standard techniques
(Stich and San, 1970; Stich et al., 1971; Lieberman et al., 1971).

                                     371

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      Acceptance specifications of each lot of tritiated thymidine should be
 carefully checked upon receipt.   Acceptable lots must also be performance
 tested with both positive and negative standards.  Source, lot number,  purity,
 concentration,  and specific activity of all preparations used should be in-
 cluded in the mutagenicity test report (U.S. EPA, 1977).

      •  Host-mediated Assay

      The host-mediated assay with mouse lymphoma cells (L5178Y) is the  second
 major mammalian in vitro cell test widely recommended for mutagenicity  test-
 ing.   The L5178Y strain has been used extensively for a number of years in  a
 variety of studies and is very well characterized.   It has an essentially
 diploid karyotype, a high plating efficiency, and grows well in vitro (Anon.,
 1975; U.S.  EPA, 1977; de Serres, 1974; Legator,  1976)  Tumorgenicity is not
 regarded as a drawback in mutagenicity testing since mutagenic mechanisms of
 tumor cells and normal cells are believed to be  essentially the same.   More-
 over, the induced thymidine kinase (TD) mutation rate with L5178Y was found
 to  be essentially the same as in other mammalian cells in cultures (de  Serres,
 1974).

      A protocol for the Mouse Lymphoma Forward Mutation Assay is given  in the
 following pages.

              EXAMPLE:   MOUSE LYMPHOMA FORWARD MUTATION ASSAY

 Purpose of Study

      •  Mutagenicity determination

 Design of Experiment

      •  Indicator Cells:   L5178Y Thymidine kinase (TK+/~)  mouse lymphoma
 cells are used.   The cells are heterozygous for  a specific autoxomal mutation
 at  the TK locus and are bromodeoxyur1d1ne (BUdR)-sens1t1ve.   Scoring  for
 mutation Jsjbased on_se_lect1ng cells  that have undergone forward mutation
 from  a TK /   to a TK /   genotype through the use of  BUdR-supplemented soft
 cloning agar.

      •   Media:  Maintenance medium -  Fischer's mouse leukemia medium with 10%
 horse serum and sodium  pyruvate.

      Cloning medium - Fischer's  medium with 20%  horse serum,  sodium pyruvate,
 and 0.37% agar.

      Selection medium - Cloning  medium  plus  0.5 mg  of BUdR/100 ml.

      •   Dosing Procedure:  All types of chemicals can  be evaluated 1n the
mouse  lymphoma assay.   Solids  are  dissolved  1n suitable solvents and added to
 test  system at seven predetermined levels.   Liquids  are added directly to the
 cultures  at seven concentrations or following  dilution  1n  appropriate solvents.
 Highly  volatile liquids  (vapor phase test required)  are added at seven dosages
 to an air-tight container of fixed volume and  allowed  to completely volatilize

                                     372

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1n presence of the exposed cell population.  Known volumes of gases are meas-
ured Into an air-tight container of fixed volume.  The volume of gas is graded
at seven dose levels.

     For most chemicals, seven concentrations are selected for assay on the
basis of a preliminary toxicity test.  At least four of the dosages are then
cloned for the mutagenicity evaluation.

     •  Vehicle:  Tissue culture growth medium or dimethylsulfoxide (at 1.0%,
or lower, final concentration) are used as solvents for the test substances.

     •  Controls

     Positive control:  Ethylmethanesulfonate (200 yg/ml), which induces
mutation by base-pair substitution, is used for nonactivation tests.  Dimethyl-
nitrosamine (500 yg/ml, which requires metabolic biotransformation by
microsomal enzymes to induce mutation, is employed in the activation test.

     Negative control:  The solvent in which the test substance is dissolved
serves as the negative control.

Microsomal activation system

     Male randon-bred mice are used as the source of hepatic microsomes.
The mice are killed by cranial blow, decapitated, and bled.  The liver is
immediately dissected from the animal using aseptic technique and placed in
ice cold 0.25 M sucrose buffered with Tris buffer at pH 7.4.  When an adequate
number of livers has been collected, they are washed twice with fresh buff-
ered sucrose and completely homogenized.  The homogenate is centrifuged for
10 minutes at 9,000 x £ in a refrigerated centrifuge.  The supernatant from
this centrifuged sample is retained and frozen at -80°C until used in the
activation system.  This microsome preparation 1s added to a "core" reaction
mixture to form the activation system described below:

          Component                           Final Concentration/ml

          1.  TPN (sodium salt)                      6 ymoles
          2.  Isocitric acid                        35 ymoles
          3.  Tris buffer, pH 7.4                   28 ymoles
          4.  MgCl2                                  2 ymoles
          5.  Homogenate fraction equivalent
               to 25 mg of wet tissue

        SUMMARY OF TESTS INCLUDED IN THE L5178Y MOUSE LYMPHOMA ASSAY

          Test                                     L5178Y	

                                             Trail A       Trial B

                                          Nonactivation  Activation

          1.  Solvent Control                 ,   X              X
                                     373

-------
                             SUMMARY  (Continued)

          Test                                     L5178Y
                                             Trial A       Trial B

              Positive  Control             Nonactivatlon  Activation
                 Ethylmethanesulfonate           X
                 Dimethylnitrosamine                           X
              Test Chemical
              Dose Level
                   1                             X             X
                   2                             XX
                   3                             XX
                   4                             XX

Conduct of  Experiment

      •  Nonactivation Assay:  A modification of the procedure of Clive and
Spector (1975)  is  used.   Prior to treatment with^ the test substance, the
indicator cells are cleaned of spontaneous TK /  mutants by growing them in
a medium containing thymidine, hypoxanthine, methotrexate, and glycine (THMG).
This  medium permits survival of only those cells that produce the enzyme
thymidine kinase and can  therefore utilize exogenous thymidine from the medium.
The cleansed cells are exposed to the test substance (solid, liquid, volatile
liquid, or  gas) at predetermined doses for five hours.  The treated cells are
then  washed, fed,  and allowed to express in growth medium for 3 days.  Daily
counts are  made.
      QUALITY CONTROL — The assay system should be calibrated with known posi-
tive  and negative mutagens and promutagens before routine testing is begun.
      Indicator cells should be free from Mycoplasma contaminants and "passen-
ger11  viruses.

      •  Activation Assay:  The activation assay differs from the nonactlvation
test  in the following manner only.  Two ml of the activation mixture is added
to 10 ml of growth medium.  The desired number of cleansed cells 1s then added
to this mixture and the flask incubated on a rotary shaker for five hours.
The incubation is terminated by washing the cells twice with growth medium.
The washed, treated cells are then allowed to express for three days as
described above for the nonactivatlon assay.
      QUALITY CONTROL — Donor animals for microsomal extract should be pre-
induced with a compound known to be effective for the class of substances to
be tested.

Observations and Tests

      •  At  the end of the three day expression period described above, TK~7~
mutants are detected by cloning the cells In the selection medium for ten days.
The surviving cell population is determined by plating diluted aliquots in
nonselective growth medium.

      •  A mutation index  is derived by dividing the number of clones formed

                                     374

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in the BUdR-containing selective medium by the number found in the same medium
without BUdR.  The ratio is then compared to that obtained from the other dose
levels and from positive and negative controls.  Compound-related toxicity
based on cell growth in suspension and cloning efficiencies are also included
in the final report.


3.4.4.5  Quality Control

     •  Control Preparations

     The laboratory should assess the sensitivity and reproducibility of all
assay systems to be used, with appropriate positive and negative controls,
prior to the beginning of mutagenicity testing.  Interlaboratory testing is
strongly recommended to determine reproducibility of the testing procedures.
In each individual assay, the laboratory must include positive controls of
the same chemical class as the test compound as well as vehicle and untreated
controls (U.S. EPA, 1977).

     •  Tissue Culture Glassware, Media, Reagents, etc.

     Mammalian cells growing in vitro are usually quite delicate and very
fastidious with respect to nutrients.  Scrupulously clean glassware as well
as properly-formulated media are required for satisfactory results.

     Most laboratories today use disposable polystyrene plastic or soda glass
containers.  Plastic containers may be used as packaged although laboratories
should be careful to use the brand specified by author of the method since
some brands have been found to be toxic.  Soda glass containers must be washed
or rinsed before use.  Most items are machine-washed with special detergents
and rinsed at least 12 times since very minute amounts of detergents or clean-
ing compounds are toxic; the final rinses should be with demineralized water.
The best quality borosilicate glass must be used for storage of cells or for
continuous cultures carried in the laboratory  (Coriell, 1973b).   All lots of
containers, pipettes, syringes, etc., should be tested in replicate with the
cell type to be used prior to mutagenicity testing.

     The water used in tissue culture work must be of the highest purity.
Conductivity should be in the range of 1 to 2 x 106 ohms.  The use of a mixed
bed ion exchanger followed by glass distillation is regarded as the best pro-
cedure.  Teflon or borosilicate glass carboys are preferable for storage
although water for tissue culture purposes should not be stored for long
periods of time at room temperature.  Great care must be taken to avoid con-
tamination by pyrogens.  Some laboratories store batch lots of water in the
refrigerator (Pumper, 1973).

     Many laboratories use commercial tissue culture media which require  only
the addition of the usual supplements such as  serum, glutamine, etc.  These
basal media from reputable manufacturers are usually of very high  quality and
require monitoring only infrequently.  Supplements and special reagents,  how-
ever, require strict monitoring.  In a one-year survey  (1968-1969)  of  fetal
calf serum, 10% of all lots were contaminated  with bovine  viruses,  bacteria

                                      375

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and mycoplasmas  (Boone, 1973).  In another survey, trypsin accounted for
almost 20% of all mycoplasma contamination (Barile, 1973).

     All tissue  culture work should be performed in laminar flow cabinets \
equipped with HEPA filters.  Masks should be worn and the hair covered.  Tilk-
ing should be reduced to a minimum and gum-chewing is taboo.  Scrupulous   '
personal hygiene should be encouraged.  Floor, walls, and work surfaces should
be scrubbed with disinfectant prior to work.  In addition to these elaboratti
precautions, a detailed performance and sterility testing program, covering
all media, reagents, and materials, should be instituted.  All instruments
and other equipment should be recalibrated at stated intervals and checked
frequently.  Freezers should be equipped with an alarm system.  All quality
control data should be entered into a bound notebook, dated, and signed by
responsible personnel.

3.4.5  Cell Cultures  - Carcinogenicity Testing

     It is estimated  that  there are at present approximately  two million known
chemical compounds, and over 30,000 of these are now in  commerce.  Our daily
exposure to many of these  chemicals in food, water, cosmetics, and in the
environment has  caused increasing  concern over their possible carcinogenic
and/or other toxic effects  (Anon., 1973).   A heroic attempt  is being made to
evaluate this hazard  through definitive tests in mammals but  the burden is too
great from the standpoint  of time, money, and personnel.  It  is becoming in-
creasingly apparent that rapid, sensitive, and reliable  in vitro methods are
needed to screen the  large number  of  new chemicals created each year in addi-
tion to the huge backlog of untested  compounds to which  society is now exposed
(Woodard, 1976).  A concerted effort  is being made in this direction and
Stich et al. (1975) in a recent survey of the problem list no less than 26
bioassays which  show  promise for the  detection of chemical carcinogens (Table
3.4.8).  Of this array, four are generally regarded as being the most promis-
ing for screening purposes  (Stoltz et al., 1974; Stich et al., 1975):

          •  Cell transformation

          •  Unscheduled DNA synthesis (DNA repair synthesis)

          •  Ames Salmonella test

          •  Drosophila melanogaster  recessive mutation  test

     Although transformation is the only recommended test which is directly
related to the carcinogenic process,  all have given good results in the de-
tection of chemical carcinogens (Anon. ,1973; Stoltz et al., 1974).  However,
it should be noted that none of these tests at this time will absolutely
identify a carcinogen.  Their main value at present is to provide a rapid main
screening system to detect "high risk potent carcinogens" and thus greatly
reduce the animal assay load in definitive carcinogenicity testing (Anon
1973).  At present, all positive cell transformation tests must be confirmed
by production of cancer in animals following the injection of the transformed
cells.  Cell transformation, at this  time, is thus an in vitro-in vivo assay
procedure (Anon., 1971).

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               TABLE 3.4.8  PROMISING BIOASSAYS FOR THE DETECTION
               OF CHEMICAL CARCINOGENS (Stich ex al.f 1975)
Test Species
   Effect
Reference
MOLECULES
- B. subtills, D. pneumonias
  H. influenza, H. streptococci
- DNA

VIRUSES
- E. coli K-12(X)

MICROORGANISMS
- Salmonella typhimurium

- B. subtills

- E. coli (exc , polA , rec )
   Genetic transfor-
   mation
   Flourescence
   Phage induction
Herriott, 1971; Maher
et al., 1970
Morgan and Pulleyblank,
1974

Heinemann, 1971
   Frame shift muta-   Ames et al. 1973a, b
   tions, base pair
   substitution, "rec"  Kada et al., 1972
   assay               Slater et al., 1971
   Differential kill-  Ishii and Konod, 1975
   ing, Mutations
EUKARYOTES
- Tetrahymena pyriformls
- Saccharomyces cerevisiae
 - Neurospora crassa

 - Aspergillus nldolans
 PLANTS
 - Vicia, Pisum, Alllum
 - Hordeum vulgare

 INVERTEBRATES
 - Drosophila melanogaster
 - Bombyx mori
   Unequal division
Mutations, gene con-
version
Mitotic crossing over
UV~, rec", differen-
tial killing
Mutations, and -3
region
Nondisjunction
Crossing over
Moutan and Fromageot,
1971
Mita et al., 1969

Zimmermann, 1975
 Koske and Stich, 1973
 Fahrig, 1974

 De Serres, 1974
                                                       Bignami et al., 1974
Chromosome aberrations  Kihlman, 1966
Chromosome aberrations  Wuu and Grant, 1966
Mutations
  Sobel,  1974;  Vogel,  1971
  Tazima  and Onimaru,  1974
                                                                Continued
                                     377

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                          TABLE  3.A.8   (Continued)
Test Species
Effect
Reference
MAMMALS
- Bone marrow  (rodents)
- Cultured cells
- Sperm

MAN
- Peripheral lymphocytes
- Lymphocyte cultures
- Cultured cells
- Urine extracts

HOST-MEDIATED ASSAY
- S. typhimurium

- N. crassa
- S. cerevisiae

- Mouse lymphoma

- Human lymphocytes
Chromosome aberrations
Micronucleus test

Chromosome aberrations
Mutations
DMA fragmentation

DNA repair

Transformation


Morphological anomalies


Chromosome aberrations

Micronucleus test
Chromosome aberrations
DNA fragmentation
DNA repair
Fish tumors
Mutations
Mutations

Chromosome aberrations
Barthelmess, 1970
Schmid, 1975; Heddle,
  1973
Barthelmess, 1970
Chu, 1972; Clive, 1974
Laishes and Stich,
  1973a,b
Stich et al., 1972a
San and Stich, 1975
Kuroki, 1974; Heidel-
  berger, 1973
Di Paolo et al., 1969
Wyrobek et al., 1975
Evans and O'Riordan,
  1975
Heddle, 1973; Schmid,
  1975

Barthelmess, 1970
Stich et al., 1973;
San and Stich, 1975
Campbell et al., 1974
Gabridge and Legator,
  1969;
Legator, 1970;
Legator and Mailing,
  1971
Clive et al., 1973;
  Fischer, 1973
Fischer et al., 1974;
  Brewen, 1975
                                     378

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3.4.5.1  Cell Transformation Assay—

     Cell transformation refers to the conversion of normal cells in culture
to malignant cells through the action of a chemical, virus, or other carcino-
gen.  Cell transformation is evidenced by the formation of colonies with the
following characteristics:

          •  Piled-up, criss-cross, spindle-shaped cells
          •  Hereditary random growth pattern
          •  Loss of contact inhibition and polar orientation
          •  Ability to grow indefinitely in vitro
          •  Relatively resistant to the toxic action of the carcinogen
          •  Stain heavily with Giemsa
          •  Cause cancer in animals

     Normal cells in culture, on the other hand, form a confluent monolayer
of polar-oriented cells of normal morphology which are susceptible to the
toxic action of carcinogens and have a limited life span in vitro; they do
not cause cancer when injected into animals.

     The mechanism of malignant transformation of cells in culture by chemi-
cal carcinogens has not been determined as yet.  Three theories, however,
with a certain amount of evidence in their support, have been advanced (Chen
and Heidelberger, 1969):

          •  Direct transformation of normal cells to cancer cells
          •  Activation of a latent cancer virus
          •  Selection of pre-existing cancer cells

     The two main physiologic abnormalities manifested by mammalian cells
transformed by chemical carcinogens in culture are:

          •  An increased aerobic glycolysis
          •  Inhibition of respiration by addition of
             glucose in presence of pyruvate (Crabtree Effect)

These changes are characteristic of many malignant cell lines and appear to
be correlated with the grade of malignancy (Sato et al., 1970).

     The species of serum employed in the cultivation of cells in vitro
appears to have considerable influence on the development of neoplastic trans-
formation.  Sanford et al. (1972) found that C3H mouse embryo cell cultures
grown in NCTC-B5 medium containing gelding horse serum regularly underwent
malignant transformation between 98-188 days in culture.

     Evans et al. (1972), in a follow-up study, found that fetal bovine
serum and mare serum delayed neoplastic transformation of the mouse cell line
whereas stallion and gelding horse serum hastened the change.  Neoplastic
transformation was found to be not associated with growth-stimulating capacity
of the sera but was believed to be related to the hormones present.

     The major disadvantages in the use of cell transformation as a carcino-

                                    379

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genicity assay method are (Anon., 1971; Anon., 1973):

          •  High rate of "spontaneous cell transformation" in certain cell
             lines, particularly mouse embryo cultures.  This does not appear
             to be a problem, however, with hamster embryo cells.
          •  The effect of culture medium constituents, such as the species
             of serum, on transformation.
          •  Lack of definitive changes in cell morphology which are indica-
             tive of transformation in some cell systems, e.g., rat liver.
             Certain other lines, however, such as hamster embryo, mouse pros-
             tate, mouse and rat embryo cultures show characteristic morpho-
             logical changes when treated with carcinogens.
          •  Necessity of confirming positive results by animal inoculation.
          •  A standard activation system is not available for piecarcinogens
             which require metabolic activation to produce malignancy.  How-
             ever, on the basis of results with rat liver cultures obtained
             by Williams et al. (1973), rodent liver mlcrosomal preparations
             such as used in the Ames mutagenicity test may be satisfactory.
          •  Methodology

     Among the cell lines in use for screening chemicals for carcinogenicity
are:  Hamster embryo cells; C3H mouse prostate cells; rat liver cells; rat
embryo cells; mouse embryo cells; Syrian hamster chondrocytes; and 3T3 cell
line (derived from C3H mouse embryos).

     A protocol for in-vitro transformation of BALB/3T3 cells is given in the
following pages.

     •  Carcinogens detected by cell transformation assays

     Carcinogenic chemicals which have been detected in vitro by cell trans-r
formation assay systems listed above are presented in Table 3.4.9*

     •  Sensitivity of cell transformation assays

     Strong  carcinogens induce cell transformation in the assay systems
described above at levels as low as 0.01 yg/ml (Table 3.4.10).

3.4.5.2  Unscheduled DNA Synthesis  (DNA Repair Synthesis) Assay—

     Unscheduled DNA synthesis or DNA repair synthesis bioassays are based
upon the fact that most cells are capable of repairing certain types of DNA
damage brought about by X-rays, chemicals, etc., by enzymatlcally excising
the damaged  portion, resynthesizing the correct sequence of components, and
inserting and sealing the new portion of the cellular DNA strand.  Nondivid-
ing cells must be used so that DNA repair synthesis is not overshadowed by
normal s-phase replicative DNA synthesis.  DNA repair synthesis can be meas-
ured by the  incorporation of DNA precursors, such as trltiated thymidine
(3H-TdR) into cellular DNA by nondividlng cells, by means of autoradiography
or scintillation counting.

     DNA repair synthesis has not been used as widely as cell transformation

                                     380

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    TABLE 3.4.9  TRANSFORMATION OF CELL CULTURES BY CARCINOGENS IN VITRO
Carcinogen
Cell Culture
Reference
3-Methylcholanthrene
ll-Methylcylopenta(a)-
  phenanthrene
Dimethylnitrosamine
N-Nitrosomethylurea
N-2 Fluorenylacetamide
N-Acetoxy-2-fluorenylacetamide
N-Hydroxy-N-2-
  fluorenylacetamlde
4-Nitroquinoline-l-oxide
4-Hydroxyaminoquinoline-N-
  oxide
Aflatoxin B.
N-Methyl-N-Nitro-N-
  Nitrosoguanidine
Methylazoxymethanol
Cigarette smoke condensate
Polycyclic hydrocarbons
Benzanthracene
10-Methylbenzanthracene
1,2,5,6-Dibenzanthracene

7,12-Dimethylbenzanthracene
9,10-Dimethylbenzanthracene
4-Fluoro-10-methyl-l,2-
  benzanthracene
3,4-Benzo(a)pyrene

3-Hyroxybenz(a)pyrene
Rat embryo

Mouse embryo

Hamster chondrocytes
Hamster embryo

Hamster embryo
Rat liver
Hamster embryo
Hamster embryo
Hamster embryo

Hamster embryo
Hamster embryo
Hamster chondrocytes
Hamster embryo

Rat liver
Hamster embryo

Hamster embryo
Hamster embryo
Hamster embryo
Hamster embryo
Hamster embryo     '
Mouse prostate
Hamster embryo
Rat embryo
Rat liver
Mouse embryo

Mouse prostate
Mouse prostate
Hamster embryo
Mouse prostate

Hamster embryo
Hamster embryo
Hamster embryo
Hamster embryo
Mouse prostate
Marquardt and Heidelberger
  (1972)
Marquardt and Heidelberger
  (1972)
Katoh (1977
Di Paolo et al. (1972)

Huberman et al. (1968)
Williams et al. (1973)
Di Paolo et al. (1972)
Di Paolo et al. (1972)
Di Paolo et al. (1972)

Sato et al. (1970)
Di Paolo et al. (1972)
Katoh (1977)
Di Paolo et al. (1972)

Williams et al. (1973)
Di Paolo et al. (1972)

Di Paolo et al. (1972)
Rhim and Huebner  (1973)
Di Paolo et al. (1972)
Benedict et al. (1972)
Berwald and Sachs  (1965)
Chen and Heidelberger(1969)
Berwald and Sachs  (1965)
Rhim and Huebner  (1973)
Williams et al. (1973)
Marquardt and Heidelberger
  (1972)
Chen and Heidelberger(1969)
Chen and Heidelberger(1969)
Berwald and Sachs  (1965)
Chen and Heidelberger(1969)

Benedict et al  (1972)
Huberman et al  (1976)
Benedict et al. (1972)
Berwald and Sachs (1965)
Chen and Heidelberger(1969)
                                                               Continued
                                      381

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                          TABLE 3.4.9  (Continued)
Carcinogen
Cell Culture
Reference
Cigarette tar
City smog

Negative Chemicals
Urethane

N-Hydroxyurethane
Diethylnitrosamine
1,2,3,4-Dibenzanthracene

2-Fluoro-10-methyl-l,2-
  benzanthracene
8-Methylbenz(a)anthracene
Pyrene

Chrysene
Hamster lung
Rat embryo
Hamster embryo
Hamster embryo
Hamster embryo
Hamster embryo
Mouse prostate

Mouse prostate

Hamster embryo
Mouse prostate
Hamster embryo
Hamster embryo
Inui and Takayama (1971)
Freeman et al. (1971)
Berwald and Sachs (1963)
Di Paolo et al. (1972)
Di Paolo et al. (1972)
Di Paolo et al. (1972)
Chen and Heidelberger
  (1969)
Chen and Heidelberger
  (1969)
Berwald and Scchs (1965)
Chen and Heidelberger(1969)
Berwald and Sachs (1965)
Berwald and Sachs (1965)
                                      382

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                       TABLE  3.4.10  SENSITIVITY  OF CELL TRANSFORMATION ASSAYS
Dose
5 yg/ml
1 yg/ml
0.05 yg/ml
0.01 yg/ml
0.01 yg/ml
0.03 yg/ml
£ 6.00 yg/ml
u>
0.015 yg/ml
1.0 yg/ml
0.4 yg/ml
Carcinogen
MCA
BP
AB1
DMBA
BP
3-HO-BP
BA
DMBA
MCA
MCA
Assay System
Hamster chondrocytes
Hamster embryo
Rat liver
Rat embryo
Hamster embryo
Hamster embryo
Hamster embryo
Hamster embryo
Mouse prostate
Mouse prostate
Treatment
Period
3 days
3 days
10 weeks
6 days
7 days
7 days
7 days
7 days
6 days
6 days
Transformation
observed
41-61 days

32 days
40-43 days
7 days
7 days
7 days
7 days
126 days
10-14 days
Reference
Katoh (1977)
Huberman et al. (1976)
Williams et al. (1973)
Rhim and Huebner (1973)
Benedict et al. (1972)
Benedict et al. (1972)
Benedict et al. (1972)
Benedict et al. (1972)
Chen and Heidelberger
(1969)
Chen and Heidelberger
(1969)
MCA - 3-Methylcholanthrene




BP  - Benzo(a)pyrene




AB  - Aflatoxin B





DMBA - 7,12-Dimethylbenzanthracene
3-HO-BP - 3-Hydroxybenz(a)pyrene




BA - Benzanthracene

-------
for carcinogenicity  testing  of  chemicals  but  two  studies  of  considerable magni-
tude have  indicated  the potential  value of  this system  in detecting  chemical
carcinogens.

     •  Human  fibroblast  cell cultures

     Stich et  al.  (1975)  tested 98 different  carcinogens,  precarcinogens,
and noncarcinogens in  a DNA  repair synthesis  assay  system consisting of
cultured human fibroblasts.

     All but two of  29 carcinogens gave positive  results  in  the assay  (6.9%
false negatives); all  28  noncarcinogens yielded negative  results  (0% false
positives); 11 of 30 precarcinogens were  negative and apparently  require
metabolic  activation to induce  malignancy.

     •  Rat Liver Cell Cultures

     Williams  (1977) used a  primary rat liver cell  culture system for  car-
cinogenicity testing of chemicals  based on  the fact that  the liver contains
all of the enzyme systems fpr metabolizing  all chemical precarcinogens known
to require metabolic activation.   All compounds were strongly positive with
the exception  of one weak carcinogen and  the  4 noncarcinogenic control chemi-
cals.

     The author points out that primary cultures  must be  used since  continuous
cell lines lose much of their metabolic activity  and would be insensitive to
precarcinogens.

                          EXAMPLE:   CELLULAR BIOASSAY
                  IN-VITRO TRANSFORMATION OF  BALB/3T3 CELLS

Purpose of Study

     •  Carcinogenicity determination

Design of  Experiment

     •  The transformation system  used is a quantitative assay that  is both
rapid and  reliable.  This method,  established by  Kakunaga  (Int. J. Cancer
12:463-473, 1973), is  not only  quantitative but scoring for  transformed clones
1s quite clear-cut and reproducible from  run  to run.  It appears  to  be an
ideal screening system for determining the potential of chemicals to induce
malignancy.

     •  An assay consists of a  positive control,  a vehicle control (negative
control),  and  four dose levels  of  the test chemical.  The  length of  time
required for testing will be 6  to  8 weeks.

Materials
     •  Assays will be performed using a subclone (obtained from Dr. Takeo
Kakunaga) derived from a clone of BALB/3T3.  The cells are grown in Eagle's

                                     384

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MEM supplemented with 10% fetal calf serum.  The cells are passaged weekly
In 60 mm culture dishes.

Conduct of the Experiment

     •  Seeding:  Approximately 10,000 cells are seeded Into a 60 mm plastic
plate and Incubated 24 hours to firmly attach the cells.  This plate will  be
used to assess transformation. Simultaneously with seeding, separate plates
will be seeded at 200 cells per plate to obtain toxldty determination.
     QUALITY CONTROL — The vehicle for the test chemical 1s used In the nega-
tive control plates.

     •  Dosing:  The positive control and four doses of test chemical are add-
ed to the transformation and toxldty plates.  Treatment with the test chemi-
cals will consist of exposing the cells 1n an airtight enclosed chamber to
either vapors or gaseous state of the test materials.  Various dose levels
will be achieved by varying the length of exposure to a fixed level  of the
vapors or gas.  Treatment will be terminated by removing the plates from the
chamber and replacing the media with fresh growth media.

     •  Incubation:  Following treatment, the cells will be Incubated for 3
to 4 weeks before scoring for transformed foci.  The toxlclty plates will  be
scored after only one week.  During the Incubation periods, growth media will
be changed twice weekly.

Observations and Tests

     •  The transformation plates are aspirated to remove media and washed
with buffered saline.  The plates are stained with Glemsa, washed, and air
dried.

     •  Transformed clones appear as darkly stained foci on a light back-
ground.  The counts of the transformation and toxldty plates are then express-
ed as foci/surviving cells for each dose level.
     QUALITY CONTROL — Confirmation of Tumorigenlclty of Transformed Clones:
Most transformed clones will produce malignant tumors when collected from an
unstained transformation plate and Injected Into syngenie  host animals.
This confirmation step can be conducted 1f desired.

Activation

     •  The BALB/3T3 cells have a limited metabolic capacity but appear to
metabolize certain classes of chemicals that have strict requirements for
metabolic activation to ultimate carcinogens.


3.4.5.3  Quality Control Aspects—

     Quality control procedures for the carclnogenicity testing of chemicals
with cell cultures have not been developed to any appreciable extent up to
this time.  Although precautions and control measures are emphasized by most
workers In their reports, no formal system of quality control or quality

                                     385

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assurance has been developed.

     General quality control measures that should be observed in all work with
cell cultures are outlined in Section 3.4.4 of this report.  Various specific
measures that are especially applicable to carcinogenicity testing with
mammalian cells are given in the following pages.

     Many chemical carcinogens are light-sensitive and must be stored in the
dark.  Light should be reduced to a minimum in preparing test solutions and
treating cell cultures.  Treated cultures, of course, must be incubated in
the dark (Katoh, 1977).

     Compounds not certified to be pure should be repurified before use.  A
number of source chemicals and reagents used in organic syntheses are strongly
carcinogenic.  However, technical grades and formulations of all commercial
chemicals should be tested as well as the purified form.  Environmental
chemicals should also be tested as mixtures since synergistic as well as
antagonistic effects may result (Anon., 1973).

     Only the cell cultures recommended here should be used and protocols
should be followed explicitly in all assays.  Primary rat liver cells should
be used in all tests involving this system with precarcinogens or unknown
chemicals since continuous cell lines lose metabolic activity (Williams,
1977).  All assay procedures should be calibrated by the laboratory before
testing is started.

     Untreated and vehicle control cultures are especially important in cell
transformation studies since "spontaneous transformation" to malignancy at a
relatively high rate is characteristic of certain cell lines (Anon., 1971;
Anon., 1973; Earle, 1943; Berwald and Sachs, 1965).  Positive controls in the
form of known carcinogens and precarcinogens should also be included in each
assay.

     Replicate testing and interlaboratory cooperative tests are also advis-
able in view of the limited data available with most of the cell culture assay
systems now being used for carcinogenicity testing of chemicals.

     All positive transformation tests must be confirmed by formation of
tumors in animals following injection of transformed cells.  Correlative data
involving transformation manifestations and tumorigenicity in animals should
be collected by a central agency or clearinghouse in an effort to define in
vitro changes which are sufficient alone as proof of the carcinogenicity of
a substance (Anon., 1971).

     Cultures used in cell transformation studies must be virus-free since
rodents used as source of cells may be parasitized by oncogenic viruses which
are capable of transforming cells in culture.  Moreover, cultures infected
with certain of these viruses are more sensitive to transformation than the
corresponding noninfected cultures (Freeman et al., 1971; Rhim and Huebner,
1973; Rapp, 1973).

     All lots of serum to be used in cell transformation assays should be

                                     386

-------
tested In untreated cultures as well as in those treated with known carcino-
gens to ascertain any effect.on transformation.  In view of results of the
studies by Sanford et al. (1972) and Evans et al. (1972), fetal bovine serum
is preferable  to calf or horse serum.

     Glassware used with carcinogens should not be used again for cell trans-
formation carcinogen assays in view of the great sensitivity of these assays
and difficulties encountered in removal of the last traces of various car-
cinogens from containers (Berwald and Sachs, 1965).

3.4.6  Cell Cultures - General Toxicity Testing

     Primary cell cultures are especially suitable in general toxicity test-
ing since they retain many of the metabolic and functional characteristics
of the original tissues for a number of passages in vitro.  The Rabbit Alveolar
Macrophage (RAM) Test, Ciliastasis assay, HeLa cell cultures, L-929 mouse
fibroblasts, WI-38 human lung fibroblasts, primary rat liver cells, and the
Clonal Toxicity Test have been proposed for toxicity testing of air pollutants,
pesticides, biomedical materials, and general toxicity of chemicals.  Results
with several of these systems have been found to correlate well with in vivo
assay results (Duke et al., 1977; Donnelly et al., 1974; Litterst et al.,
1969; Felling et al., 1973).

3.4.6.1  Air Pollutants - Test Methods—

     •  Rabbit Alveolar Macrophage (RAM) Assay

     Alveolar macrophages represent a "first line" defense of the mammalian
pulmonary system due to their ability to phagocytize and remove particulate
material.  Consequently, maintenance of viability and phagocytic activity of
these cells is essential in protecting the lungs from effects of bacteria
and other harmful substances.  The Rabbit Alveolar Macrophage (RAM) Assay has
been developed as a rapid and convenient in vitro assay for the detection of
toxic airborne particulates and associated chemicals.  A protocol for this
assay is given in the following pages.

     Date may be collected on the form illustrated in Figure 3.4.11.

     The arc-sine transformation is used in regression analysis (Finney, 1972)
since cell viability may be considered to be a binomial response.  Viability
is plotted against the natural logarithm of the molar concentration.

              EXAMPLE:  RABBIT ALVEOLAR MACROPHAGE ASSAY (RAM)

Purpose of Study

     •  Cytotoxicity - Employ rabbit alveolar macrophage to measure quantita-
tively cellular metabolic impairment and death resulting from exposure in
vitro to soluble and particulate toxicants.

Design of Experiment
                                     387

-------
     •  Materials.  New Zealand white rabbits (Including both sexes) weighing
1.5 to 2.0 kg are sacrificed for acquiring alveolar macrophages.  Lung lavage
1n situ 1s carried out according to the procedure of Coffin et al. (1968) us-
ing prewarmed (37°C) sterile 0.85 percent saline.  Before conduct of the
experiment, make absolutely certain that the following two elements have been
properly controlled:

          •  Rabbits must be clinically healthy.
          •  Determine the cellular composition of the pooled lavage fluid
             and Insure there 1s routinely 95% alveolar macrophages, 2% to 3%
             polymorphonuclear leukocytes, and 2% lymphocytes.Discard
             lavage fluid found to contain blood or mucus.

Conduct of Experiment

     QUALITY CONTROL -- Establish regular audits of performance throughout
the experiment.

     •  Cell Culture:  The alveolar cells are washed once by centrifugalion
at 365.3 for 15 minutes at 25°c and resuspended in prewarmed (37°C) tissue
culture medium 199 in Hanks' balanced salt solution.  Supplements added to the
medium Include heat-Inactivated fetal bovine serum (10%), penicillin (100 units/
ml), streptomycin (100 vg/ml), and kanamydn (100 yg/ml).
     QUALITY CONTROL— Use supplements 1n a consistent way.  These biologi-
cal s will be available from:  G1bco, Grand Island, NY.

     •  Cell Count:  The cells are counted by a hemocytometer or automatic
cell counter and diluted to approximately 1 x 106 cells per ml with supple-
mented medium.
     QUALITY CONTROL — Maintain the Instruments properly and calibrate them
as required.

     •  Dosing:  One ml of the cell suspension is added to each well of 100 x
100 mm 4-place cluster dishes (Falcon Plastics) containing effluent sample,
and sufficient medium 1s added to bring the total volume per well to 2.0 ml.
(1) Solid samples:  final particle concentrations are 10, 30, 100, 300 and
1000 yg/ml of culture medium, and a control.  (2)  Liquid samples are added
with and without sterile filtration to give a final concentration of 6, 20,
60, 200, and 600 yl/ml, and a control.
     QUALITY CONTROL — All samples are assayed In a concentration tested in
duplicate.
     Randomization.

     •  Cell Incubation:  The cultures are  Incubated, with rocking, for 20
hours at 37°C 1n a humidified atmosphere containing 5% C02.  The pH of the
final Incubation mixture Is recorded before and after Incubation.
     QUALITY CONTROL — No pH adjustments are made for the Initial testing.
When pH adjustments are made, the sample 1s tested both with and without
adjustments.

     •  Cell Trypsinization:  At the end of the incubation period, the culture
medium 1s poured off and retained separately 1n a culture tube.  Cells are

                                      388

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dissociated by using 0.25% trypsin in Gibco solution A.   The suspended cells
are recombined with the original culture medium and chilled.  This trypsin-
ized cell suspension is ready for cell counts, cell viability, total protein,
and ATP determination as described below.

     •  Cell Counts and Viability:  (1) Dilute appropriately, usually 4-fold,
with cold 0.85% saline to yield a suspension of no more than 2 x 105 cells/ml,
(2) Add trypan blue, freshly diluted with 0.85% saline to 0.01%, to an equal
volume of cell suspension for determination of cell viability.  Use a hemo-
cytometer or Cytograf (Biosphysics Systems, Mohapac, NY) to perform simul-
taneously determinations of cell viability and cell counts.
     QUALITY CONTROL — All determinations are performed in duplicate.
     Adequate calibration and proper maintenance of the instrument is
essential.

     •  Protein Determinations:  (1) Wash cells twice with 0.85% saline.
(2) Lyse cells washed in 1.0% sodium deoxycholate (Schwarz-Mann, Orangeburg,
NY) and assay 0.1 ml aliquots of these lysed cells according to the method of
Lowry et al. (1951) by using a bovine serum albumin standard (Nutritional
Biochemicals Corp., Columbus, Ohio).
      8UALITY CONTROL -- All analyses are made in duplicate.
      se standards.

     •  ATP (Adenosine triphosphate) determinations:  Follow DuPont Model  760
Luminescence Biometer procedure.  (1) Extract ATP from 0.1 ml aliquot of
trypsinized cell suspension containing 0.3 to 0.4 x 105  cells with 0.4 ml  of
dimethylsulfoxide.  (2) After 2 minutes at room temperature, buffer the
extracted sample with an addition of 5.0 ml cold 0.01 M morpholinopropane
sulfonic acid (MOPS) at pH 7.4.  (3) Place the tube containing the buffered
sample in an ice bath.  (4) Inject 10 yl aliquots from (3) into the lumines-
cence meter's reaction cuvette containing 0.7 mM luciferin, 100 units luci-
ferase, and 0.01 M magnesium sulfate in a total volume of 100 yl of 0.01  M
MOPS buffer, pH 7.4 at 25°C.  (5) Light emitted from the reaction cuvette is
measured photometrically in the luminescence meter and proportional to the
ATP concentration of the sample.
     QUALITY CONTROL — All determinations are made in duplicate.
     Require adequate calibration and proper maintenance for Biometer, as
described by manufacturer.
     •  Phagocytic Activity:   (1) Add 1.1  ym polystyrene latex particles (DOW
Diagnostics, Indianapolis, Indiana) to alveolar macrophage cultured in Labtek
(Miles Laboratories, Inc., Naperville, IL) four-chambered micros!ides
(approximately 25 particles per cell  in 1  ml of supplemental  medium).  Prep-
aration and maintenance conditions are as  previously described.  (2) One hour
after the addition of latex particles, drain slides, air-dry, and  expose for
3 minutes to concentrated Wright stain.  (3) Expose the slides for an addition-
al 5 to 6 minutes with 1:1 aqueous solution of Wright stain.   (4)  A1r-dry
again, and place the slides in xylene for  1 hour to dissolve extracellular
particles according to the procedure of Gardner et al. (1974).  (5) Air-dry
again and mount the slides in permount.  (6) Determine the phagocytic activity
under oil immersion by scoring a minimum of 200 cells.  Each cell  which con-
tained at least one particle  is considered phagocytically active.   Typically,

                                      389

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80% to 90% of control cells ingested one or more latex particles.
     QUALITY CONTROL -- Duplicate analysis is made.
     Needs a quality compound microscope for excellence of work.

Data Collection and Handling

  Parameters Measured      Unit                     Calculations

  Cell counts          Number of cells
                       per milliliter of
                       cell suspension

  Cell viability       Percentage (%)     Viability index   No. cells
                                          = Viability (%) x experimental
                                                            No. cells
                                                            control

  Total protein        Percentage (%)                       Experimental
                                                     inn* v total protein
                                                     IUU* x Control protein

  ATP                  Percentage (%)                       Photometric
                                                     inn* v reading of expt.
                                                     IUU* x Photometric read-
                                                            ing of control

  Phagocytic activity  Percentage (%)                       Experimental
                                                            phagocytic
                                                     100% x cel1 counts
                                                            Controlcounts

     Samples found  in the  initial screening to significantly affect the para-
meters being measured are  retested for confirmation.

     Since cell viability  could be considered a binomial response, the arc-
sine transformation  is employed in the regression analysis.  Linear relation-
ships of data can be obtained by plotting the transformed viability versus
the natural logarithm of the molar concentration.  The prediction can be made
on the concentration of the test toxicant that yielded a 50% response for any
measure parameter (EC50) using a simple regression line.  Fifty percent end-
points (EC50) for the various test parameters are obtained through inverse
prediction of the simple regression  line.  All positive samples are retested
for confirmation.

References

     •  The discussion here is principally derived from Section 3.3.2.1 Rabbit
Alveolar Macroptiage (RAM)  Assay in Chapter III, Level  1 Bioassay Techniques of
the following report:  Duke,  K. M.,  M. E. Davis, and A. J. Dennis.  1977.
IERL-RTP Procedures Manual:   Level 1,  Environmental  Assessment Biological
Tests for  Pilot Studies.   EPA-600/7-77-043, April 1977.


                                     390

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 Sample No.	     DIFFERENTIAL

 Date Rec'd	       Macrophages_

 Description of Sample	       Neutrophils_

 	       Other
 Date Tested                          Incubation Time
 Date Report Out	     EC50 Value
 No. Rabbits Used	       Cell Count

 Remarks About RabbitsJ	       Viability
                                        Viability Index
 Total No. Cell Recovered               Protein
 Seeding Population of Cells	       Other
                              TEST RESULTS

       Cone.
       (ug/ml  	pH                             Via-
 Tube    or             After   Cell No. as   Viable  bility
 No.    vil/ml)  Initial  Incub.   % of Control  Cells   Index  ATP* Protein
* ATP/106 cells as » of control
   Figure 3.4.11  Data sheet for alveolar macrophage toxicity testing
                           (Duke et al., 1977)
                                   391

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     Other references are:

     •  Coffin, D. L., et al., 1968.   Influence of ozone on pulmonary cells.
Arch. Environ. Health 16:633-636.

     •  Gardner, D. E., et al., 1974.  Technique for differentiating particles
that are cell-associated or  ingested by macrophages.  Appl. Microbiol. 25:471.

     •  Lowry, 0.  H., N. J.  Rosebrough, A. L. Farr and R. J. Randall.  1951.
Protein measurement with folin phenol  reagent.  J. Biol. Chem.  193:  265-275.

     •  Mahar, H.  1976.  Evaluation of Selected Methods for Chemical and
Biological Testing of Industrial Particulate Emissions.  EPA-600/2-76-137,
or PB-257-912/AS,  U.S. Government Printing Office, Washington, D.C.

     •  Waters, M. D., et al.  1975.   Metal toxicity for rabbit alveolar
macrophages  in vitro.  Environ. Res.   9:32-47.

     •  Waters, M. D., et al., 1974.   Screening studies on metallic salts
using the rabbit alveolar macrophage in vitro.  Environ. Res.  10:342.
     •  Other Tests

     Other cell culture tests for air pollutants are outlined in Table 3.4.11.



             TABLE 3.4.11  CELL CULTURE TESTS FOR AIR POLLUTION
Assay
Test Substance
Reference
Rabbit alveolar macrophage
 (RAM)

Human alveolar macrophage

Human respiratory epithe-
 lial cells (Ciliastasis
 assay)

Rabbit ciliated epithe-
 lial cells

Rabbit cat tracheal cilia-
 in vitro/in vivo

Rat trachea ciliated
 epithelia
Soluble; particulate   Coffin et al., 1968
Cigarette smoke

Smoke




Cigarette smoke



Tobacco smoke



Chromates
Pratt et al., 1971

Ballanger, 1960
Kensler and Battista,
 1963

Dalhamn, 1970
Mass and Lane, 1976
                                                                 (Continued)
                                    392

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                           TABLE 3.4.11 (Continued)
Assay
          Test Substance
Reference
Freshwater mussel ciliated   Cigarette smoke
 cells (Anodonta cataracta)
Hamster tracheal rings
          Tobacco smoke
                                 Walker and Klefer, 1966
Donnelly et al., 1974
     In ciliastasis assay a number of experimental factors have been identi-
fied as being critical (Donnelly et al., 1974):

          o  Species of experimental animal
          o  Variations in sample preparation
          o  Test temperatures (pre-chilling of cells is Important)
          o  Control of the ciliostat
          o  Age of the test sample (loss of volatiles, oxidation, etc.)
          o  Animal-to-animal and operator-to-operator variations

     A randomized complete block design, balancing animal/operator combina-
tions within treatments is recommended.

3.4.6.2  Pesticides—

     Pioneer studies on the use of cell cultures for determining pesticide
toxicity were made in the middle 1960's.  HeLa, KB, human diploid fibre-
blasts, human Chang liver, and monkey kidney cultures have been employed with
a variety of assay techniques and a wide spectrum of pesticides.  Comparative
studies with HeLa and KB, HeLa and Chang liver, and HeLa and human diploid
skin fibroblasts, indicated that sensitivities of the various pairs were very
similar for a number of pesticides.  Table 3.4.12 outlines tests in use.

               TABLE 3.4.12  CELL CULTURE TESTS FOR PESTICIDES
Assay
Test Substances
Reference
HeLa cells
 KB cells

 Human diploid
  fibroblasts
Chlorinated, organophos-
 phorous, or carbamate
 insecticides
DDA
Various insecticides

DDA

Insecticides and
 metabolites
 Chang human liver  Insecticides
  cells
Litterst et al., 1969
Johnson and Weiss,  1967
Gablicks,  1965

Johnson and Weiss,  1967

Litterst and Lichten-
 stein, 1971

Gablicks and Friedman,
 1965
             (Continued)
                                     393

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                          TABLE 3.4.12  (Continued)
Assay              Test Substances                  Reference


Monkey kidney      Malathion                        Desi et al., 1975
 cells
3.4.6.3  Biomedical Plastics—

     Plastic containers and devices are widely used in the field of human
medicine, e.g., containers for transfusion blood, saline, glucose, and other
products of injection, in dwelling catheter tubes, heart valves, tracheostomy
tubes, transfusion sets, prosthetic devices, etc.

     The standard toxicity test method for bioplastics for a number of years
has been the rabbit implantation test involving insertion of a small strip of
the plastic into the muscle of the animal for 3 to 7 days and examination of
the tissue macroscopically and microscopically for evidence of toxicity.
Since the method is time-consuming and somewhat expensive, a search has been
made for rapid, convenient, and sensitive cell culture tests.

     •  Leachates from Polymers

     In the 1960's, Rosenbluth, Guess, Autian and coworkers developed the
L-929 Mouse Fibroblast Cell Culture Assay which correlates well with the in
vivo test and is actually more sensitive than the latter (Rosenbluth et al.,
1965; Guess et al., 1965).

     •  Biodegradation Products

     Hegyeli and coworkers have developed rapid and quantitative cell culture
assays using radiolabelled polymers to predict rates of decay and liberation
of toxic substances from plastics intended for use in the body over a very
long period of time and which undergo slow biodegradation in vivo (Hegyeli,
1972; Hegyeli et al., 1974).  Two procedures used are the Plasma Clot Method
and the Organ Culture Method.

     Biodegradation rates with a group of cyanoacrylate polymers were as
follows:

  Polymer                  Method      Exposure Period    Degradation Rate

  Poly(methyl-2-        Organ culture       24 hr              52.2
   cyanoacrylate)       Plasma clot         72 hr              47.4

  Poly(ethy1-2-         Organ culture       24 hr               3.1
   cyanoacrylate)

  Poly(propyl-2-        Organ culture       24 hr               1.99
   cyanoacrylate                                               (Continued)

                                     394

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  Polymer                  Method      Exposure Period    Degradation Rate

  Poly(butyl-2-         Plasma clot         72 hr              0.66
   cyanoacrylate)

  Poly(isobutyl-2-      Organ culture       24 hr              3.60
   cyanoacrylate)

  Poly(1(+)-lactic      Plasma clot         72 hr              3.50
   acid)

     •  Lysosomal Acid-Phosphatase Assay

     Grasso et al. (1973) compared the lysosomal acid-phosphatase assay with
the agar-overlay cell culture method for toxicity testing of plastics.  Pri-
mary neonatal rat kidney cell cultures were used in place of L-929 mouse
fibroblast cells for both assays.  Test plastics employed were samples of
polyvinylchloride^ containing 0, 0.17%, 0.5%, or 1.4% dibutyltin diacetate.
Endpoint responses were plaques of dead cells with loss of Neutral Red in the
agar-overlay method and increased lysosomal acid-phosphatase activity and
loss of Neutral Red in lysosomal assay.

     The extent of cell necrosis and other responses was directly proportional
to the concentration of dibutyltin diacetate toxicant in both assays.  The
agar-overlay method was found to be the more sensitive procedure in detecting
low concentrations of the toxicant.

     •  Evaluation of Assays for Toxicity Testing of Medical Plastics

     Pelling et al. (1973) evaluated the three major methods for toxicity
testing of medical plastics: agar-overlay method, with either (1) L-929
mouse fibroblasts or (2) primary rat kidney cells, and (3) the rabbit im-
plantation test.  A variety of plastics used in medical devices were used
for the comparative tests.

     The L-929 mouse fibroblast assay was the most sensitive method tested.
Positive controls produced 2 cm (approx.) plaques of dead (unstained) cells.
Negative controls caused no toxicity.  The rabbit implantation assay
(Sacrospinalis muscle) was more sensitive than implantation in rat subcu-
taneous tissue.  There was good correlation between the L-929 tissue culture
assay and the rabbit implantation test although the tissue culture method was
considerably more sensitive.  The authors suggested that since the L-929 assay
is so highly sensitive, positive results should be checked by rabbit implanta-
tion tests.

3.4.6.4  General Cellular Toxicity—

     •  WI-38  Human Lung Fibroblast Assay

     The WI-38 strain of human lung fibroblasts is regarded as the best
characterized diploid human cell strain available at present for general
toxicity testing.  All major DNA, RNA, and protein synthesis pathways common

                                     395

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to all  dividing  cells have been  found  in the WI-38 strain and these cells
also possess a number of  inducible enzyme systems.  WI-38 cell cultures are
used by the U.S. EPA Industrial  Environmental Research Laboratory, Research
Triangle Park, for Level  1 testing of  all solid and liquid effluents wherever
possible (Duke et al.,  1977).  A protocol for this assay is given in the
following pages.  Following  the  protocol, Figure 3.4.12 gives a form for data
collection.  Table 3.4.13 outlines other general tests available.

                 EXAMPLE:  HUMAN  LUNG FIBROBLAST (WI-38) ASSAY

Human Lung Fibroblast (WI-38) Assay

Purpose of Study

     •   Cytotoxicity -  Employ human lung fibroblasts in culture to measure
quantitatively cellular metabolic impairment and death resulting from exposure
in vitro to soluble and particulate toxicants.

Design  of Experiment

     •   Normal human diploid WI-38 cells, available from the American Type
Culture Collection, Rockville, Maryland, are seeded with 1.75 x 10s cells/ml
(4.0 ml  total volume) in  25 cm2  Falcon flasks.  These cells are grown to
confluency in Eagle's basic medium (BME) plus 10% fetal calf serum (FCS).
They are then fed with  BME plus  0.5% FCS for 5 days.

     •   Dosages  will be determined from preliminary toxicity curves estab-
lished  from treatment with 1.0,  0.10, 0.01 and 0.001% levels of the test
compound.  Three dose levels of  each compound will be selected.  A positive
and a negative control  will also be run.

Conduct  of Experiment

     QUALITY CONTROL -- Establish regular audits of performance throughout
the experiment.

     •   Culturing:  Subcultivate the cultures twice weekly by use of 0.25%
trypsin  in Gibco solution A (Gibco, Grand Island, NY) with a 1:2 split ratio.
     QUALITY CONTROL -- Cultures should not be employed beyond the 35th sub-
cultivation.
     Use standard culture media.

     •  Seeding:   Cultures or any other subcultures are seeded at 1.75 x 105
cells/ml (4.0 ml  total   volume) in 25 cm2 Falcon flasks and maintained in
Basal  Medium Eagle (BME) with Earle's salts plus 10% fetal bovine serum,
2 ymole/ml  L-glutamine,  100 units/ml  penicillin, 100 yg/ml streptomycin, and
2.5 yg/ml amphotericin-B.   Cells maintained under these conditions show a
period of rapid growth from 24 to 72 hours after subcultivation during which
time the experiments are performed.
     QUALITY CONTROL --  Fetal bovine serum must be virus-screened.  Routinely
antibiotics should be removed from the maintenance to determine the presence
of contaminating  microorganisms and mycoplasma.

                                     396

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   Sample No. 	      EC50 VALUES

   Date Rec'd 	       Cell Count

   Description of Sample	       Viability
                                         Viability Index
   Date Tested 	       Protein

   Date Report Out 	       ATP	
   Passage of Cells 	       Other

   Seeding Population of Cells 	

   Incubation Time
        _                       TEST RESULTS
        cone.
                      PH _                        Via-
          or               After   Cell No.  as   Viable bility
   0>    yl/ml)     Initial  Incub.   % of Control  Cells  Index  ATP  Protein
Figure 3.4.12  Data sheet for WI-38 cellular toxicity testing (Duke et al, 1977)


                                     397

-------
      •  Dosing:   (1) Plant 1.5 to 2.0 x 105 cells per flask in 24 cm2 Falcon
flasks.

      (2) Add dilutions of the effluent test material 24 hours after the cells
have  adhered to the flask surface, as described from the RAM (rabbit alveolar
macrophage assay).
      QUALITY CONTROL —  Each concentration is tested in duplicate.

      •  Incubation:  The culture-effluent mixture  is incubated with closed
caps  for 20 hours at 37°C.

      •  Trypsinization:  At the end of  this incubation  period, the cells
are trypsinized and cell counts, cell viability, protein, and ATP determin-
ations are performed.

      •  Sample Analysis:  Perform cell counts, cell viability, protein, and
ATP determinations as described in RAM assay.
      QUALITY CONTROL --All analyses are performed in duplicate.
      All instruments must be adequately calibrated and in proper maintenance.

Data  Collection and Handling

      •  See description  for RAM assay.

Reference

      •  Duke, K. M., M.  E. Davis, and A. J. Dennis.  1977.  IERL-RTP Pro-
cedures Manual:  Level 1 Environmental Assessment Biological Tests for Pilot
Studies.  EPA-600/7-77-043, Office of Research and Development, U.S. Environ-
mental Protection Agency, Washington, D.C.

             TABLE 3.4.13  OTHER GENERAL CELLULAR TOXICITY TESTS

Assay                    Test Substance                   Reference

Clonal toxicity          Environmental toxicants          Duke et al., 1977
 (L929 mouse
 fibroblast)

Human KB cells           Misc. chemicals                  Smith et al., 1963
3.4.7  Statistical Analysis

     An important feature of sound experimentation involves statistical
analysis of the data obtained.  Since the purpose of a biological assay is
to obtain an accurate estimate of the potency of a substance, frequently in
relation to a standard, two main types of statistical analysis are involved •
estimation of the endpolnt and evaluation of results obtained (Finney, 1964)
The statistical methods to be applied are determined largely by the general


                                     398

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type of experiment, kind of data, results obtained, and so forth.

3.4.7.1  Methods for Calculating a Median Effective Dose—

     The biological assays discussed in this section of the report are mainly
quantal (yes or no) assays, e.g., death or survival of cells in a treated
culture, appearance or absence of a mutant, cessation of ciliary activity,
and so forth.  The designated endpoint in most quantal bioassays is a median
effective dose (ED50, LD50, etc.), i.e., the amount of substance under test
which produces a response in fifty percent of the experimental subjects
(Finney, 1964).  The fifty~percent endpoint is usually chosen because it is
more accurate statistically than any other.  A number of statistical methods
have been developed for estimating fifty-percent endpoints in biological
studies.  Procedures applicable to quantal assays which have been widely used
are:

     Reed-Muench Method (Reed and Muench, 1938)
     Spearman-Karber Method (Spearman, 1908; Karber, 1931)
     Probit Method (Finney, 1964)
     Logit Method (Berkson, 1944)
     Angle Distribution (Knudsen and Curtis, 1947)
     Litchfield-Wilcoxon Method (Litchfield and Wilcoxon, 1949)
     Moving Average Method (Thompson, 1947)
In addition to an accurate estimation of the median effective dose, the
statistical procedure employed should also permit calculation of 95% con-
fidence limits from the data (Stephan, 1976).

     The Reed-Muench method is the least involved procedure for estimation of
the ED50, LD50, etc., but, unfortunately, is valid only when the tolerance
distribution is symmetrical and requires an unlimited range of doses (Finney,
1964).  Moreover, it does not calculate confidence limits or give validity
tests.  Some leading authorities at least, regard it as statistically infe-
rior to the other methods and state that it should not be used (Finney, 1964;
Stephan, 1976).

     The original Spearman-Karber method is a rapid and convenient procedure
for determination of the median effective dose but cannot always be used in a
routine manner since results are biased in some cases (Stephan, 1976).  A
rather specialized experimental design is also required.  The number of sub-
jects per dose should be constant and the doses should be equally spaced
(Finney, 1964).  For LC50 determinations, the number of doses required is
usually large and must cover the complete range from 0% to 100% kill (Stephan,
1976).  A modification of this procedure (Armitage and Allen, 1950), however,
may be used which does not require a geometric series of doses or equal
numbers of test species at each concentration.  The method is reported to
give approximately the same results as the Probit method if appropriate
formulas are used and doses of the test substance cover the complete range
from 0% to 100% lethality (Stephan, 1976).  Finney  (1964) states that the
Spearman-Karber method may actually be better than maximum likelihood methods
for estimation of the ED50 when the number of subjects per dose is very small.

     In quantal assays, the ED50 can also be estimated by converting doses to

                                     399

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 logarithms  and  percent  effects  to  probits,  logits,  or angles  followed by
 application of  the curve  fitting technique  (Litchfield and Wilcoxon, 1949).
 These  three transformations  according  to  Finney  (1964) are quite  similar over
 a wide range of responses.   In  one series of  comparative  estimations of the
 ED50 with 12 sets  of  data, the  probit  and logit  methods agreed very well by
 the X2 test, with  the angle  transformation  being somewhat less satisfactory
 (Finney, 1964).

     The Probit method  (Finney,  1964)  uses  the integrated normal  curve and
 the maximum likelihood  curve fitting technique.   It can be used regardless
 of the dosage or number of subjects per dose  (Ashton,  1972; Finney, 1964).
 It also calculates 95%  confidence  limits  and  provides  validity tests.  If the
 ED50 and standard  deviation  of  the tolerance  distribution can be  estimated
 beforehand,  Finney (1964) states that  the use of the Probit method will give
 all available information that  can be  extracted  from the  records.  Stephan
 (1976) points out  that  for calculation of the LC50, however,  the  Probit
 method can  be used only with data  which include  at  least  two  "partial kills"
 unless certain  of  the data are  "adjusted".  He goes on to state that adjust-
 ment of data cannot be  justified by any statistical theory.   Stephan (1976)
 also points out that  the  method is very tedious  if  a computer or  minicomputer
 is not employed.

     The Logit  method (Berkson,  1944)  uses  the logistic curve instead of the
 integrated  normal  curve (Ashton, 1972).   Ashton  (1972) states that although
 the two curves  agree  well over  the range  usually involved in  bioassays, the
 normal curve is the better of the  two  for this type of study.  The logistic
 curve  is best for  physicochemical  studies (Ashton,  1972).  Stephan (1976)
 points out  that for estimation  of  the  LC50, the  Logit  method  like the Probit
 transformation,  is applicable only for data with two or more  partial kills,
 unless the  data are "adjusted".

     The Angle  Transformation (Knudsen and  Curtis,  1947)  may  be used with
 reasonable  confidence that the  results will usually be practically the same
 as if  probits or logits had  been used  provided that extremes  of dose, which
 would  cause  complications due to the limited  range  of  the tolerance distri-
 bution, are   not used (Finney,  1964).  However,  if  the number of  subjects per
 dose is either  very large or very  small,  probits or logits are preferable
 (Finney, 1964).  Computations involved in the Angle transformation method are
 relatively  simple,  an important  feature in  routine  assays (Finney, 1964).

     The Litchfield-Wilcoxon method (1949)  can be used, according to Stephan
 (1976), to  estimate the LC50 and its 95%  confidence limits from data with one
 or no  partial kills.  He points  out, however, that  it  should  not  be used in
 cases where  the Probit  method is not applicable  since  it  was  devised as a
more convenient method  to be used  in place  of the latter.  Furthermore, since
 it is  a semi-graphical  method, variation  in judgement  between individuals may
 be considerable.   Finney  (1964)  feels  that  although the Litchfield-Wilcoxon
method cannot be recommended without reservation for general  use, it may be
 useful for the  professional  statistician who  will recognize situations in
which  it will give  valid results.

     The Moving Average method  (Thompson, 1947)  is  regarded by Stephan (1976)

                                    400

-------
as being usable with more sets of data than any of the other methods, without
using adjusted or assumed data.  Moreover,  it can be performed relatively
easily either manually or by computer (Stephan, 1976).  Finney (1964), how-
ever, calls attention to the fact that a large number of doses are usually
required and that the doses should be equally spaced and the number of sub"
jects per dose constant.  Also, the tolerance distribution must be symmetrical
for a valid estimation of the ED50.  Confidence limits cannot be calculated
for an LC50 if there are no partial kills,  unless "adjusted" data are used
(Stephan, 1976).

     Finney*s recommendations on the choice of a method are as follows:

     •  If nothing is known about the ED50  beforehand, the following may be
        used:

        o  Spearman-Karber
        o  Moving Average (with largest possible span)
        o  Probit

        Moving average method is preferred  over the Spearman-Karber but is
        more laborious and probably will give almost the same result as the
        latter.  Probit method will give validity tests which the others do
        not provide but precision will be little or no greater and it is more
        laborious.  Dose range must be very wide to be certain of bracketing
        the ED50, and doses should be spaced fairly closely.

     •  If the experimenter is fairly certain that the ED50 lies between
        known limits which are not very far apart:

        o  Moving average method (span 3 or greater) is preferable to the
           Spearman-Karber.  In analysis of results, the largest span allowed
           by data should be used.
        o  Probits may be used if data are  unsuitable for the Moving Average
           method due to an unwise selection of doses.  Probits must be used
           if validity tests are desired.

     •  If both ED50 and the standard deviation of tolerance distribution can
        be "guessed" prior to assay, Probits must be used if the experimenter
        wishes to extract all available information from the records.

The above recommendations assume that statistical advice was followed during
planning of the assay (Finney, 1964).

     Stephan (1976) makes the following recommendations in his review of
methods for calculating an LC50.  He points out that they apply also to esti-
mation of LD50, ED50, and EC50 in quantal assays:

     •  Moving Average method and log concentration is method of choice with
        one or more partial kills.  This method may be used also with no
        partial kills but confidence limits cannot be calculated under these
        conditions.
     •  Probit, Litchfield-Wilcoxon, and Logit methods should be used only

                                     401

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        with two or more partial kills.
     •  Spearman-Karber method should be employed only if both 0% and 100%
        kill are included in the data.

3.4.7.2  Methods for Assessing Significance of Data—

     Procedures for evaluating significance of results obtained are discussed
in Section 2.2.3.
                                     402

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3.4.8  References

Akin, E. W., and W. Jakubowski.  1976.  Viruses in finished water:  The
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3.5  MAMMALIAN BIOASSAY

3.5.1  Experimental Design Aspects

     The principal goal of any mammalian bloasaay experimental design Is to
ensure, as far as possible, Chat no agents, physical factors, or biologic
organisms, except those under test or used as treatments, contribute to or
Influence the observed result.  This Ideal Is generally very difficult to
achieve.  In practice In mammalian bloassays, the best that can be done Is
to ensure that all factors, Influences, or conditions, except one, the treat-
ment agent, act equally on two populations, the exposed, test or treatment
group and the control group (that group not exposed to the treatment agent).
It Is Important to emphasize that no valid conclusions can be drawn from an
uncontrolled experiment.

     The control group ought to be handled wherever possible In the exact same
manner as the test group.  The very act of administering an unknown or toxic
agent may Influence the course of a bloassay and the observed results, even
though the agent may be Inert or only weakly toxic.  A simulated treatment
using a known Inert substance, termed a placebo control, Is often used to avoid
this difficulty, since the method of administration or dosing of an animal may
produce the dominant adverse effect If the stress is overly great on an animal.
Placebo controls In a pilot study should be Included to elucidate such potential
problems (Goldstein, 1964).

     Good experimental design with mammals generally requires that the controls
be run concurrently with the treatment groups within a simultaneous experi-
mental situation.  "Before-after" comparisons generally make for weak logic
and Inferences.  For example, If a previous group of animals had a high acute
mortality against a specific agent while a second group of control animals
observed later showed little or no mortality, It may be argued that the test
group could have died mainly from secondary causes (e.g., Infections, contam-
inated water or feed, or Intracage fighting).

     In some unique Instances It may be possible to use each animal as Its own
control.  Such designs which Incorporate each animal as Internal controls have
been used In skin and eye Irritancy or allergenlclty tests where more than one
area or organ Is available to the Investigator, one of which receives placebo
treatment.  There Is alwvn a formal requirement that the test agent In pilot
studies show only localized adverse effects.   Any agent which readily migrates
from the area of treatment via systemic transfer within a specified time period
cannot be validly bloassayed using this internal control method.  Such is the
case for chemicals which distribute within an animal's body following rapid
percutaneous absorption (e.g., solvents such as acetone or ethanol), but not
the case for more reactive or more polar agents such as formaldehyde or
diethylthalamide which have been demonstrated to produce positive skin
irritations in the Dralze (Internal control) dermatitis assay (Steinberg
et al., 1975).

     In some Instances where the toxic responses, particularly In a chronic
toxlclty or carcinogenic!ty test, may vary with time or be brought Into question


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due to changing physiology with age, it may be necessary to assay a positive
control group along with the placebo controls and test groups.  Positive controls
may also be necessary in the case of potentially weak carcinogens where a clear-
cut interpretation of the histopathologic observations requires concurrent
analysis of results of a known carcinogen for comparison in a given species and/
or strain of animal (Weisburger, 1975).

     In all the above examples, the objective of using control populations has
been to isolate the effects, whether they be acute toxicity, mutagenicity, or
carcinogenicity, resulting from the interaction of only the test agent on the
organism.  It is thus necessary that there be equivalence, as nearly complete
as possible, between the housing, feeding, watering, dosing, examination
schedule, and posting  in both the control and the test animals.  Even then,
the resulting observations are strictly valid only for the conditions of the
original bioassay.  The strength of any correlations and inferences with
respect to human health evaluations and exposures must, therefore, rest on the
relative merits of the experimental designs and techniques employed in the
bioassay and not just on the actual statistical numbers or observations made.

     Randomization in the selection of animals for use as test and control
groups is another means of achieving the necessary concurrent testing equiv-
alence between these groups.  Randomization prevents most subconscious bias
which has been demonstrated to play a surprisingly large role in the assignment
of mammals in a given trial.  For instance, if the investigator chooses only
those animals that are the slowest and easiest to catch for a test group, while
the remaining  friskier animals are used as controls, one may point to the
possibility that the two populations are not equivalent.  In fact, the test
group may be fatter or metabolically impaired in some unknown way allowing
them to be more easily caught.  Goldstein  (1964) succinctly observed that "no
characteristic of a subject whatsoever shall play any part in his assignment
to a group" in a properly run trial, since sound statistical inferences rely
on the assumption of random distribution or assignment of subjects or individual
organisms.

     The blind design is a preferred approach because bias in the conduct of
the trial or bioassay and in the evaluation of results is eliminated or mini-
mized.  In the blind design the personnel who administer, observe, score and/or
evaluate the results are "blind" as to which individual animals belong to a
control set and which belong to test sets, usually by means of coding of dosage
materials.  The strength of the blind design lies in the high degree of objec-
tivity that it affords the personnel and investigators directly involved in the
trial progress and evaluation.

     The basic design in bioassay is a single factor design (a single toxic
substance is under test at a given time) with replication (treatment applied
to more than one animal in a group).  Usually the treatment (factor) is applied
at more than one level and these levels are fixed (not chosen at random).

     All the considerations mentioned above apply also to experiments with more
than one factor and where some additional considerations apply.
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     The general definition for balanced design is one involving two or more
factors in which comparisons are made between treatment groups in such a way
that all factors except the treatment of interest affect the treatment groups
equally.  A special case of the balanced design called a cross-over design is
useful when a bioassay is to be repeated or when several different exposures
are to be assessed over an extended period of time.  In the cross-over design,
the test group and control group in the first trial are exchanged in the second
trial.

     Obviously trials involving acute toxicities which lead to permanent injury
or death of the test subjects cannot be performed in this manner.

     When more than a single control set and test set is involved, then more
elaborate designs such as the Latin square, randomized block, or factorial
designs may be advantageous.  The placement of the animal cages within an
animal room may be moderated via one of these designs so as to eliminate the
possibility of skewed effects due to adverse lighting, noise, vibration,
temperature, etc., at one end of a room.  Factorial designs have been discussed
in Sect. 2.2 which also given references to sources of more elaborate designs.

     When a sequence of exposures, treatments, or observations is needed within
a trial (not necessarily a time sequence) a nested or hierarchical design is
recommended whereby a tier of trials is performed in a specific sequence.   This
method might be used to assess the performance of several instruments, tech-
nicians and/or laboratories in terms of the accuracy and precision through
replication of procedures and assays.  The enhanced reliability of the data
obtained may be crucial for discussions involving safe limits of human exposure
or in determining "no-effect" levels to wildlife.

     The test sample size is also of primary importance in an experimental
design since the statistics based on this sample will generally be used as
estimators of the corresponding parameters and data in larger populations.
For a specific level of significance (e.g., P<0.05), the Type I, or alpha
error (i.e., the probability of assigning a significantly positive cause-and-
effect relationship, when in fact the reverse is true) is specified at the
beginning of a trial by the investigator.  By contrast, the Type II, or beta
error (i.e., the probability of assigning a non-significant cause-and-effect
correlation, when in fact this correlation is significant) is a complex function
of several factors, one of which is the alpha error chosen.  If one can tolerate
a larger alpha error (say P<0.05  instead of P<0.01), then the beta error is
made concomitantly smaller.  Secondly, as the sample size is increased, the
distributions between the control sample and the test sample will become
narrower due to the "central limit" theorem and less overlap between these
distributions will result.  As stated by Sokal and Rohlf (1969), the "central
limit" theorem predicts that as the sample size increases, the means of samples
drawn from a population of any distribution will approach a normal or Gaussian
distribution.  Moreover, if the sample values already fit a Gaussian distribution,
then increasing the sample size has the effect of narrowing that distribution
(i.e., reducing the variance) about the central tendency (i.e., the mean of the
sample values).

     One final approach to reducing the beta error is to choose that measurement

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parameter which is most readily influenced by the treatment, drug, toxicant,
etc.  Thus, for example, if the earliest and most sensitive measures for in-
halation intoxication are cardiac and respiratory rates, while recording of
electroencephalographic activity recordings are slower to change, then the
former, more sensitive responses will yield the least beta error when cause-
and-effect correlations are drawn.

3.5.2  Conditions of Test

3.5.2.1  Compounding the Test Material—

     The general principles involved in the preparation of various dosage forms
are discussed here with reference to the in vivo delivery of test materials.
Besides route of administration, a number of factors influence the extent of
test agent bioavailability, that is, the degree to which the test agent in a
specific dosage form is available for absorption, distribution, biotransfor-
mation, and physiologic action.  Among the factors that affect bioavailability
are:
         o  Stability and chemical purity
         o  Particle size and/or crystalline form
         o  Diluents and/or excipients including fillers, binders,
            disintegrating agents, lubricants, coatings, solvents,
            and suspending agents
         o  Method of manufacture and/or compounding which may cause
            chemical and/or physical degradation, introduces metal or
            packaging contaminants (active or inactive)

The U.S. Pharmacopeia XIX explicitly states that "the maintenance of a demon-
strably high degree of bioavailability requires particular attention to all
aspects of production and quality control that may affect the nature of the
finished dosage forms"  (U.S. Pharmacopeia, 1975).

     The setting of specification standards for products requires a statement
of the expected shelf-life for each product or preparation, which in turn
requires knowledge of stability (i.e., the time lapse from initial preparation
during which a dosage form continues to fulfill specifications for identity,
strength, quality and purity).  It has been recommended (U.S. Pharmacopeia,
1975) that analytical methodology should be cited capable of differentiating
between intact preparations and the degradation products thereof.  In addition,
stability is prolonged by storage at optimum environmental conditions (i.e.,
generally low temperature, humidity, air and light).  Four types of stability
criteria should be checked in order to certify that a specific shelf-life is
accurate:

          o  Chemical - each active ingredient retains its chemical
             integrity and labeled potency, within the specified limits
          o  Physical - the original physical properties, including
             appearance, uniformity, dissolution and suspendability,
             are retained
          o  Microbiological - sterility or resistance to microbial growth
             is retained according to the specified requirement; anti-
             microbial agents that are present retain effectiveness within

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             the specified limits
          o  lexicological - no significant increase in toxicity occurs

     Though not all these criteria are fully applicable to the testing of all
toxicants (e.g., it might in fact be the objective of a study to determine
how and what toxicants are chemically formed when storage conditions for a
pesticide are far from optimum), proper toxicologic investigations should
require delineation of these stability criteria, especially before and at the
end of a chronic feeding experiment when the possibility is very high that a
toxicant is chemically, physically, or microbiologically altered with time.

3.5.2.2  Vehicles-

     Capsules and tablets are the two most common forms of oral administration
of test compounds to humans, while other mammals are orally dosed chiefly by
water or feed supplemented with test compounds or less often by gavage with
liquid formulations (Weisburger, 1976; Goodman and Oilman, 1975).  In most
animal tests, the oral route (supplementation of water or feed or dosing by
gavage) is generally the most practical and reliable approach in bioassaying
for acute, subacute or chronic toxicity, or carcinogenicity.  Gavage or per os
(po) administration offers the advantage (or disadvantage) of quantitative
dosage delivery using one entire dose at a time.  Absorption of test compounds
from dietary water or feed, on the other hand, has the advantage (or disadvantage)
of protracting the animals' exposure, leading to a greater possibility of
biotransformation.  An important consideration here is that the animals may
be offended by the taste of the test compound and reduce their intake of food
and/or water, making for poor comparisons with control animals.  Moreover,
checks should be made of the uniformity of distribution of the test chemical
in feed by quantitative chemical analyses before exposing the animals in the
proposed bioassay.

     Dosing via inhalation can be quite complex requiring exposure of nasal,
respiratory and/or oral tracts in a uniform (between animals) and/or consistent
(within an animal) manner before a valid assessment of local and/or systemic
effects can be made.  Nebulizers are suitable for the administration of in-
halation solution only if these instruments can be certified to give droplets
of requisite and uniform size distribution so that the dosing mist is assured
of reaching the bronchioles.   Devices that release a metered dose via  aero-
solization with a liquified propellant of "inert" gas should first be tested
without test substance to determine side effects, if any, of the propellant.
Upon addition of the test agent, the device should again be tested to ascertain
whether the specified particle size distribution and amounts released by the
aerosol are achieved.

3.5.2.3  Routes of Administration—

     Administration of test substances to test  mammals should incorcorate
those routes that most closely approximate the routes whereby humans become
exposed (Weisburger, 1976; U.S. FDA, 1959).  If the chosen route of absorption
is to be via the gastrointestinal tract (enteral), then three oral routes of
administration to animals are possible:


                                      421

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          o  Gavage  (gastric  intubation)
          o  Mixing  into  solid  diet
          o  Mixing  into  drinking water

The  gavage method has  the advantages  of  (a)  ease of  quantification,  (b) use of
minimal  test agent,  (c) provision of  fresh  test preparations, and  (d) ease of
storage, but the disadvantages  of  (a)  a  large  amount of animal handling,  (b)
high hazard of  lung  and/or esophageal  damage within  the test animals with
subsequent increases in non-toxicant-induced mortality, (c) a requirement for
small and sometimes  concentrated volumes of  toxicant,  (d) use of a solvent,
and  (e)  need for close weight-matching of test animals.  Solid diet mixing
affords:   (a) a greater total intake  of  test agents, (b) close simulation of
the  mode of human exposure, (c) lessening of the hazards to the animals' lungs
and  esophagus,  (d) reduction  of the requirement for  solvents, and  (e) avoidance
of the requirement for close  weight-matching of animals.

When the animal feed is to be supplemented with the  test agent, a number of
potential problems,  however,  must be  taken into consideration such as (a) lack
of homogeneity  of the  mix,  (b)  decomposition and/or  interaction of the toxicant
with the feed during storage, (c) adverse palatabllity of the mix, (d) varying
quantity of ingestion  between individual animals yielding only average esti-
mates, and (e)  contamination  of the feed with  synergistic or antagonistic
substances that may  go unnoticed or be uncontrollable.  Compared with dietary
exposure, similar advantages  and disadvantages exist for mixing toxicants
into the animals' drinking water except  that inhomogeneity is less of a problem,
while decomposition  due to hydrolysis  may be a major objection to  dosage  via
drinking water.

     Some common parenteral routes of  administering  liquid test substances,
which obviate problems associated with the  gastrointestinal tract  (e.g.,  slow
absorption, gastric  decomposition, or  precipitation), are compiled below in
Table 3.5.1 along with the major uses, limitations and precautions involved in
each technique.  The instantaneous or  prompt absorption and complete systemic
distribution via these parenteral routes ensure complete and uniform dosing of
the  test animals, but  require great care in  their handling and in avoidance of
overdosing.  The above routes all have in common that the toxicant or test
substance will  rapidly (parenteral routes) or  eventually (enteral routes) reach
a nearly uniform systemic  distribution in the  test organisms' bodies prior to
being concentrated in  various tissues  and organs, causing reversible or irre-
versible physiologic changes, and usually resulting  in a termination of bio-
logic action by means  of  tissue-organ-specific biotransformation and elimination.

     Localized  pharmacologic or toxic  actions, however, may be produced by
application of  test  substances to various specific locations on the skin and
eye,  by inhalation into the lungs and  nasal  passages, and by other mucosal,
especially sublingual  (beneath the tongue),  applications.   Cutaneous or dermal
entry of test agents generally requires  the  use of a solvent which has been
demonstrated to be nonirritating.   Elicitation of a dermal reaction by the
combined toxicant-solvent  formulation  is thus proof positive of absorptive
cutaneous toxicity,  whereas the lack of a dermal reaction may indicate poor
or nonabsorption across the epidermal  stratum corneum rather than lack of


                                      422

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       TABLE 3.5.1  CHARACTERISTICS OF COMMON ROUTES OF TOXICANT ADMINISTRATION (Goodman and Oilman, 1975)
     Route
Pattern
Special Utility
Limitations and Precautions
     Intravenous
      (iv)
     Subcutaneous
      (sc)
     Intramuscular
      (1m)
fo
     Intraperito-
      neal (ip)
Absorption instantaneous
with potential for im-
mediate physiologic
effects.
Prompt absorption from
aqueous solution, but
permitting slow and sus-
tained distribution from
repository preparations.

Prompt absorption from
aqueous solution, but
permitting slow and sus-
tained distribution from
repository preparations.

Same as intramuscular,
but can be quicker.
Permits titration of dose
and use in an emergency.
Suitable for large volumes
and irritating substrates
and drugs if diluted.

Suitable for some insoluble
suspensions and for implan-
tation of solid pellets.
Suitable for moderate vol-
umes, oily vehicles, and
some irritating substances.
Same as intramuscular.
     Oral ingestion  Variable absorption de-
      (per os, po)  pending on gastric pH,
                    gastric emptying rate,
                    dissolution rate of
                    solids, powders, crystals,
                    coatings or capsules, etc.
                          Most convenient, safe and
                          economical dosing method.
Must introduce substances slowly, as
a rule, and watch for increased risk
of adverse effects. Not suitable for
oily solutions, particulate materi-
als, or insoluble substances.

Not suitable for large volumes. May
cause slough from irritating
substances.
May interfere with interpretation
of some diagnostic tests (e.g.,
creatine phosphokinase).
May cause infections and/or ad-
hesions.  Not used on man.

Absorption potentially erratic and
incomplete for agents that are
poorly soluble and absorbed slowly.
Agents which are degraded or
destroyed by gastric acids and
enzymes are precluded from dosing
in this manner.

-------
cutaneous toxicity.  A primary irritant has been defined by the FDA as a sub-
stance producing an injury on first contact.  The resultant injury will depend
on:

          o  the nature of the irritant-solvent combination
          o  the concentration of the irritant
          o  the total duration of the first exposure

Primary dermal irritation may be measured via the patch-test technique on
intact or abraded skin clipped free of hair.  The irritation process, an
incipient inflammation, may vary from barely perceptible hyperemia, to edema
and vesiculation, to erythema, and finally to intense suppurative processes.
Numerous methods of primary irritancy (PI) quantification have been reported,
but the Draize approach is widely accepted as the method of choice (Steinberg
et al., 1975).

     Ocular PI is also of great concern.  Because of the vital role they play
in vision, the FDA recommends that injuries to the cornea and iris be weighted
more heavily than injury to bulbar and palpebral conjuctivae (U.S. FDA, 1959).
The cornea, having 40% of a score, is rated on the basis of the density of
induced opacity and the amount of area involved while the iris, also weighted
40%, is scored on the intensity or degree of inflammation exhibited.  The
conjunctivae, including the cornea, iris, palpebral and remaining bulbar
mucosae, are scored for a total of 20% and are rated on the basis of the degree
of chemosis, redness, and discharge.

     Other mucosae, i.e., oral, genito-urinal and rectal, are subject to wider
variations than ocular muscosa in toxic responsiveness due to changes in tissue
pH, contact with food and microbes, secretions, excretions, and absorptive
capacity, on an hourly, daily, or monthly basis making intertest comparison
difficult.  These mucosae, however, generally exhibit faster absorption due
to the lack of a cornified barrier (i.e., the stratum corneum) than absorption
through intact skin, with the result that the necessity of compounding of the
test agent with a solvent may be avoided.

     In contrast to the single localized exposure  PI bioassays described
above, sensitization or contact allergy studies on skin, eye or mucosae are
studies which result in:

          o  tissue reactions that are remote from the original site of
             test application
          o  enhancement of responsiveness with each subsequently applied
             equal dosage
          o  potentiation of the cell-mediated, as well as the anti-
             body-mediated, immune systems
                                     424

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Reactions such as erythema and/or edema are generally not observed upon first
contact with the toxicant but become increasingly manifest when further
exposures occur.  The number of prior exposures to application of agents
required to produce these allergic responses may vary greatly depending on
sensitizing potential of the agents, the species susceptibility and the number
of exposures to dosages at a wide range of concentrations.  Sensitization is
scored on the basis of the number of positively reactive animals and on the
degree of their reactivity.  It should be noted, however, that many classes
of substances exist (e.g., poison ivy allergens) which induce contact
sensitivity in humans but for which natural animal models are rare or non-
existent.

      Systemic dermal toxicity, chiefly measured in terms of acute or subacute
lethality following cutaneous exposure, is generally more difficult to assay
than toxicity studies resulting from ingestion or inhalation due to the fact
that the animal must be restrained from licking the skin or inhaling toxic
vapor arising therefrom.  In acute single exposure studies, the agent may be
held in skin contact by means of a rubber sleeve with a reservoir containing
test agent for periods varying up to 24 hours.  Multiple dosage 20-day and
90-day subacute dermal toxicity studies are most difficult to execute properly
due to the requirements for preventing exposure via inhalation or ingestion
during the entire course of these assays.  Progressive deterioration of the
skin may thus be investigated together with protracted damage to other tissues
and organs as a result of the eventual systemic distribution of cutaneously
absorbed toxicants.

     Gases or aerosol preparations are best tested by acute or subacute in-
halation toxicity tests, in addition to skin mucous membrane assays.  In the
case of aerosols, an Important quality control criterion would be the size
distribution of the aerosolized particles.  Particles in the range of less
than 3 urn readily reach and deposit in the alveolar sacs of mammalian lungs,
whereas particles of 3 to 10 ym  arrive at the lung parenchyma with great
difficulty.  Particles larger than 10 ym are effectively prevented from reach-
ing the parenchyma and alveoli and would therefore provide false tests of
lung intoxication.  A closely confined test space or exposure chamber is a
necessity for each control and test animal.

     In acute inhalation testing the objective is to assay the test animals
using single or multiple dosages of gas or aerosol within a short period
(e.g., less than 8 hours).  The U.S. FDA (1959) recommends that the animals
be immobilized within the test chambers with a suitable covering placed over
their eyes.  Immobilization allows the investigator to direct the release of
aerosol or gas around the head and upper trunk of the animal, while avoiding
the release of spray directly into the animal's respiratory passage with the
possibility of causing a bolus toxic effect.  A minimum of 4 test animals is
required by the FDA for determining acute effects of aerosol preparations.
                                     425

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Their recommended procedure, following immobilization of each animal (one per
chamber), is to dose for 30 seconds of continuous spray release concomitant
with a 15-minute continuous ensuing exposure before the 30-second dosage is
repeated.  At 30-minute intervals the spray release is repeated until a
minimum  of 10 successive exposures has been completed.  In order to control
extraneous routes of exposure, it is recommended that each animal's fur and
body be  cleaned of extraneous test substances before placement in a standard
maintenance cage.  Over the ensuing 4-day period, observations of symptomatology,
food intake, body weight changes and hematology are recommended at a minimum,
to be followed by sacrifice of the animals and histopathology of their tissues
and organs.

     Subacute inhalation assays are required once or several times in daily
repetitive testing over a considerable length of the animals' life.  U.S. FDA
(1959) mandated that this be a period of 90 days for subacute toxicity assess-
ments.   As above, the same exposure chamber and animal cleanup procedure is
used, this time with a minimum of five animals.  At least two daily 30-second
continuous spray exposures several hours apart are required.  The parameters
of symptomatology, food intake, body weight changes and hematologic morphology
are measured daily.  At the conclusion of the trial, histopathologic examination
of tissues and organs of the sacrificed animals is performed.

3.5.3  Good Animal Care Laboratory Practices (QLP's).

     A complete set of good animal care laboratory practices for use in
carcinogen bioassay based on the DHEW Guide (ILAR, 1974a) will be found in
Appendix B.  The following sections comment on the main aspects of the practices
useful for all mammalian bioassay.

3.5.3.1  Sources of Animals—

     According to the U.S. Department of Health, Education and Welfare, it is
recommended that only commercial suppliers that are accredited by the American
Association for Accreditation of Laboratory Animal Care (AAALAC) should serve
as sources of laboratory-bred animals.   These suppliers must meet  AAALAC's
criteria based on published standards for good quality, health,  housing,  hygiene,
overall  care, feeding, watering, and care by competent veterinarians, breeders
and animal care personnel before accreditation is granted.  See ILAR (1974a)
and associated references (1960 to 1977).

3.5.3.2  Physical Facilities of Quarantine Area—

     The quarantine area should be located in rooms physically separated from
existing testing areas.  Separate rooms should be provided for each species.

     Except for relaxed caging requirements prior to distribution, physical
conditions during quarantine shall be of the same quality as that provided
animals under test.

     If an epizootic disease or parasitic infection is found among the animals
upon arrival, or at any time during quarantine, the entire shipment should be
discarded and the room disinfected prior to the receipt of additional animals.

                                     426

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3.5.3.3  Examination Upon Receipt—

     Animals shall be received, in their unopened shipping containers, in the
designated quarantine area.

     Discard substandard animals on receipt for size, health or other reasons.

     Examine all animals for general health.  Sacrifice a random sample of
1/20 of the animals and examine for parasites.  Palpate all animals and dis-
card any with an abnormality.

3.5.3.4  Caging Before Distribution for Test—

     A shipment may be caged together during quarantine, acute toxicity test
and repeated dose study according to the weight-space requirements in Table
3.5.2.
                                      427

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TABLE 3.5.2  SPACE RECOMMENDATIONS FOR LABORATORY ANIMALS  (ILAR, l974a)
Species '
Mouse



Rat



Hamster



Guinea Pig

Rabbit


Cat

Dog-




Primates*-4
Group 1
Group 2
GroupS
Group 4
Groups
Pigeon"
Chicken*



Sheep and
Goat

Hog

Cattle









Hone

Weight
Up to 10 g
10-15g
16-25g
Over 25 g
Up to 100 g
100-200g
201- 300 g
Over 300 g
Up to 60 g
60-80g
81- 100 g
Over 100 g
Up to 250 g
250- 350 g
Over 350 g
Up to 2 kg
2-4kg
Over 4 kg
Up to 4 kg
Over 4 kg
Up to 15 kg
15-30 kg
Over SO kg
Up to 15 kg
15 -30 kg
Over 30 kg

Up to 1 kg
Up to 3 kg
Up to 15 kg
Over 15 kg
Over 25 kg
—
Up to ^ kg
Vi~ 2kg
2-4 kg
Over 4 kg
Up to 25 kg
25 to 50 kg
Over 50 kg
i Up to 50 kg
50-100 kg
Over 100 kg
Up to 350 kg
350 -450 kg
450 -550 kg
550 -650 kg
Over 650 kg
Up to 75 kg
75-200 kg
200-500 kg
500-600 fcg
600-700 kg
Cfvcr 700 kg
^_
—
Type of
Housing
Cage

"
11
Cage
it
(I
Ii
Cage
• i
Ii
*t
Cage
il
11
Cage
•I
II
Cage
Ii
Pen or Run
it
••
Cage
Ii
11

Cage
„
"
Cage
Cage
"
"
11
Pen
11
II
Pen
II
Stanchion
11
••
11
Pen
N
II
II
IJ
II
Tic Stall
Pen
Floor Area/Animal
(Square)
39 cm ( 6 in)
52cm ( 8in)
77 cm (12 in)
97 cm (15 in)
110cm (17 in)
148 an (2.1 in)
187 cm (29 in)
258cm (40 in)
643cm (10.0 in)
83.9cm (13.0 in)
1032cm (16.0 in)
122.6cm (19.0 in)
277 cm ( 43 in)
374 cm ( 58 in)
652 cm (101 in)
.14 m ( 13 ft)
28 ra ( 3.0 ft)
J7 m ( 4.0 ft)
28 m ( 3.0 ft)
.37 m ( 4.0 ft)
.74 m ( 8.0 ft)
1.12 m (12.0 ft)
223 m (24.0 ft)
.74 m ( 8.0 ft)
U2m (12.0ft)
Height1
12.7 cm ( ; in)
12.7 cm ( 5 in)
12.7 cm ( 5 in)
12.7 cm ( 5 in)
17.8 cm ( 7 in)
17.8 an ( 7 in)
17.8 cm ( 7 in)
17.8 an ( 7 in)
152cm ( 6 in)
152 an ( 6 in)
152 cm ( 6 in)
152 an ( 6 in)
17.8 an ( 7 in)
17.8 an ( 7 in)
17.8cm ( 7 in)
35.6 cm (14 in)
35.6 an (14 in)
35.6 an (14 in)
61.0 an (24 in)
6 1.0 cm (24 in)
—
—
81.3cm (32 in)
91 .4 an (36 in)
REFER TO FOOTNOTE NO. 2

.15 m ( 1.6 ft)
28 m ( 3.0 ft)
.40 m ( 4.3 ft)
.74 m ( 8.0 fr)
2J3 m (25.0 ft)
742cm (115 in)
232.3 cm ( 36 in)
464.5 an ( 72 in)
1090.4cm (169 in)
1651.7 an (256 in)
0.93 m ( 10 ft)
1.40 m ( 15 ft)
1.86 m ( 20 ft)
.56 m ( 6 ft)
1.12m ( 12ft)
2.79m ( SOft)
1.5 ra ( 16ft)
1.7 m ( 19 ft)
2.0 m ( 21 ft)
22 m ( 24 ft)
2.5 m ( 27 ft)
22 in ( 24 ft)
4.7 m ( 50 ft
94 m (100 ft)
112m (120ft)
13.0m (140ft)
14.0m (150ft)
•4.1 m C -14ft)
13.4 m (144 ft)

50.8 an (20 in)
762 an (30 in)
76.2cm (30 in)
91.4cm (36 in)
213.4 an (84 in)
—
__
_
—
—
__
_ _

—
—
—
_
—
—
—
—
— -
—
—
—
—
^^
                                 428

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                               TABLE  3.5.2   (continued)
                1 Height meant from the resting floor to the cage top.
                ' The* recommendation* may require modification* according to. the bodv con-
             formation* of particular breed*. As a further general guide, the height of a dog raqv
             should be equal to the  height of the dog over  the shoulder* (at the withers), plus at
             least six inches, and the width and depth of the cage should be equal to the length
             of the dog from the tip of the nose to the base of the tail, plus at least six inches.
                •The primate* -ire grouped according to approximate size with  examples of
             specie* that may be included in each group:
                Croup  1—Marmosets, tupaias. and infants of various specie*.
                Croup 2—Cebu* and similar species.
                Group 3—Macaques and large African specie*.
                Croup 4—Baboon*, monkeys larger than 15 kg. and adult members of brachiating
                         specie* such as gibbons, spider monkeys and woolly monkeys.
                Group 5—Great Ape*.
                * Where primate* are housed in groups in  pen*, only compatible animal* should
             be kept. Minimum height of pens  should be six feet. Resting  perches, nesting boxes
             and  escape  barrier* necessary  for  rhe well  being of the particular animal* should
             also be provided.
                1 Sufficient headroom mun be provided so bird* can stand erect without crouching.
3.5.3.5  Quarantine Period—

    o Animals  should be quarantined  for  a minimum  of  7 days.

3.5.3.6  Reexamlnation of Animals—

      At the end of the quarantine period,  the  animals should be  reexamined
for health (and palpated) and any additional substandard  ones discarded.
                                            429

-------
     If a sufficient number of healthy animals to satisfy test protocol
requirements is on hand after reexamination, they may be distributed for
testing.  If the number is insufficient, a new supply of animals may need to
be obtained and the quarantine and examination repeated.

3.5.3.7  Disposal of Animals Dead on Receipt or During Quarantine—

     All procedures involved in the disposal of dead animals shall be in
conformance with Federal, State and local laws and regulations pertaining
to pollution control and protection of the environment.

     Waste cans for use in removal of dead animals should be equipped with
leakproof disposable liners and tight-fitting lids.

3.5.3.8  Quality Controls at Quarantine—

     Shipments containing dead, moribund, or unsatisfactory animals must be
reported immediately to the program management and in writing to the animal
supply house concerned, with a copy to the program management.

     Results of examination for parasites in individual animals in the sample
sacrificed, including all negative findings, should be recorded in a bound
laboratory notebook by the clinician performing the examination and witnessed
by the laboratory supervisor.  It shall be the responsibility of the laboratory
supervisor to verify that a complete record has been made for each shipment
within 8 days of receipt of the shipment.

3.5.3.9  General Health Requirements—

     Hinkle (1977) has stressed that "a good laboratory animal medicine program
should provide effective preventive medicine" since there is little reason for
using diseased animals in any type of bioassay.  Thus, the following quality
control checks should be made:

     Observation - All animals should be observed regularly by properly
qualified personnel for signs of diseases.  Animal care should be under direction
of veterinarians with specialized training and experience in laboratory animal
medicine, especially those certified by the American College of Laboratory
Animal Medicine.  Sick or moribund animals or animals found dead should be
removed from the colony, and a proportion should be examined by laboratory
procedures (including pathology) to determine the cause of morbidity or death.

     Routine Monitoring Methods - At regularly scheduled intervals, water
bottles and feces should be cultured in order to determine whether the pre-
dominant organisms present are similar or identical to those previously
established and that pathogens are not present.  At regularly scheduled
intervals, normal-appearing animals should be removed from the colony for
laboratory tests.  Serum samples should be obtained and tested for antibodies
to murine viruses.  Bacteria, mycoplasms, protozoa, and metazoa should be
identified, if present.  Tissues or organs should be examined histologically
to determine the presence or absence of lesions.


                                      430

-------
     Record Keeping - Daily records should be maintained on morbidity, mortality,
and laboratory findings by room, species, and strain.  This information should
be reviewed weekly.

     Parasitology - Microscopic examination of specimens obtained from fresh
feces by concentration procedures and Scotch tape impressions of the perianal
region from representative animals should be examined for the presence of
parasitic ova.

     At the time of sacrifice, in addition to routine methods described above,
urine should be examined microscopically for nematode eggs, and the intestinal
tract, cecum, and bladder opened and examined with appropriate magnification
for internal parasites.

     In addition, histologic examination of the tissue and organs will assist
in determining whether selected protozoan or metazoan parasites are present.
Special attention and selective stains are recommended for the lower respiratory
tract and brain for Pneumocystic and Nosema, respectively.

     Reference should be made to the Diagnostic Guide (Section I) (ILAR, 1971)
and Disease Outlines (Section II) (ILAR, 1971) of "A Guide to the Infectious
Diseases of Mice and Rats", for description of clinical and pathologic features
of diseases plus appropriate diagnostic procedures.  Positive and negative
findings should be reported for each animal examined.  It should be the respon-
sibility of the laboratory supervisor to monitor the examination to assure its
completeness and correctness.

3.5.3.10  Optimization of Facilities and Housing Conditions—

     General Design - It is important wherever possible to provide access into
the animal quarters from a "clean" corridor and egress via a "dirty" corridor.
The traffic pattern should prohibit backflow from any area into a cleaner area.
The animal quarters, in particular, should be well protected as well as cor-
ridors leading into them so as to prevent, for example, the movement of dirty
cages down the "clean" corridor.

     Temperature and Humidity - Each animal room or group of rooms with a
common purpose should have individual temperature and humidity controls.  The
heating-cooling-ventilation system of the animal facility should be sensitive
enough to permit adjustments within ± 1°C for any temperature within the range
of 18°to 30°C (65°-85°F).  A temperature of 23°C ± 1°C  (74°F ± 2°F) should be
maintained in all mouse and rat rooms.  The optimum temperature for hamsters
is 20°to 24°C.  According to Federal regulations, the ambiant air temperature
in rooms where these rodents are quartered should not be less than 16°C  (60°F)
or greater than 30°C (85°F).  A relative humidity of 40% ± 5% should be main-
tained in all mouse and rat rooms.  The relative humidity for hamsters should
be 40 to 45%.  An automatic recording and alert system should be used to monitor
the ambient temperature and relative humidity in each animal room.  An emergency
power source should be available with a capacity sufficient to operate the air
conditioning and light systems of the animal facility.

     The temperature and relative humidity record charts  for each 24-hour period

                                      431

-------
throughout each bioassay  test  should be  dated,  signed, and filed for audit.
The automatic devices  for recording temperature and relative humidity should
be recalibrated monthly.   All  pertinent  data  should be entered in a bound note-
book and signed by  the technical  personnel who  performed  the work and by the
supervisor.  The  alert and emergency power systems should be tested monthly
and the results recorded.

     Ventilation  -  Each animal room should have 10 to 15  fresh-air changes
per hour without  drafts.   All  air should be adequately filtered before enter-
ing and before discharge  from  the animal facility in order to lower the risk
of transmitting viral  or  bacterial infections to the animals.  HEPA (high
efficiency particulate air)  filters having a  99.9% to 95% efficiency for re-
taining particles of 0.3  micrometer or greater  diameter are strongly recom-
mended .

     The general  exhaust  air from areas  where chemical carcinogens are used is
subject to Federal  regulation.  Recirculation of exhaust  air from rooms where
chemical carcinogens are  used  is  not permitted.  Air pressure should always
be adjusted so that all animal rooms are slightly positive to the "dirty"
corridor and negative  to  the "clean" corridor.   Rooms bordering a single
access corridor shall  be  kept  under negative  pressure with respect to the
corridor.  The animal  facility and human occupancy areas  should have separate
ventilation systems.

     A maintenance  check  on  all mechanical ventilation equipment (air condi-
tioner, blowers,  fan motors, etc.) should be  made monthly.  Air intake and
discharge filters should  be  inspected at least  monthly and replaced when
necessary.  Air pressure  of  animal rooms with regard to entrance and egress
corridors should  be checked  and adjusted, if necessary, each day.  The number
of fresh-air changes per  hour  in  animal  rooms should be monitored at least
weekly and appropriate adjustments made  when  indicated.   All data pertaining
to the above must be entered in a bound  notebook and signed by personnel
involved.

     Lighting - Housing quarters  for laboratory animals should have ample
light which is uniformly  diffused throughout  the area.  Light intensity at
the cage level shall be a  minimum of 100 foot-candles.  Examination and
animal treatment  areas  should have a minimum  light intensity of 125 foot-
candles at the work surface.   Continuous strip  fluorescent lighting mounted
flush in the ceiling is recommended.  Fixtures  must be properly sealed to
prevent the harboring  of vermin.  Animal cages  and other  primary enclosures
should be positioned so as to protect the animals from excessive illumination.
A time-controlled system  to provide a regular diurnal lighting cycle should be
provided.  Controls should be located in the  main control room.  Provisions
must be made to provide hamsters with a  lighting period of approximately 12
hours which is somewhat less than  the optimum for other small rodents.  Light
switches should be  located outside each  room  in  both clean service and evac-
uation corridors,  and  lights should be serviced, if possible, via a crawl
space or other method which does not necessitate entering the room.  Convenience
outlets should be waterproof, recessed in walls  and partitions, and located a
minimum of 0.6m (2  ft) above the  floor.


                                     432

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     Light intensity at cage level and at the work surface in animal
ation and treatment areas should be determined weekly and adjusted if neces-
sary.  Instruments for determining light intensity should be calibrated month*-
ly.  The light cycle should be monitored regularly and adjusted if necessary
to provide the optimal diurnal cycle for the species in question.  The position
of animal cages with respect to the light source should be checked regularly
to make certain that animals are not subjected to excessive illumination. All
test results and observations above must be entered in a bound notebook and
signed by personnel involved.

     Noise Control - Laboratory rodents, particularly mice, must be protected
from noise, especially high-pitch noise (upper limits of human auditory range
and beyond).  Audiogenic strains must be maintained at very low noise levels.
All noisy operations in the animal facility, such as cage and rack cleaning
and washing, etc., must be carried out in an area separate from rooms where
laboratory animals are housed.  Animals should not be caged near incompatible
species whl'ch disturb or distress them.  Carts, trucks, racks, and other move-
able equipment used in animal quarters should have rubber-tired casters and
rubber bumpers.  Concrete walls are preferred over metal or plaster construc-
tion to contain noise in animal quarters.   Acoustical tile and similar materials
should be used wherever possible to reduce the effect of "noise pollution" in
animal rooms.
     Evaluation of noise control practices should be included in all inspec-
tions of the laboratory animal facility and remedial measures instituted where
necessary.  A permanent record of these evaluations should be kept together
with data recorded for the temperature, humidity, ventilation and lighting.

3.5.3.11  Drinking Water for the Animals—

     In providing drinking water to the animals, watering bottles may be used
although an automatic watering system is preferred.  They must have an adequate
supply of fresh and treated water ad libitum.  Checks should be made to ensure
that the water bottles are accessible to all animals and that sanitized water
bottles, stoppers, and sipper tubes are supplied at least twice weekly.  The
animal care workers should routinely examine the watering device to assure
its proper functioning.

     Samples of the drinking water on a weekly basis as supplied into the
animal quarters should be directly quality controlled by immediate chemical
analysis (e.g., by gas chromatography, atomic absorption analysis, etc.) and
by microbiologic culturing to screen for pathogenic microbes and viruses.
Potential pathogens carried in the water should be killed or removed through
appropriate treatment, such as sterilization, pasteurization, filtration,
and/or chlorination.  The methodology and standards described by Rand et al.
(1975) should be the minimal criteria followed.

3.5.3.12  Laboratory Animal Feed and Bedding—

     Feed should be accessible to all animals at all times.  The feed containers
should be durable and should be kept clean by sanitizing at least once a week,
at which time remaining feed in these containers should be discarded.  The


                                     433

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 containers  should  be mounted on the animals'  cages  so as  to  prevent  or minimize
 contamination by excreta.

      Date of  manufacture  and delivery of all  feed supplies should  be recorded
 upon  receipt.  Products delivered 90 days or  more after manufacture  should
 not be  accepted.

      Feed and bedding shall be stored in a clean area and protected  from
 spoilage or deterioration and infestation or  contamination by  vermin.  A
 continuous  pest control program is essential.   Containers should be  stored
 off the floor on pallets,  racks,  or crates.   The area should be physically
 separated from refuse areas.

      Feed should be stored in receptacles with tightly fitting lids  or covers
 which can be  sanitized before reuse, or  in original containers as  received
 from  the supplier.   The storage area should be cool (10°C or less)'  dry and
 airy.

      All supplies  of feed  and bedding as well  as equipment in  storage should
 be carefully  protected against contamination  by pesticides.  No pesticides
 should  be used inside buildings unless specifically agreed to  by program
 management.

      Temperature in the feed storage area should be recorded continuously by
 an automatic  recording thermometer.   Temperature recordings  should be inspected
 daily and adjustments made when necessary to maintain a temperature  of 10°C or
 less.   All  charts  should  be dated,  signed,  and filed for  audit by  program
 management.

      The automatic  temperature recorder  should be recalibrated at  least monthly
 and data recorded  and signed by technical personnel performing the work.

      All storage areas should be  inspected weekly for the presence or evidence
 of vermin and appropriate  action  taken when necessary.

      Feed in  containers found open  during inspections should not be  used.

      If  possible,  feed should be  sterilized consistent with  a  disease control
 program.  It  is  recommended  that  periodic sterilizer runs be monitored to
 assure  that vegetative forms  of microorganisms have been  killed.  This may be
 most  easily accomplished by  placing  a  filter paper  strip  impregnated with
 Escherichia coli in  the center  of load.   The strip  is then incubated in a
 suitable  medium  and  examined  for  growth.  Food may  be held in  a clean storage
 area until culture  results are  available.

     Care must be taken to ensure that nutrients are not  degraded or that the
 palatability  of  the  feed is  reduced.   Random feed samples should be  collected
with each new batch  of  feed  and analyzed  in accordance with  the Association
 of Official Analytical Chemists  (AOAC, 1975).   Samples (500-800 g) of each
batch should be  stored in a  freezer  for  the duration of each bioassay so as
 to permit back-referencing against a control feed sample  should this be required.


                                     434

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Feed that is older than 90 days may be considered unsatisfactory due to loss
of essential nutrients.
     As each new batch is received, samples of the feed should be chemically
analyzed for pesticides, mycotoxin, industrial chemicals and biologic contam-
inants in accordance with the procedures described by the Association of
Official Analytical Chemists.  If unacceptable levels of contaminants are
detected, the feed should not be used and a change in source might be investi-
gated.
     If pesticides are used in the animal facility, supplies of feed and
bedding shall be analyzed at monthly intervals.  Results of all analyses
should be recorded and reported immediately to program management who will
notify the bioassay laboratory of any lots unsuitable for use.

3.5.3.13  Vermin Control—
     A safe and effective program for the control of insects, ectoparasites,
avian  and mammalian pests in and around the animal facility should be
established and maintained under the supervision of a veterinarian or other
qualified person.
     The animal facility should be inspected weekly for the presence or
evidence of vermin and remedial measures instituted if necessary.  Results of
inspections and remedial action taken should be recorded in a bound notebook,
dated, and signed by inspector and supervisor.
     Wild rodents and other vermin carry a variety of bacteria, viruses, and
parasites which may be transmitted to experimental animals should they gain
entrance to the facility.  The population of wild rodents in the vicinity of
animal buildings should be reduced or eliminated if possible.

3.5.3.14  Changing of Litter or Bedding, Changing of Laboratory Animal
          Cages and Disposal of Waste—

     Cages should be program- and chemical-specific.  They should be returned
to the same chemical group and dose level to prevent test-chemical contamina-
tion.

     Provision should be made for prompt removal and disposal of all food
wastes from laboratory animal cages so as to minimize vermin infestation,
odors, and disease hazards.

     Measures must be taken to prevent molding, contamination, deterioration
or caking of feed.  Uneaten fruit or vegetable supplements must not be allowed
to accumulate in animal cages.

     Catch-pans for animals caged in exposure chambers should be cleaned and
relined with new absorbent paper daily.

     Animal cages should be inspected daily and litter or bedding changes as
frequently as necessary, but not less than once per week, to comply with good
animal laboratory practices.

     Animal care personnel should be responsible for changing animals to
sanitized cages with fresh bedding on at least a weekly basis.
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     Supervisors should monitor removal and disposal of all wastes containing
chemical carcinogens or infectious agents to make certain that all procedures
are in compliance with applicable Federal, State and local laws and regulations
of the U.S. EPA.

     Commercially available spore strips should be included in all autoclave
loads of infectious waste and subsequently cultured to monitor the efficacy
of the sterilization procedure.

     Data pertaining to the disposal of infectious wastes or wastes containing
chemical carcinogens must be entered in a bound notebook, dated, and signed
by personnel involved and the supervisor.

     Food and other wastes contaminated with known or suspected chemical
carcinogens should be placed into separate plastic bags or other suitable
impermeable containers for each carcinogen, closed, sealed, and labelled with
both name of carcinogen and "DANGER — CHEMICAL CARCINOGEN", before being
transported to storage or disposal area.  Final disposal should be in con-
formance with Federal, State and local laws, and with the NCI Office of
Research Safety Regulation (NCI, 1976).

     Wastes which are not contaminated with carcinogens or infectious agents
may be disposed of at a public incinerator or burned at the facility.  In-
cineration should comply with U.S. EPA regulations.

     Infectious wastes should be autoclaved or rendered noninfectious by other
effective measures before removal from the animal facility.

     Waste disposal must comply with all Federal, State, and municipal laws,
statutes, or ordinances.

3.5.3.15  Sanitation of Equipment and Supplies for Laboratory Animals—

     Cages, racks, feeders, water bottles, catch-pans, exposure chambersf an
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     All sanitized cages, feeders, water devices, racks, catch-pans, and
exposure chambers shall be inspected for physical cleanliness prior to reuse.
Unsatisfactory items shall be resanitized.

     Frequency of sampling of cages and other items for microbiological
monitoring of the sanitization procedure will depend upon type of decontam-
ination and level of protection desired.  No gram-negative organisms should
be detected, especially Pseudomonas spp., but sporeformers and heat-resistant
non-pathogens will be found occasionally.

     Detection of gram-negative organisms should result in an immediate shut-
down of washing equipment and correction of the defect or institution of
better room sanitization procedures, depending on probable source.

3.5.3.16  Disinfection and Sanitation—

          o  If an epizootic disease occurs among animals in quarantine or
on test, the area shall be disinfected before use for new animals.

          o  Disinfectants for use in any part of the bioassay facility must
be approved by EPA's program management.

          o  If formaldehyde gas is used, the room should be sealed and then
treated by evaporating 500 ml of Formalin (37% solution of formaldehyde in
water and stabilized with methyl alcohol) for each 27 m3 of space.  The
temperature should be at least 21°C (70°F) and the relative humidity 75 to 80%
during fumigation.  The exposure period should be 24 hours.

          o  Disinfected animal rooms should not be reused until results of
microbiological analyses indicate the absence of microorganisms pathogenic
for humans and domestic animals.  In this regard, the effectiveness of all
disinfection procedures should be evaluated by sample swabbing of tables,
benches, racks, walls and floor (at least one swab per area) and culturing
(cell cultures, embryonated eggs, or laboratory animals).  Acceptable diagnostic
practices of the American Society of Clinical Pathologists or an equivalent
organization should be used.

          o  Room and corridor floors, sinks, and pipes should be washed with
a microbicidal solution weekly.  Ceilings, walls, and partitions shall be
treated in a like manner at regular intervals.

          o   After a room has been emptied of animals, all surfaces and fixed
equipment should be washed with a microbicidal solution.  Portable equipment
for the room should be sanitized and/or sterilized, returned to the room, and
the room and equipment fumigated.  Paraformaldehyde is recommended for this
purpose.

          o  The primary animal testing enclosures should be cleaned and
sanitized often enough to prevent an accumulation of excreta, debris, dirt and
harmful contamination.

          o  These enclosures should be sanitized by washing with hot water

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(82°C) and soap or detergent, or by washing all soiled surfaces with a detergent
solution followed by a safe and effective disinfectant, or by cleaning all
soiled surfaces with live steam.

          o  All wastes should be collected and removed regularly and frequently
in a safe sanitary manner.  For example:  highly infectious waste should be
rendered noninfectious, by autoclaving or other effective means, before re-
moving them from the animal facility.

3.5.3.17  Disposal of Dead or Sacrificed Animals and Their Tissues—

          o  All procedures involved in disposal of dead animals and tissues
should be in conformance with Federal, State, and local laws and regulations
pertaining to pollution control and protection of the environment.

          o  All dead animals should be subjected to full gross and histologic
examination in accordance with EPA's bioassay program.  Carcasses may be dis-
carded immediately following necropsy and fixation of all required tissues
needed for histopathology.

          o  Contaminated wastes, cleaning devices, and animal carcasses
should be transported to the disposal area in a closed impermeable container
and disposed of by methods approved by the EPA.

          o  Refrigerated storage should be available for holding dead animals
until necropsy.  The area should be separate from all other cold storage and
should be used exclusively for refuse storage.  The temperature should be kept
below 7°C (45°F).  The animals shall not be frozen.

          o  Supervisory personnel should monitor the disposal of all dead and
sacrificed animals and tissues to make certain that all procedures are in
accord with Federal, State, and local laws as well as with regulations of the
EPA.

          o  Containers, liners, covers, etc., used in storage and disposal of
sacrificed animals and tissues should be inspected during operations to main-
tain conformance with EPA's safety regulations.

3.5.3.18  Disposal of Radioactive Biologic Wastes—

          o  Radioactive biologic waste must be disposed of in accordance with
applicable Federal and State regulations and licenses. *

          o  A regular schedule for the collection of this waste should be
set up.

          o  Leakproof disposable liners in waste cans must be used for burial
or disposal of such radioactive waste.

          o  The storage area for radioactive waste should be physically
separated from other storage facilities and animals.


                                      A38

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          o  In some instances, the ordinary storage facilities may be used
for holding the waste, if properly monitored, until removal for proper burial
or disposal.

          o  Special shielding of the storage area may be required.

          o  Cage washing equipment should be of a type capable of decontam-
inating cage and accessories without accumulating radioactive waste.  Machines
should not recirculate the wash solution.

          o  A system of radiation monitoring must be instituted so as to
prevent the spread of radioactive waste.

3.5.4  Bioassay Methods

     The purpose of direct toxicity testing is to establish the potential
harmfulness or safeness of single substances or mixtures of substances to
humans and other animals via correlation of the biologic effects in test
animals at different concentrations of the test substance (Hayes, 1975;
Loomis, 1974).  The ultimate response by a test animal is often an all-or-none
effect (i.e., death, permanent neurologic damage, etc.), while some minimal
lower concentration produces no measurable effect, each extreme varying from
one animal to another.  Such quantal or all-or-none responses differ from
graded responses (i.e., body temperature, pulse or breathing rate changes)
in that the latter can be continuously altered while the former cannot.  The
experimental determination of the range of dosages causing quantal effects
in a group of animals is the basis of the quantal dose-response relationship
that is often mathematically determined by probit analysis (Litchfield and
Wilcoxon, 1949).  Graded responses are analyzed using normal distribution
theory.

     Since the susceptibility of different individual animals and different
species must be based on a common parameter of comparison, by convention the
body weights of the test organisms are taken into account in dosing by
establishing the mass of the organism as the basic unit of dosage  (i.e.,
generally units of mg/kg or yg/kg of body weight).  Dosage, the amount of
test agent provided in relation to the weight of the test animals, should
always be specified instead of the dose, which relates only the amount of
agent given, independent of the animals'weights.  It is often useful to
denote the time dimensions with the dosage  (i.e., mg/kg/hour), especially
when it is necessary to repetitively dose these animals in the short interval
of the acute bioassay.

3.5.4.1  Acute, Single, and Multi-Dosage Toxicity Bioassays—

     The most widely accepted unit for expressing the results of acute,
quantal, single-dose mammalian toxicity assays is the "1-ED50",  the statistical
estimate of  the single dosage of test substance that produces the  measured
effect in 50% of a population of test species under stated assay conditions
and routes of exposure.  The measured quantal effect can be physiologic
(i.e., sleep induction, total ataxia) or lethal.  In the latter  case,  the unit
is retitled  the "1-LD50", the statistical median  lethal dosage.  Acute,  quantal,

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multi-dose assays, generally performed within a 5-to 10-day interval in which
dosages are administered daily, are less common but are sometimes necessary
in order to test for an acute physiologic build-up of toxicants, especially
substances which are lipidsoluble  (i.e., vitamins A and D).  In either the
single-or the multi-dosage bioassays, randomly chosen groups of animals are
established and each group is given one of a series of increasing dosages
selected in such a way that the smallest dosage will produce the biologic
effect in only a small number of each group receiving that dosage while the
largest dosage yields the same effect in the majority of animals receiving
the test substance at that level.   Any effect measured in this way must be
an all-or-none response and can thus be easily statistically analyzed by
Litchfield and Wilcoxon's probit analysis technique.  An outline of a sample
protocol is given in the following pages.

                  EXAMPLE:  ACUTE  IN VIVO TEST IN RODENTS

Purpose of Study

•  Mammalian acute toxicity determination

Materials

•  Young adult rats (approximately 250 g each) can be purchased from the
   supplier.

Design of Experiment

•  It is recommended that a two-step approach be taken to initial acute in
   vivo toxicology evaluation of unknown compounds.  These two approaches
   are:  The quantal (all-or-none) response and the quantitative (graded)
   response.  Normally, the quantal assay is used to determine the necessity
   to carry out the quantitative assay.

Quantal Assay

•  Number of Animals:   Five male and five female rats.

•  Quarantine Period:   A minimum of five days.

•  Dose:  A single dose of 10 g per kg of test material  undiluted if a liquid,
   diluted with a biologically inactive solvent if a solid.  A control is
   required.

•  Route of Administration:  By gavage.  If no mortality occurs in the quantal
   study, no further work need be done on the test substance.  If a single
   animal in this study dies in the 14 day observation period, then a quan-
   titative study will  be performed.

Quantitative Assay

•  Number of Animals:   One hundred animals equally divided by sex will be used
   for this study.

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•  Quarantine Period:   7 days.

•  Health of Animals:   Must be good when ready to be experimented upon.

•  Randomization:   The animals  will be randomly divided into five groups of
   five male and five  female animals per group.

•  Test Material:   Selected as  in quantal  study.

•  Dosing:  3.0, 1.0,  0.3, 0.1  g per kg and a control.   This dosage schedule
   will be selected depending greatly on the results of the quantal study in
   regard to the numbers of animals that died and severity and type of signs.
   Require randomization.

•  Route of Administration:  By gavage.

Conduct of Experiment

   QUALITY CONTROL —  Establish regular audits of performance throughout the
   experiment.

Quantal Assay

*  Observation:
   (1)  Immediately following administration of test material and at frequent
   intervals during the first day, observe all toxic signs or pharmacological
   effects indicated in Table 1.
   (2)  Score the frequency and severity of the signs.
   (3)  Pay particular attention to time of onset and disappearance of signs.
   (4)  Make daily observation on all animals through the test period (14 days).
   (5)  Investigate further the test materials which produce harmful effects
   in vivo and do not  result in deaths.
   QUALITY CONTROL —   Signing and witnessing of all records.

•  Pathology:  At end  of observation period, kill all surviving animals and
   perform necropsies.  Likewise, perform necropsies on all animals that die
   during the course of the assay.
   QUALITY CONTROL —   Signing and witnessing of all data collected.

Quantitative Assay

•  Observation:  Same  as indicated in quantal assay.
   QUALITY CONTROL ~  Signing and witnessing of all records.

•  Pathology:  Same as indicated in quantal assay.
   QUALITY CONTROL ~  Same as above.

Data Analysis

Quantal Assay

•  Should no mortality occur in the quantal study, the LD50  should  be reported

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   as greater than 10 g per kg.
   QUALITY CONTROL — Use statistical expertise in analysis of results.

Quantitative Assay

•  Calculate LD50 by methods of Horn (1956) or other reliable methods
   QUALITY CONTROL — Use statistical expertise in analysis results.

•  If the data are not suitable for a precise LD50 calculation, i.e.
   total mortality occurs in the lower dosage level, make an estimate of
   the LD50 or express LD50 as greater than 3 g per kg or less than 0.1
   g per kg.
   QUALITY CONTROL — Use standard statistical techniques at all  times.

•  Depending on the results of the assay of higher dosages, lower dosage
   or another series at intermediate dosages may be a necessity.

Reporting Data

•  The report should include:  (1) a statement of methods, (2) the results
   obtained, (3) a statement of conclusion.
   QUALITY CONTROL -- Adopt a good system for review and publication  of reports.

References

Horn, H. 1956.  Simplified LD50 (or ED50) calculations.  Biometrics
   Vol. 12, pp. 311-322.

    The discussion in this example is principally derived from the following
report:

    Duke, K.M., M.E. Davis, and A.J. Dennis.  1977.   IERL-RTP Procedures
Manual:  Level 1 Environmental Assessment Biological Tests for Pilot  Studies.
EPA-600/7-77-043, April 1977.
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       TABLE 1.  PHYSICAL EXAMINATIONS IN ACUTE TOXICITY TESTS IN RODENTS
   Organ System
    Observation and
       Examination
     Common Signs of Toxicity
CNS and
somatomotor
Autonomic
nervous system

Respiratory
Cardiovascular

Gastrointestinal



Genitourinary



Skin and fur

Mucous
membranes
Eye

Others
Behavior
                   Movements
Reactivity to various
stimuli
Cerebral and spinal
reflexes
Muscle tone
Pupil size

Secretion
Nostrils
Character and rate
of breathing

Palpation of cardiac
region
Events
Abdominal shape
Feces consistency
and color
Vulva, mammary
glands
Penis
Perinea! region
Color, turgor,
integrity
Conjunctiva, mouth

Eyeball
Transparency
Rectal or paw skin
temperature
Injection site
General condition
Change in attitude to observer,
unusual vocalization, restless-
ness, sedation
Twitch, tremor, ataxia, cata-
tonia, paralysis, convulsion,
forced movements
Irritability, passivity,
anaesthesia, hyperaesthesia
Sluggishness, absence

Rigidity, flaccidity
Myosis, mydriasis

Salivation, lacrimation
Discharge
Bradypnoea, dyspnoea, Cheyne-
Stokes breathing, Kussmaul
breathing
Thrill, bradycardia, arrhy-
thmia, stronger or weaker beat
Diarrhea, constipation
Flatulence, contraction
Unformed, black or clay colored

Swelling

Prolapse
Soiled
Reddening, flaccid skinfold,
eruptions, plloerectlon
Discharge, congestion,
hemorrhage cyanosis, jaundice
Exophthalmus, nystagmus
Opacities
Subnormal, increase

Swelling
Abnormal posture, emaciation
3.5.4.2  Subacute Toxicity Bioassays—

    Subacute toxicity testing involving protracted dosing has been performed,
more or less by convention, up to one-tenth of an experimental animal's life-
span.  For rats a 90-day Interval is considered adequate, while dogs are tested
for a 1-year period, in each case with daily dosing.  Boyd and Boyd (1962)
appear to have been the first to report subacute toxicity in the form of a
                                      443

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90-LD50 (i.e., a 90-day median lethal dosage).  Boyd (1968) later pointed
out that subacute tests using rats would be reduced to 70 days (i.e., 70-LD50)
essentially without loss of important information.  The results of 90-day
studies not only in rats but also in dogs have been shown by Weil and McCollis-
ter (1963) to be similarly comparable to corresponding lifetime studies in
these species for a wide variety of compounds.  As reviewed by Hayes (1975)
the 90-LD50 (or 90-ED50) is statistically comparable to the 1-LD50 (or 1-ED50)
in that the dosage expressed in logarithms is linearly related to the percent-
age biologic effect expressed as probits.

    An example of a subacute bioassay protocol is given below.

                    EXAMPLE:  MAMMALIAN SUBACUTE BIOASSAY

Purpose

    The purpose of subacute studies 1s to determine what cumulative effects
might become manifested during prolonged subacute exposure to the test sub-
stance and to provide pilot chronic study information through one-tenth of
the species' lifespan prior to performing a longer term (chronic) bioassay.

Design of Experiment

    The preferred method (Zbinden, 1973) of assessing subacute toxicity is to
challenge both sexes of at least two species of animals (one rodent and one
non-rodent) with diets dosed with at least three levels of test agent, and to
compare the biologic responses of these test animals after prolonged exposure
with the responses in control animals fed the same but undosed diet over the
same interval.  The Maximum Tolerated Dosage, or MTD, estimated to cause zero
deaths during the preceding acute animals study should be the highest level
of exposure incorporated into an average dietary feeding portion (Sontag et
al., 1976).  Thus, the concentration of test substance at the highest test
level should afford the animal not more than 1-MTD in each average daily
feeding portion (e.g., 1-MTD per 25.0 g of feed per rodent, assuming it has
been demonstrated that this amount of feed is the average amount consumed by
each rodent).  At least two other lower dietary test levels (e.g., 1/3-MTD
and 1/9-MTD, or 1/5-MTD and 1/25-MTD) also should be bioassayed with separate
randomly picked groups of animals.

Summary of Experiment Design

Species     Group     No. Females     No. Males      Dosage      Duration
ecies     Group     No. Females     NO. Males      Dosage      uuratior
Rat         1           ^0            20          Control      90 days
            2            20            20          MTD         90 days
            3            20            20          1/3 MTD     90 days
            4            20            20          1/9 MTD     90 days
Dog         155           Control      1  year
 "2            5             5           MTD         1  year
 "35             5           1/3 MTD     1  year
 "45             5           1/9 MTD     1  year
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 Route of Administration of Test Substance

 •   Dosing of test animals on a long-term basis  (e.g., 90 days to 1 year) 1s
    feasible only through the animals' diet, since other routes such as Intra-
    muscular Injection or stomach tube gavage are less convenient and afford
    more trauma to the animals.

 •   Accurate and up-to-date record keeping of body weight changes and amount
    of food ingested are essential for later interpretation of comparative
    mortality and pathology data between control and test populations.

 •   At the end of the prescribed dietary exposure period, it is required that
    surviving rodents be fed an uncontaminated diet for an additional 2 weeks
    or longer while surviving dogs be given uncontaminated feed for an added
    4 weeks or longer prior to sacrifice, necropsy, and histologic examination.

 Exposure Schedule

 •   The test substance should be ingested daily by all test animals for the
    duration of the bioassay as described in the above experimental design.

 Design Outline

 •   Two species of mammals each consisting of four groups (20 rodents per
    group or 5 dogs or cats per group); one group serves as the negative
    (vehicle) control, while the remaining three groupings ingest geometrically
    decreasing dosages, the highest being the MTD; rodents observed a minimum
    of 2 weeks further on the uncontaminated diet following the full exposure
    duration; dogs further studied a minimum of 4 weeks.
    QUALITY CONTROL — Same as that outlined for acute toxicity bioassay.
    Analyze periodically freshly mixed diet samples and maximally aged diet
    samples, as used in the study to assure stability of the toxicant.

 Observations and Bioassays

 •   Twice daily records should be kept on all animals during and after the
    exposure with respect to relevant clinical  signs (see list below), all
    function tests and hlstopathologies, morbidity rate, necropsy observations,
    etc.; all survivors including controls should also be examined clinically
    (see list below), by histopathology and by necropsy for comparison with
    non-survivors.
    QUALITY CONTROL — Same as that outlined for acute toxicity bioassay.

 Clinical Laboratory Studies in Subacute Bioassays

•   The following clinical laboratory assays should be performed on a minimum
    of 5 animals of both sexes of both species  from the control and all the
    test groups (selected randomly 1n the cases of survivors) on a scheduled
    basis (i.e., at 0,45 and 90 days and at termination for rodents, and at 0,
    26, and 52 weeks and at termination for dogs or cats, where the Oth day or
    week immediately precedes exposure of the test groups).
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   Hematology

     Hematocrit                      Total  leukocyte count
     Hemoglobin                      Differential  leukocyte count
     Erythrocyte count               Reticulocyte count

   Clinical Chemistries

     Blood sugar                     Serum glutamic oxaloacetic transaminase
     Blood urea nitrogen             Serum glutamic pyruvic transaminase
     Alkaline phosphatase

   SMAC determinations will be made at termination, including in addition
   to the above:

     Creatinine           Albumin            Total bilirubin
     Cholesterol          Calcium            Lactic acid dehydrogenase
     Uric acid            Phosphorus         Iron
     Total protein        Globulin           Triglycerides
     Creatine phospho-    C02                Sodium chloride
      kinase              Potassium

   Ur^'nalysis

     pH                   Glucose            Specific gravity
     Ketones              Albumin

Autopsy and Terminal Necropsy Examinations

•  Gross examination should be done as called for in the acute bioassay.

Histopathologic Examinations

•  Histopathologic examinations should be performed on selected tissues and
   organs.

Records and Reports

•  Records will be maintained on:

     Sampling assays      Feed consumption   Body weights
     Daily observations   Mortalities        Necropsies
     Pathology

Changes or Revisions

     Any changes or revisions of this approved protocol will be documented,
     signed by the study director, dated, and maintained with this protocol.
     The sponsor will be notified prior to any revision of this protocol.
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3.5.4.3  Chronic Toxicity Bioassays—

     It is not valid to assume that chemicals which produce a finite toxicity
in one month will, at one-half the concentration, produce the same toxicity
in two months.  Consequently, if human exposure has been or will be protracted
over several months or years, then animals should be bioassayed for equivalent
lengths of their lifespans by  comparable routes of administration and at
comparable as well as excessive dosages.  Otherwise, extrapolation of these
animal results to mankind would be invalid.

     In order to assess the potential long-term hazards of low levels of
suspected toxic substances in our food, water, environment, etc., the results
of acute and subacute animal studies must be extended in chronic, low-level
exposure bioassays in other animals so as to provide sufficient data to permit
an intelligent evaluation of all possible dangers at a minimum cost in time
and money.  In general, chronic mammalian toxicity studies are directed at
investigating the chemical induction of specific phenomena such as carcino-
genesis, long-term cytotoxicity, mutagenesis, and teratogenesis  and reproduc-
tive retardation.  This subsection, however, will be limited to discussion of
currently accepted techniques of evaluating the carcinogenic potential of
chemical substances and mixtures.

     At present, the assessment of the carcinogenic potency of a substance
is based on the subjective judgment of a qualified pathologist making histo-
pathologic evaluations of tissues taken from exposed or treated animals.  The
experience of the pathologist with tissues from many different animal species
under various test conditions is critical since there is no objective means
for correlating observed long-term effects with chronic treatments or exposures
at present, other than by direct chemical analysis for the tested substance in
target tissues and organs.  The histopathologic observer must be able to rec-
ognize the interfering effects of concurrent infections, intracage fighting,
numbers and types of "spontaneous" tumors, chronic degenerative diseases,
dietary deficiencies or any other condition, unrelated to the chronic exposure
or treatment, that may bias the carcinogenicity results.  The answers, at
present, cannot be found in statistical data alone, but must be combined with
pathologic evaluations before a safe exposure limit level for mankind can, if
possible, be established.

     Because of the duration, effort, and expense involved in conducting
chronic toxicity studies in mammals, any preliminary effort is well spent
when expended in selecting the most pertinent dosage levels, the best conditions
of animal husbandry and hygiene, the accuracy of the dietary exposures or
chronic dermal or inhalatory treatments, quality of mixing vehicle, avoidance
of bias from contaminants in water, air and food, etc.  To control contaminant
interferences, poor hygiene, etc., the National Cancer Institute has recommended
(Sontag et al., 1976) that a second set of untreated control animals, independent
of the vehicle control animals, be incorporated into the overall bioassay.
These untreated animals should, moreover, be housed in a separate room with a
separate air and water supply so as to reduce inadvertent exposure to the test
agent.  These design controls have been summarized in outline form in the
sample protocol on the following pages.


                                     447

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                    EXAMPLE:  MAMMALIAN CHRONIC BIOASSAY
Purpose

    Carcinogenicity determination.

Summary of Experimental Design

                                        No. Males
                                            60
                                            60
                                            60
                                            60
                                            60
                                            30
                                            30
                                            30
                                            30
                                            30
   Control-A is the untreated separated control group; Control-B is the
   vehicle-treated control group; MTD is the maximum tolerated dosage as
   determined in the preceding subacute bioassay;  1/3 and 1/9 MTD are
   fractions of the MTD.
   Dogs or cats may be required because some of their metabolic and physiologic
   characteristics of interest may be closer to humans than those of rats or
   mice.
Speciest
Mouse
ii
it
ii
ii
Dog
ii
n
ii
ii
Group
1
2
3
4
5
1
2
3
4
5
No. Females
60
60
60
60
60
30
30
30
30
30
Dosage*
Control -A
Control -B
MTD
1/3 MTD
1/9 MTD
Control -A
Control -B
MTD
1/3 MTD
1/9 MTD
Duration
30 months
30 months
30 months
30 months
30 months
48 months
48 months
48 months
48 months
48 months
Chronic Exposure Schedule and Routes of Administration

•   Three dosage levels are recommended (Grice and Da Silva, 1973) in chronic
    bioassays beginning with the MTD (maximum tolerated dosage) as the highest
    level and geometric reductions in dosage used at two lower levels.

•   The test substance should be ingested daily by all test animals for the
    duration of the bioassay as described in the above summary.  Dermal dosing
    of the test agent in a chronic bioassay might also be conducted in a
    similar manner, but only for those substances where cutaneous contact is
    the main route of human exposure.

•   A minimum of 60 smaller mammals (rodents, rabbits, hamsters, guinea pigs,
    etc.) per sex per test and control group is required for observing a mini-
    mum of a 5% incidence of chronic toxic effects with a confidence probability
    of 0.95 (Zbinden, 1973).

    A minimum of 30 larger mammals (dogs, cats) per sex per test and control
    group is required for observing a minimum of a 10% incidence of chronic
    toxic effects with a confidence probability of 0.95 (Zbinden, 1973)

    The test agent should be given for a length of time sufficient to yield
                                     448

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    the maximum response in the test animals.  For this reason it is recommended
    that rodents and rabbits be tested for as long as 30 months, and dogs and
    cats for as long as 48 months.  When 20% survival of test animals and/or
    20% tumor-free incidence are reached, however, consideration must be given
    to earlier termination of the investigation.

•   When exposure is through the feed or drinking water on a daily basis, the
    amount of the test substance consumed must be quantified via measurement
    of the food or water intake.  Body weights must also be recorded at least
    on a weekly basis to determine if eating patterns have been altered.

•   If more than 20% mortality occurs in the first 18 months among the smaller
    mammals (mice, rats, hamsters) serving as vehicle-treated controls,
    consideration should be given to declaring this particular trial invalid.
    Similar invalidation should be considered if 20% mortality results in the
    first 30 months among the larger mammals (dogs, cats).

•   No recovery period should be permitted; instead, all survivors should be
    sacrificed at termination for necropsy and histopathologic examination.

Design Outline

•   Two species of mammals of each sex and each consisting of 5 groups (60
    rodents per group or 30 dogs per group); one group serves as untreated,
    separated controls; another group serves as vehicle-treated controls;
    remaining groups are dosed with geometrically decreasing amounts of test
    agent, the highest being the MTD via mixing into feed or drinking water.
    QUALITY CONTROL — Same as that outlined in acute toxicity bioassays
    especially periodic analyses of the feed and water for extraneous or
    contaminating substances which may interfere.

Observations and Bioassays

•   Twice daily recordings of morbidity rate and autopsies, if any; daily
    recordings of quantities of water and feed ingested; weekly recordings of
    body weight and general health observations; monthly recordings of relevant
    clinical signs and function tests (see list below); all recordings of
    gross anatomical and histopathologic investigations or autopsies and  final
    necropsies as occur for all non-survivors and for all animals sacrificed
    at termination of each chronic study.
    QUALITY CONTROL — Same as that outlined in acute toxicity bioassays
    especially with respect to record keeping, signing and witnessing of
    notebooks, "blind status" of examining histopathologist, and survival
    of at least 80% of control animals within first 18 months for rodents and
    30 months for dogs.
                                     449

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Clinical Laboratory Studies in Chronic Bioassays

•   Clinical laboratory assays should be performed as in the Subacute
    Bioassay.

Autopsy and Terminal Necropsy Examinations

•   Gross examinations should be made as in the Acute Bioassay.

    If mortality in any test group after 18 months for rodents or 24 months
    for dogs or cats approaches 50%, consideration should be given to early
    termination of the bioassay.

Histopathologic Examinations

•   Histopathologic examinations will be performed on selected tissues and
    organs.

Records and  Reports

•   Records  will be maintained on:

               Sampling assays              Mortalities
               Feed consumption             Necropsies
               Body weights                 Pathology
               Daily observations

    In  addition to the final report,  interim  reports may be made available to
    the sponsor if required.  The frequency of  such  reports will be determined
    prior  to study initiation.  The report will  be a complete  scientific
    presentation of results  and conclusions.

Changes or Revisions

 •   Any changes or revisions of this  approved protocol will be documented,
    signed by  the study director, dated, and  maintained with this protocol.
    The sponsor will be notified prior  to any revision of  this protocol.

 3.5.4.4  Chronic Inhalation  Study in  Rats with  In Utero Exposure—

  EXAMPLE:   EFFECT ON LITTER  OF CHRONIC  INHALATION BY PARENTS BEFORE BREEDING

 Purpose of Study

    Determination of effects on reproduction  process
    Chronic toxicity determination
    Carcinogenic evaluation
                                       450

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Design of Experiment

•  Phase I - Reproduction:
               Group No.

                   2
                   3
                   4
                   5
                   6
 90 male and 180 weanling Fischer rats, 70 to 90 g
 in weight.

No. of Animals
Male    Female
IS-
 IS
 15
 15
 15
 15
	    Dose Levels
 30       Control
 30       1/100 MTD
 30       1/30 MTD
 30       1/10 MTD
 30       1/3 MTD
 30       MTD
   Breed to obtain 25 litters per group (estimate  85%  of bred  females will
   litter).  At weaning, randomly select 3 males and 3 females from each
   litter for Chronic Toxicity Phase.

   Phase II - Chronic Toxicity and Carcinogenicity
               Group No.
                   1
                   2
                   3
                   4
                   5
                   6

Route of Administration
No. of Animals
Male    Female
-75-
 75
 75
 75
 75
 75
         Dose  Levels
"75       Control
 75       1/100 MTD
 75       1/30  MTD
 75       1/10
 75       1/3
 75       MTD
o  Inhalation exposure in stainless  steel  chambers  with  effective  exposure
   areas of 5.5 x 5.5 x 6.0 feet.

Exposure Schedule and Subsequent Handling

o  Phase I - Reproduction

   - Expose parent generation in individual  cages 23 hours  per day,
     7 days per week for approximately 100 days  or  for at least 60 days
     prior to breeding.
   - At end of exposure period,  one  male and two females will  be placed
     in each breeding cage for one week.
   - Females will then be placed in  individual  cages and allowed to  litter
     and nurse their offspring for 28 days.   The Utters will  be reduced
     to eight pups, half of each sex,on day 4 of lactation.

•  Phase II - Chronic Toxicity
                                    451

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   - One week after weaning (day 28) of the first litter,  all  excess  rats
     (above 8 from each of 25 litters) will be discarded and remaining  rats
     will be identified by ear tag.  This will be considered Week 0 for re-
     cording purposes.  The actual data of birth and heritage of each rat
     retained for the chronic study will be recorded for possible further
     evaluation. These rats will be individually housed.

Conduct of Experiment

   QUALITY CONTROL -- At experimental design stage, advice of a statistician
   should be sought on group size and on methods of statistical analysis to
   be used.

   QUALITY CONTROL — Hold in quarantine for 1 week.

•  Assign animals to study group as removed from shipping  crates.
   QUALITY CONTROL — Use randomization procedures.

•  Identify animals by cage, group and individually.
   QUALITY CONTROL — Achieve an equivalent mean body weight between  groups.
   QUALITY CONTROL — Use ear tags and durable cage markers.
   QUALITY CONTROL — Accurately determine body weight and food consumption.

•  House individually.

•  Supply food and water ad libitum.
   QUALITY CONTROL — Use commercial rodent ration.

•  Analyze basic laboratory diet for contaminants  PBB's and PCB's, antibiotics,
   estrogens, aflatoxins, lead, arsenic and mercury and nutritional content.
   QUALITY CONTROL -- Selected frequency, rejection of contaminated feed,  and
   inter- and intra-lab control tests.

•  Analyze water for heavy metals and coliforms.
   QUALITY CONTROL —  Quarterly.

•  Generate atmospheres by method appropriate to the test  material.   For
   volatile liquids generate high concentrations by passing compressed  air
   through the liquids at constant rates.  Reduce to dilution with filtered
   warm air drawn through the chambers which operate under negative pressure.
   QUALITY CONTROL —  Monitor continuously; prior to exposure of animals
   calibrate the monitoring equipment (such as hydrocarbon analyzer)  with
   the substance being tested.  The range of calibration points will  encompass
   the selected dosage levels.  Aliquots of the test substance will be
   introduced into large gas sampling bottles of known volume.   After vapor
   concentration reaches equilibrium the aliquot will be introduced into the
   analyzer.
   QUALITY CONTROL — Analyzer should be equipped with 10-point timed solenoid
   sampling system:  1 to 8, level of substance in eight chambers; 9, room
   atmosphere;  10, combined stack effluent.
   QUALITY CONTROL — Sample four times each day for 10 minutes per sampling
   point.

                                     452

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    QUALITY CONTROL — Adjust flow as required.

Observations and Tests

•   Phase I - Reproduction:   The following information will be collected:

      1. Parent body weights and food consumption at 1, 4, and 8 weeks of
         treatment.
      2. Observations on parents tabulated weekly.
      3. Live and dead pups and external appearance at birth.
      4. Number, sex, appearance, and individual weight of pups at day 4.
      5. Number, sex, appearance, and individual weight of pups at day 28.

    Any abnormal pups will be killed and prepared for examination by fixing in
    Boulin's solution or by clearing for skeletal examination.  The males will
    be killed and gross necropsy performed after weaning (day 28).  The uteri
    of nonpregnant females will be inspected for resorption sites.  At 100 days
    of age, one male and one female from each litter (total 25 males and 25
    females) will be sacrificed.  Organ weights will be recorded for each rat
    as at termination.
    QUALITY CONTROL — Good form design.
    QUALITY CONTROL — Signing and witnessing of all records.

•   Phase II - Chronic Toxicity:  Individual body weights and food consumption
    will be recorded at monthly intervals.  Observation of gross signs of
    toxicity, pharmacologic effects, and the incidence, size, and location
    of tumors will be recorded at the same intervals.  All animals will be
    observed daily for mortality.  Starting at 18 months and continuing until
    termination, mortality observations will be made twice daily. Necropsies
    will be performed on all animals that die during the course of the study,
    and tissues will be taken.  Should an animal be moribund and not
    anticipated to live to the following day, it will be killed, necropsy
    performed, and tissues taken and preserved as described below for terminal
    examinations.
    QUALITY CONTROL — Good form design.
    QUALITY CONTROL — Signing and witnessing of all records.

Clinical Laboratory Studies

•   The following clinical laboratory studies will be performed on five male
    and five female animals from the control and each test group (selected
    from among those individually housed):

      - Hematology - at 26 and 52 weeks, and termination includes:

        Hematocrit                          Total leukocyte count
        Hemoglobin                          Differential leukocyte count
        Erythrocyte count                   Reticulocyte count

      - Blood Chemistry - at 26 and 52 weeks, includes:
                                     453

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       Blood sugar                        Alkaline phosphatase
       Blood urea nitrogen                Serum glutamic pyruvic
       Serum glutamic oxaloacetic          transaminase
        transaminase

       SMAC determinations will be made at termination, including in
       addition to the above:

       Creatinine            Phosphorus              Chloride
       Cholesterol           Globulin                CO?
       Uric acid             Total bilirubin         Potassium
       Total protein         Albumin                 Lactic acid
       Creatine              Calcium                  dehydrogenase
        Phosphokinase        Iron                    Triglycerides
       Sodium

     - Urinalysis - Using pooled samples from the five rats per group per
       sex at 26 and 52 weeks, and termination includes:

       pH                    Specific gravity
       Glucose               Albumin
       Ketones

Termination and Postmortem Examination

•  The study will be terminated at 30 months or 20% survival, whichever comes
   first, and all surviving rats will be killed and necropsied.  The weights
   of the following organs will be recorded for each rat and the organ weight
   and body weight ratios will be calculated:

       heart                 kidneys                 lungs
       liver                 adrenal glands          brain
       spleen                testes (with            thymus
                              epididymides)
Histopathologic Examination

•  Histopathologic examination will be performed on all of the following
   tissues from all  rats in the two highest dosage groups having adequate
   survival, all controls, and those dying during the course of the study.
   Three target organs from all animals in the remaining groups will be
   examined histopathologically.  Selected body tissues and organs will be
   analyzed for content of the toxicant and/or biotransformation products.

•  Appropriate samples of each will be preserved in 10% neutral formalin:

       all gross lesions                             esophagus
       brain (cerebrum,cerebellum, brainstem)        stomach
       spinal  cord (2 sections)                      small intestine (duodenum,
       eye                                            ileum, jejunum)
       pituitary                                     large intestine (colon,
       salivary gland                                 cecum)


                                     454

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       heart                                         adrenal  glands
       thymus                                        pancreas
       thyroid                                       liver
       lungs (with mainstern bronchi)                 kidneys
       trachea                                       urinary  bladder
       spleen                                        ovaries/testes
       bone (with marrow)                             prostate
       lymph nodes (2)                               uteri (corpus, cervix)
       skeletal  muscle                               skin (mammary area)

   A peripheral  blood smear will  be made and maintained for possible future
   examination in the event that  other histopathologic findings suggest
   leukemia or other blood-related alterations.

Records and Reports

•  Records will  be maintained on:

       sampling assays                               mortalities
       feed consumption                              necropsies
       body weights                                  pathology
       daily observations

•  In addition to the final report, interim reports may be made available to
   the sponsor if required.  The  frequency of such reports will be determined
   prior to study initiation.  The report will be a complete scientific
   presentation of results and conclusions.

Changes or Revisions

•  Any changes or revisions of this approved protocol will be documented,
   signed by the study director,  dated, and maintained with this protocol.
   The sponsor will be notified prior to any revision of this protocol.
Approval of Protocol


Date:	  Sponsor:
Date:	  Study Director:
                                     455

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                EXAMPLE:  24 MONTH INHALATION STUDY IN MICE

Purpose of Study

     Carcinogenic effects determination

Design of Experiment

•  300 male and 300 female young B6C3F1 strain mice less than 20 g in weight.

                                No. of Animals
                  Group No.     Male    Female     Dose Levels
                      1          50       50       Control
                      2          50       50       1/8 MTD
                      3          50       50       1/4 MTD
                      4          50       50       1/2 MTD
                      5          50       50       3/4 MTD
                      6          50       50       MTD

•  All animals will be housed by sex and dosage, five animals per cage.

Route of Administration

•  Inhalation exposure in stainless steel chambers with effective exposure
   areas of 5.5 x 5.5 x 6.0 feet.

Exposure Schedule

•  Exposure for 23 hours per day, 7 days a week.

Conduct of Experiment

   QUALITY CONTROL -- Advice of a statistician should be sought on group size
   and on methods of statistical analysis to be used.
   QUALITY CONTROL — Hold in quarantine for 1 week.

•  Assign animals to study group (as removed from shipping crates) following
   quarantine.
   QUALITY CONTROL -- Use randomization procedures.

•  Identify animals by cage, group, and individually.
   QUALITY CONTROL — Achieve an equivalent mean body weight between groups.

•  House animals individually.
   QUALITY CONTROL --  Use ear tags and durable cage  markers.
   QUALITY CONTROL --  Accurately determine body weight and food consumption.

•  Supply food and water ad_ libitum.
   QUALITY CONTROL -- Commercial rodent ration.

•  Analyze basic laboratory diet for contaminats; PBB's and PCB's; antibiotics,
                                    456

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   estrogens, aflatoxins; lead, arsenic, mercury; nutritional content.
   QUALITY CONTROL -- Selected frequency.
   QUALITY CONTROL — Reject if contaminated.
   QUALITY CONTROL — Inter- and intra-lab control tests.

•  Analyze water for heavy metals and conforms.
   QUALITY CONTROL - Quarterly.

•  Generate atmospheres by method appropriate to the test materials.  For
   volatile liquids generate high concentrations by passing compressed air
   through the liquids at constant rates.  Reduce to dilution with filtered
   warm air drawn through the chambers which operate under negative pressure.
   QUALITY CONTROL — Monitor continuously; prior to exposure of animals
   calibrate the monitoring equipment (such as hydrocarbon analyzer) with
   the substance being tested.  The range of calibration points will encompass
   the selected dosage levels.  Aliquots of the test substance will be intro-
   duced into large gas sampling bottles of known volume.  After vapor concen-
   tration reaches equilibrium the aliquot will be introduced Into the analyzer.
   Analyzer should be equipped with 10-point automatically timed solenoid
   sampling system:  1 to 8, level of substance in eight chambers; 9, room
   atmosphere; 10, combined stack effluent.
   QUALITY CONTROL — Sample four times each day for 10 minutes per sampling
   point.
   QUALITY CONTROL — Adjust flow as required.

Observations and Tests

•  Individual body weights and the number of survivors will be recorded for
   each group weekly until the weights of the animals stabilize (13 weeks).
   Body weights thereafter will be done on a monthly basis.  Feed consumption
   for each cage group will also be recorded at the same intervals.

•  All animals will be observed at least every 8 to 10 hours for deaths and
   morbidity.  Observations of gross signs of toxicity, pharmacologic effects
   and incidence, size, and location of tumors will be recorded at weekly
   intervals.  Should an animal appear moribund, have an obvious lesion, or a
   grossly evident tissue mass, it will be housed separately to prevent
   cannibalism.

•  Should an animal be moribund and not anticipated to live to the following
   day, it will be killed, a necropsy performed, and the tissue taken and
   preserved.  Necropsies will be performed on all animals that die during the
   course of the study, and the tissues will be taken and fixed.

Termination and Postmortem Examination

•  The study will be terminated at 24 months and all surviving mice will be
   necropsied.  If the mortality 1n any dose sex group between 18 and 23 months
   approaches 50%, consideration should be given to early termination of the
   study.

Histopathologic Examination

                                     457

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   Hlstopathologic examination will be  performed on all of the following
   tissues  from  all mice  in  the  two highest dosage groups having adequate
   survival, all controls, and those dying during the course of the study.
   Three  target  organs  from  all  remaining groups will be examined histopatho-
   logically.  Appropriate samples of each will be preserved in 10% neutral
   formalin:
          all  gross  lesions
          brain  (cerebrum, cerebellum,
            brainstern)
          spinal cord  (2 sections)
          eye
          pituitary
          salivary gland
          heart
          thymus
          thyroi d
          lungs  (with  mainstern bronchi)
          trachea
          spleen
          bone (with marrow)
          lymph nodes  (2)
     esophagus
     stomach
     small intestine (duodenum, ileum,
      jejunum)
     large intestine (colon, cecum)
     adrenal glands
     pancreas
     liver
     kidneys
     urinary bladder
     ovaries/testes
     prostate
     uteri (corpus, cervix)
     skin (mammary area)
     skeletal muscle
 •  A peripheral blood smear will be made and maintained for possible future
   examination in the event that other histopathologic findings suggest
   leukemia or other blood-related alterations.

 Records and Reports
   Records will be maintained on:

          sampling assays
          feed consumption
          body weights
          daily observations
     mortalities
     necropsies
     pathology
•  In addition to the final report, interim reports may be made available to
   the sponsor if required.  The frequency of such reports will be determined
   prior to study initiation.  The report will be a complete scientific
   presentation of results and conclusions.

Changes or Revisions

•  Any changes or revisions of this approved protocol will be documented,
   signed by the study director, dated, and maintained with this protocol.
   The sponsor will be notified prior to any revision of this protocol.
Approval of Protocol

Date:	

Date:
Sponsor:
Study Director:
                                     458

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3.5.5  Gross Observations

3.5.5.1  Mortality-

     Death of a whole animal is generally defined (Gove, 1976) as "the
ending of all vital functions without possible recovery".  These vital
functions or signs consist of voluntary movements, breathing, blood pulse,
eye blinking and other involuntary reflexes, righting reflex, and
electroencephalographic activity.  As a quality control procedure, it may
be suggested that an explicit definition should be made of the criteria of
declaring occurrences of mortality in each bioassay protocol.  Requirements
should also be established as to the technical backgrounds and/or licenses
of those investigators  who shall function to certify deaths and perform
autopsies, sacrificing of animals and necropsies.  It has been recommended
(Hinkle, 1977) that veterinarians be accredited by the American College of
Laboratory Animal Medicine (ACLAM) and that animal laboratory technicians
be accredited by the American Association for Accreditation of Laboratory
Animal Care (AALAC) prior to establishing bioassay programs.

     In a similar vein, the criteria for cellular death, necrosis, and
autolysis ought to be delineated in each proposal for a bioassay investigation
where gross and microscopic pathologic examinations are to be made.  Here also,
the need for qualified personnel, particularly pathologists and technicians
certified by the American Society of Clinical Pathologists (ASCP), the College
of American Pathologists (CAP), the American Association of Experimental
Pathologists (AAEP), the American Industrial Hygiene Association (AIHA) or
the above ACLAM, should be apparent.

3.5.5.2  Body Weight, Growth, and Nutrition—

     Routine evaluation of the growth and development of large numbers of
animals receiving a test compound has been a crucial means of predicting
possible toxic effects, especially in subacute and chronic studies (Barnes
and Heath, 1964: McLean and McLean, 1969; Case et al., 1976).  The quality
control aspects to be applied to these studies should, of course, involve a
means of certifying the accuracy of equipment used in measuring the animals'
weights.  To this end, the following recommendations are made:

     •  All animals used in an EPA bioassay program should be weighed
individually at these times:  At time of receipt, at time of assignment to
treatment groups, and periodically during the actual bioassay.

     •  Weights should be determined to the nearest gram using an appropriate
animal weighing scale.

     •  Balances employed for determining animal weights should be recalibrated
against NBS approved standards at least monthly and calibration data recorded
in a bound notebook and signed by the responsible personnel.

     •  Supervisors should be responsible for making certain that all animal
weights are accurately determined and recorded to insure validity of the
bioassay test results.

                                    459

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     With regard  to  the effects of  toxicants on animal weight and growth it
has been found  that  restricted feeding, enriching the diet in protein, altering
nutrition, etc. has  important influences on the expression of toxic phenomena
by animals.  The  National Academy of  Sciences has made recommendations concern-
ing the nutrient  requirements of common laboratory animals (i.e., mouse, rat,
hamster, guinea pig,  cat and dog) (SLAN, 1972).  Often commercial feeds will
provide a considerable excess of these nutrients so as to diminish the effects
of degradative  loss  or altered bioavailabillty.  Some minerals and vitamins,
however, may be toxic when  given in excess or may act to synergize the effects
of the test agent.   Thus, it is not really possible to make generalizations
concerning exact  levels of  nutrients  required in each bioassay.  In chronic
and subacute bioassays, however, it is crucial that an accurate specification
of the animals' nutrient requirements not only be made but checked periodically
by chemical analysis.  This is especially true if the test compound is to be
mixed in the feed and an ongoing program of measuring and recording the amount
of feed (and concomitant test agent)  must be performed.  Homogeneous distribution
erf nutrients and  test agents is, therefore, of ultimate importance in chronic
and subacute studies.

3.5.6  Reproduction  and Teratology  Studies

     Methods used to  presently estimate reproductive and teratogenic hazards
involve:

     •  Treatment span encompassing all or most of the period of organogenesis

     •  Use of  rodent-rabbit species  largely because of convenience in
handling and low  cost

     •  Multi-generation testing in order to screen in several ways for toxic
effects at specific  points  in the mammal's reproductive cycle

     A laboratory animal should be  chosen by evidence that it metabolizes and
distributes a test substance, transfers the substance across the placental
barrier, and biotransforms  the substance in utero in nearly the same manner
as humans.  Since a  priori  knowledge  of the reproductive and teratologic
effects of new  test  substances in humans may not be available, mammaHan models
must be chosen which  approximate, to  the best of our knowlege, human physiology,
drug and toxicant metabolism, reproduction and embryology.  A "3-generation
bioassay" was suggested by  the FDA  in the mid-1960fs in order to test for
teratogenic effects resulting from  low-dosage , long-term exposure to chemicals
(e.g., food additives, pesticides,  drugs, contaminants, etc.) in food.  Animals,
usually rats, are treated continuously through 3 reproductive cycles so as to
provide opportunity  for evaluating  a  multitude of parameters measuring repro-
ductive performance,  embryonic development, fetal and neonatal survivability
in several successive generations.  Although this is at present the best gen-
eral screening procedure for judging  test agents within a chronic, low-dosage
bioassay,  Wilson  (1975) is  of the opinion that more specialized tests are
necessary for scrutinizing  teratogenic and mutagenic potentials.  Teratogenic
short-term risks, for example, in the period of highest susceptibility (i.e.,
fetal organogenesis stage) may be masked due to maternal homeostatic dispersal


                                      460

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and early induction of maternal hepatic enzymes which may protect the fetus in
later stages.  Wilson lists five separate tests for assaying developmental
abnormalities that depend upon the duration of treatment:

    •  Testing throughout entire reproductive cycle — whole generations

    •  Testing throughout pregnancy — conception to term

    •  Testing throughout organogenesis — primitive node to palatal closure

    •  Short-term (3 to 4 days) testing sequences during organogenesis

    •  Testing aimed at specific parameters — mutagenesis, postnatal toxicity

    The FDA guidelines of 1966 specified the following characteristics for
3 types of studies:

             Type I - Fertility and general reproductive study

                  o   Males given MTD (maximum tolerated dose) for 60
                      days before mating

                  o   Females given MTD for 2 weeks before and during
                      mating, pregnancy and lactation

                  o   Young examined at 13 days gestation, term, and
                      nursing

             Type II - Teratology study

                  o   Pregnant females treated days 6 through 15

                  o   Young examined 1 to 2 days before term

             Type III - Perinatal and postnatal study

                  o   Dam treated last third of pregnancy and throughout
                      lactation

                  o   Young evaluated for survival and growth

    The exact timing of maternal conception and implantation is very crucial
in a well designed teratology study.  When this knowledge of the maternal
status is sketchy, the results may be misleading or highly variable due to a
number of interferences or missed critical parameters which are listed in
Table 3.5.3.

    In an effort to circumvent these difficulties. Wilson  (1975) proposed a
number of new assay designs for improving current teratogenicity testing
techniques.  These included:   (a) the use of some short-duration dosages,
besides dosing throughout organogenesis, in order to avert enzyme induction
and other adaptive responses on the part of the mother,  (b) running tests in

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TABLE  3.5.3  WAYS IN WHICH REPEATED TREATMENT  PRIOR TO THE PEAK  SUSCEPTIBLE
    PERIOD OF  THE EMBRYO  MAY PRODUCE MISLEADING RESULTS  (Wilson, 1975)
        Time of treatment
                                 Primary effect
                                Secondary effect capable of
                                  altering test results
     Before implantation
     Early organogvnesis
     ••fore peak susceptibility

     Before peak susceptibility

     Before peak susceptibility

     Before peak susceptibility

     Before peak susceptibility
interference with implantation
early embryonic death
induction of catabolizing enzymes

inhibition of catabolizing enzymes

liver pathology or reduced function

kidney pathology or reduced function

saturation of protein binding sites
no Issue
no issue
reduced blood level during susceptible
 period
Increased blood level during susceptible
 period
increased blood level during susceptible'
 period
Increased blood level during susceptible
 period
increased blood level during susceptible
 period
species that more closely approximate  human  biotransformation mechanisms
than  do the rodent-rabbit species,  (c) determining the  embryo-toxicity
threshold levels in animals so as  to permit extrapolation downward  to accept-
able  levels for humans,  (d) expanding and improving postnatal function
evaluations (e.g., sensory modalities, muscular coordination, and learning
capabilities),  and (e) reserving primates for agents needed, likely to be
used,  or of especially significant  risk during human pregnancy.

3.5.6.1  Parental Observations—

      In designing an experiment or  bioassay to test the potential embryo-toxic
or  teratogenic  effects of a substance, it is  most desirable to effectively
control the following variables to  which the  test parents are to be exposed:

    •   Quality  of feed, bedding and drinking  water

    •   Temperature, humidity, barometric pressure, amount and periodicity
        of light and noise

    •   Cage size, material, type of racks

    •   Group size unless  animals are caged individually

    •   State of health and standard of laboratory care

    •   Species  and strain of test animals

    •   Proof of male fertility, and fecundity

    •   Parity and time of mating of test females

    •   Accuracy of female exposure  times to test substance and date of
        first insemination

    •   Treatment of concurrent control animals in all respects equal except
        for exposure to test substance

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     Teratogenicity tests in particular require meticulous record keeping
throughout the assay period, careful necropsy of any female dying spontaneously
and reporting of all dead and resorbed conceptuses.  A pertinent report (U.S.
EPA, 1977) stresses the importance of:  (a) Running contemporaneous control
animals chosen at random from the initial population of assay animals and
coded along with the test animals in a manner that is "blind" to all except
the principle investigators, (b) having a knowledge of the degree of spontan-
eous malformations and the range of variants (skeletal and visceral) character-
istic of the chosen strain of test animal, and (c) availability or morphologic
atlases of this chosen strain by which accurate comparisons may be made of the
type and degree of malformation induction.  Although the FDA required only 2
dosage levels be tested, Wilson and others have recommended the testing of a
broader range of levels such as:  0.5, 0.25, and 0.125 times the acutely toxic,
maternal LD50 dosage.  The degree of variability and level of spontaneous
malformation, which is evident in all species to some extent, further supports
the contention that not just rats and mice, but also dogs, cats, pigs, sheep
and other mammals ought to be assayed since their placental arrangement is
more akin to human than is that of rodents and rabbits.  Since a single ideal
animal does not exist which would satisfy all these criteria, Wilson has
suggested successive levels of evaluation in different animals as depicted in
Table 3.5.4:

TABLE 3.5.4  A NEW CONCEPT IN TERATOGENICITY TESTING BASED ON MULTILEVEL TESTS
                IN DIFFERENT TYPES OF ANIMALS (Wilson, 1975)
Order of
test
First level
Second level
Third level
Fourth level
Purpose
find embryotoxic dote range
confirm or adjust above
only if second level results ere equivocal
only If use in human pregnancy needed or
likely
Suitable species
rat. mouse, hamster, or rabbit
a carnivore or an ungulate
alternate to that used in second level
macaque monkey or baboon
No. of
pregnant
animals
130-150
40-60
40-60
40-50
   a. Tests would terminate at second or third level in most instances.
   b. Whenever possible selection should be made on the basis of metabolic
      similarity to man.

Large numbers of inexpensive animals, such as rats or mice, could be first
used for finding the general embryotoxic dosage range.  The second-level
tests would utilize large animals (either carnivore or ungulate) whose re-
productive anatomy and physiology is more like that of humans, and upon whose
results a more accurate estimate of potential human teratogenicity might be
based.  A third level of testing in another subprimate mammal might be necessary
only if the results of the first-and second level bioassays remain question-
able.  Fourth-level tests involving nonhuman primates have to be restricted
to   substances which pregnant women are inadvertently exposed to in signif-
icantly large quantities and for which an epidemiologic suspicion has been
deduced, and (b) medicants and drugs which are necessary for control of disease
or severe discomfort during pregnancy.  Extrapolation of these results to the
human condition, however, will always be difficult even with retrospective
epidemiologic surveillance of human populations.
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 3.5.6.2   Fetal Manifestations—

      Several uncertainties exist in the suggestion of any inferences  between
 the results in animal models and the expected effects in human infants  due  to
 developmental differences between species.   An Important consideration  made
 (U.S.  EPA,  1977)  is that of the time course of intrauterine development,
 irrespective of life span or gestation period.  In this review of  terato-
 genicity screening assays, it is pointed out that  the preimplantation period
 (during  which cells are undifferentiated and hence not yet demonstrating
 teratogenic responses) varies from 4.5 days in hamsters to 6.15 days  in man
 to  10 days  in sheep.   Organogenesis takes place in the first 6 weeks  in human
 development, while a proportionally longer  or shorter period out of the total
 gestation time is evident in other mammals.   The chronology of structural
 developments in the central nervous system  (CNS),  a prime target for  terato-
 genesis, when measured histologically varies greatly among species with the
 final  steps for humans occurring much later during the postnatal period.
 Other  major differences between the fetal development of humans versus that  of
 other  mammals as  documented are:

     •  Single implantation in humans versus  multiple implantations  in  many
       test  animals which can cause variations in the proportion of embryos
       and fetuses resorbed, in  number of abortions, still births,  etc.

     •  Differences in  endocrinology,  metabolism,  pharmacology,  phartnacokinetics
       and nutrition

     •  Inbred nature and genetic characteristics  of laboratory  animals as
       compared with the more randomly bred  and larger,  more chronically
       exposed human populations.

     Another potential  fetal variability factor causing dosage  fluctuations
 and  resulting from maternal homeostatic adaptions  may be avoided by testing
 only short-duration dosages.  Fewer animals  are  needed in each short-term
 assay  group than  is the case when  the dosages are  given throughout organo-
 genesis.  Later,  it is  recommended  that the  highest dosage that  produces no
 increase  in embryotoxicity during  the short-term treatment spans should be
 given  to  another  test  group for assay over  full  period  of  organogenesis.
 According to  Wilson,  the  few animals  that are required  in  these  separate short-
 duration  assays still  provide more  precise  information  than those  from  a larger-
 scale  single  experiment.

    Demonstration  of a  level  of test  substance which is embryotoxic independent
 of levels which are teratogenic has received  little attention  and  needs to
 have more emphasis  in  the  future.  A  threshold of  teratogenic  potential for a
 particular  toxicant, in  fact, may be  substantially close  to the  embryotoxic
 level, in which case the chemical may still be used  as  long as  concentrations
do not exceed  these threshold limits.   If applied  in  sufficient  dosage  at a
 susceptible developmental  stage of a  laboratory mammal,  a  toxicant may  prove
 to be embryotoxic, while higher doses  at  later or  earlier  periods  would be
needed to demonstrate a teratogenic effect.   This  information  should  be
required before setting safe  tolerance  limits.


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     Postnatal functional capabilities must receive greater emphasis in the
future due to the fact that:   (a) nervous and endocrine systems or organs are
not fully functional at birth in humans and other species, and (b) functional
deficiencies in these systems or organs may not readily be reflected in poor
growth and/or survivabillty postnatally.  Thus, it is crucial that postnatal
functional evaluations be extended beyond weaning and be included in growth
and survival records.  Specific tests of sensory modalities, muscular
coordination, learning capabilities need to be refined so as to allow for
Improved quantitation and concomitant quality control specification.    .

3.5.6.3  Mathematical and Statistical Analyses—

     As a result of these differences in fetal responsiveness between species,
there is a lack of consensus as to the best experimental design or approach.
Although it would be deemed ideal, a program of single dosages to test each
specific organ and tissue sensitivity at the time of the most rapid pro-
liferation would be far too costly.  Multiple doses, however, have been re-
ported to yield cumulative effects and/or few malformations due to development
of metabolic tolerance by the parent female.  So-called "equivalent dosages"
(i.e., dosages weighted for known tissue sensitivities) have been advocated as
a result.  The notion of a "threshold" concept of dosage-responsiveness has
been recommended, as well as the concept of a "teratogenic ratio" (the ratio
of the maternally-measured toxicity of a chemical to the embryotoxic level of
this chemical) has been proposed as the best predictor of teratogenic potential
(Robson, 1970).  Thus, there appear to be several types of mathematical analyses
which have achieved varying degrees of acceptance by the scientific community,
no one of which provides answers to all questions.

     Collins and Collins (1976) recommend that a table of random numbers be used
in order to assign animals to control or experimental groups, ensuring that
each pregnant female is treated as an independent sampling unit.  Percentages
of affected fetuses per litter and incidence of affected fetuses per total
number of fetuses should be reported.  The best experimental design is one in
which the evaluators are "blind" as to which dams are of the test group and
which are of the control through a labeling code known only to the principal
investigators.  At each dosage level quantitation must be made of each specific
anomaly and of the total anomalies that appear in each grouping, since the
sum of all anomalies considered together in a specific grouping may indicate a
teratogenic effect.  A series of at least three dosage levels, allows for
preliminary dose-response correlations.  It should later be required that both
the embryotoxic level and the no-observed-effect level be determined, as both
are valuable in the regulation of potentially toxic substances.  Safe human
exposure levels should be a fraction (e.g., 1/1000, 1/100, 1/20) of the
calculated embryotoxic dosage.  Although absolute certainty is impossible to
achieve, the estimated risk of teratogenic effects from new substances or
chemical combinations must be assessed if we are to avoid subjecting our off-
spring to unknown hazards.

3.5.7  Mammalian Mutagenicity Tests

3.5.3.1  Dominant Lethal Bioassay—


                                     465

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     Gross genetic damage has been induced by x-rays and chemical mutagens
and has been measured by a technique termed the dominant lethal test.  The
association of zygote lethality with dominant chromosomal aberrations, such
as chromosomal translocations that result in nonviable zygotes, has been used
as evidence in the correlation of chemically-induced damage with heritable
mutation(s), which in this case are lethal.  In their recommendations of the
general practice of dominant lethal assays, Green et al. (1976) suggested the
following calculations and measurements be utilized "to evaluate statistically
the results of dominant lethal studies":

     •  Fertility Index - This index is used to analyze the number of
        pregnant females per number of mated females via the chi-square
        comparison of the values for each treatment group versus the
        calculated control values.  A trend for linearity of proportions
        may be used to ascertain whether this index is linearly related to
        arithmetic or logarithmic dose of test agent.

     •  Total Number of Implantations Index - Significant differences
        between average numbers of implantations per pregnant female in
        each treatment group against control values are assayed statistically
        via the t-test.  Linear regression analysis is used to determine if
        this index is related to arithmetic or logarithmic dose.

     •  Total Number of Corpora Lutea - Again, significant differences are
        ascertained by t-test comparison between control and treatment
        values, and linearity by regression analysis.

     •  Preimplantation Losses Count - These losses are measured by direct
        counting of the number of corpora lutea.  The preimplantation losses
        for each female are mathematically transformed to the Freeman-Tukey
        arc-sine values and the t-test is then used to compare each treatment
        value with those controls.

     •  Dead Implantation Count - These counts are statistically analyzed in
        the same manner as preimplantation counts.

     •  Proportion of Females with One or More Dead Implants - The chi-square
        test is suggested for comparing control and treatment values, while
        the trend for linear proportions may be used to determine if these
        proportions are linearly related to arithmetic or logarithmic dose.
        Alternatively, probit regression analysis may be employed to test
        for these types of linear correlation.

     •  Proportion of Females with Two or More Dead Implants - Mathematic
        analyses of these proportions are carried out in the same manner as
        that for the previous proportions.

     •  Dead Implantations per Total Implantations - Using the count data
        from each female, a Freeman-Tukey arc-sine transformation is obtained
        for each control and treatment value.  Comparison of these values is
        then made via the t-test.


                                     466

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     •  Variation between Males with Time - Using a nested model, the analysis
        of variance between individual males and between results obtained in
        individual test weeks or pairs of test weeks.

     Although the dominant lethal bioassay is thought to reflect chromosomal
aberrations, it cannot by definition measure heritable chromosomal effects
other than those that produce lethality.  Many agents which induce dominant
lethality, however, are known to cause heritable chromosomal aberrations when
investigated by non-mammalian or in vitro bioassays.  The actual measure in
the dominant lethal test is early fetal loss (Green et al., 1977; Embree et al.,
1977 ; Epstein et al., 1972).  The working hypothesis of this test is that
abnormalities produced in sperm may lead to developmental errors causing
early death of a zygote.

     There is some scientific concern that dominant lethality as measured by
fetal death or wastage may occur for reasons other than chromosomal mutation.
As a result, problems may result in the interpretation of the observations
made in using this system in a definitive manner.  The utility of the dominant
lethal assay, however, stems from its ease of performance and the positive
correlation that has been established between it and other animal systems.

                     EXAMPLE:  DOMINANT LETHAL BIOASSAY

Experimental Design

•    Dosing and mating schedules have generally been designed so as to permit
     a sampling of potential effects on all sperm cell stages through meiosis.
     A shortened approach to this bioassay has also been presented (Green et al.,
     1977) which is outlined below along with suggested quality control steps.

Design Outline

•    Adult male rodents treated for 5 consecutive days with acute or subacute
     dosage; each is then mated to 2 virgin females each week for 8 to 10
     week periods; each female is sacrificed 14 days from the midweek of co-
     housing; inclusion of untreated males separately tested as negative,
     vehicles control animals and a second group of males dosed via the same
     route with known mutagen as positive control animals.
      lUALITY CONTROL — Same as that outlined in acute toxicity bioassays
      Section 3.5.4.1).  Especially important are age, weight, good health
     and proven fertility checks of males prior to bioassay.  If known,
     the spontaneous frequency of dead or abnormal implantations should be
     specified for each strain and species tested.

Bioassay and Statistical Analyses

     The number of corpora lutea, as well as the count of the dead implantations
     per pregnant female is transformed by Freeman-Tukey square rooting and
     subsequently subjected to t-test analysis; the count of females with 1 or
     more dead implantations and the number of females with 2 or more dead im-
     plantations are subjected to chi-square analysis.
     QUALITY CONTROL — Same as that outlined in acute toxicity bioassays

                                     467

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     (section 3.5.4.1) particularly with respect to record keeping, "blind
     status" of examining pathologist, and substantial results with positive
     control animals.

     Results obtained in a test of triethylenemelamine (TEM) showed that this
shortened approach reduced the amount of data usually required with no loss
of pertinent information concerning inherited lethality.  Information concern-
ing what stage spermatogenesis is adversely affected, however, requires the
more elongated bioassy.

3.5.7.2  In Vivo Cytogenetics and Cytotoxicity Bioassays—

     Cytologic and cytogenetic bioassays utilizing the techniques of visible
light and transmission electron microscopy have been available for detecting
chromosomal anomalies in mammals exposed to potential toxicants in vivo.
These procedures fall into two categories, those that detect damage (trans-
mittable and non-transmittable) expressed in germ cells during early embryo-
genesis, and those whereby somatic tissue cells are assessed for nuclear,
chromosomal, cytoplasmic and mitochondrial damage within cells of critical
tissues and organs.  The somatic tissue used is usually bone marrow (Legator
et al., 1969; Georgian, 1975; Majumdar et al., 1976), while testes have been
assayed in germinal cell studies.  Human lymphocytes may also be tested if
accidental or chronic use exposure has occurred and information is available
on the cytomorphology of the lymphocytes prior to exposure (Lubs and Samuel-
son, 1967).

     Regarding quality control aspects, Cohen and Hirschhorn  (1971) have
stressed that replicates of each cytologic sample in the form of multiple
slides be examined by cytopathologists in a "blind" manner  (i.e., coded) on
different days in randomized sequences.  To overcome any potential observer
biases, these slides should, if possible, be further examined and scored by
2 other microscopists, and their scores averaged.  It was also suggested that
all repeated experiments be statistically tested for "homogeneity of variances,1
while tests of significance between results of treated and untreated groups be
based on statistically significant differences in the mean scores and variances
thereof via t-test and F-test techniques, respectively.  A summary of these and
other critical points is here presented in outline form:

     Summary of Experimental Design Outline

     •  Acute Studies - 3 groups of 5 male albino rats are used for each
        agent at each dosage level; and equal number of rats (negative
        controls) treated with vehicle only, a third group (positive controls)
        dosed with triethylenemelamine (TEM); test agent(s) orally administered
        at one of 3 dosage levels (LD5, LD25 or LD50, and "usage level1); 2 to
        4 hours prior to scheduled sacrifice, 4 mg/kg of colcemid is injected
        intraperitoneally; of the 3 groups at each dosage level, one is
        sacrificed at 6 hours, one at 24 hours, and the last at 48 hours
        after treatment.
        QUALITY CONTROL — Seek advice of biostatistician on group size and
        methods of statistical analysis to be used in scrutinizing data.
        Additionally, the quality control steps outlined for acute toxicity

                                     468

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        bioassays (Section 3.5.4.1) should be performed.

     •  Subacute Studies - 3 groups of 5 males are used for each test agent
        with negative controls as in the acute studies, but here the oral
        dosage is applied for 5 days at 24 hour intervals at the same 3 levels
        (LD5, LD25 or LD50, and "usage level").  The assays are terminated
        as described above for acute studies.
        QUALITY CONTROL — Same as above.

     •  Observations and Bioassays —• Bone marrow cells are taken from the
        femurs of each rat and prepared in a routine manner; the percentage
        of single and multiple metaphase chromosomal aberrations (i.e.,
        frequency of chromatid breaks, fragmentations, chromosomal inter-
        changes and ring formations) as well as incidence of swollen mitochondria,
        increased cytoplasmic granulation, vacuolizations, growth of abnormally
        large cells, and/or cellular membrane damage is recorded.
        QUALITY CONTROL — All scoring of bone marrow cells is performed on
        coded slides.  In addition to the steps outlined for acute toxicity
        bioassays (Section 3.5.4.1), the following data should be recorded:
        (a) Vernier and magnification settings used in the microscopic
        observations, (b) written definitions of each chromosomal aberration
        accompanied by an example photo, and (c) identification of chromosomal
        gaps separately.

3.5.7.3 Specific Locus Bioassay for Detecting Gene Mutations—

     The mouse-specific locus test has been used for the detection of specific
gene mutations that have been induced in the germ cells of rodents (generally
mice) exposed to chemical or physical (x-rays) mutagens (Russell, 1951; Searle,
1975; Cattanach, 1971).  The test requires stocks of at least 2 strains of
mice, one that is homozygous for dominant wild-type alleles at 7 loci and one
that is homozygous recessive at these same loci.  Mice of the wild-type stock
are treated with the presumptive mutagen, and after a specific duration,
allowing for metabolism and induction of mutational events in the males' germ
cells, the treated males are mated with mice of the homozygous recessive
tester strain.  The chosen specific duration between treatment and mating
determines the stage of the developmental germ cells which are assayed.
Induced mutations at these loci can be scored as changes in coat color, eye
color, ear size in 1 week old progeny, etc.  Dominant mutations at other loci
may also be observed in these litters if they are of a visible type.

     A few disadvantages of this bioassay are that it requires large numbers
of animals in order to obtain marginal sensitivity, and laboratories with the
required mouse colonies and expertise are limited.  Moreover, it is of concern
that not all types of mutagenic events (e.g., base-pair substitutions) can be
readily detected by simply phenotypic observation.  Future electrophoretic
typing of sera proteins may help in detecting these other events.  The bioassay
at present, however, has the great advantage of estimating germ cell risks to
genes in response to acute and subacute dosages in an intact animal via routes
analogous to the possible pathways of human exposure.  Critical points per-
taining to the.se problems are summarized below:


                                     469

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     Summary of Design Outline

     •  Mice of the inbred strains C3H and 101 and of the multiple recessive
        T-stock, each of 7 to 8 weeks of age are required.  As outlined by
        Searle  (1975), 200 C3H females per test or control group should be
        mated with 100 male 101 mice in trios, and the 3H1 male offspring
        mated with 200 T-stock females in a scheduled manner to yield at
        least 50,000 progeny at each of 2 C3H-parental dosage levels, as well
        as for  controls.
        QUALITY CONTROL — Seek advice of biostatistician on group sizes.
        Additionally the quality control steps outlined for acute toxicity
        bioassays  (subsection 3.5.4.1) should be performed.  To detect a
        true doubling above the natural mutation frequency, Searle (1975)
        estimated that at least 30,000 classifiable offspring (preferably
        more) be examined.

     Bioassay and Statistical Analyses

     •  All young from the mating of 3H1 males with T-stock females should be
        counted, sexed and carefully examined with respect to the 4 early
        phenotypes, and after 18 to 19 days, 10 final phenotypes.  The
        calculated mutation for each parental dosage or control group per
        locus is given by total number of specific locus mutations divided
        by the  product of the number of young classified times the number of
        loci.   Statistical comparison of control and test results may be
        performed by Fisher's "exact treatment" method from a 2 x 2 table as
        described by Searle (1975).  A chi-square test may also be used, but
        Yates correction for continuity should be applied.
        QUALITY CONTROL — The "blind" status of the examiners, the care in
        recording all examinations and the confirmation all genetic loci tests
        against example mouse photos are key quality control steps.

     The specific locus bioassay can provide direct information relevant to
assessing human genetic risks.  Unfortunately, large numbers of offspring
must be examined since this bioassay:  (a) Detects only 6 or 7 recessive loci
out of many thousands, (b) assays only forward mutations at these loci which
yield visible phenotypic changes, (c) permits determination of the nature of
these mutations only with difficulty unless they involve both the d_ and se
loci, (d) may allow non-mutational events to be scored as mutations, and (e)
may miss counting heterozygous mutatants that do not survive long enough to be
recognized.  It is likely that future research, however, will improve the
number of loci that can be assayed and will allow further reductions in the
detectability limitation of this bioassay technique.
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3.5.8  References

AOAC.  1975.  Official Methods of Analysis, 12th Edition.  Association of
     Official Analytical Chemists, Washington, D.C.

Barnes, J. M., and D. F.  Heath.   1964.  Some toxic effects of dieldrin in
     rats.  Brit. J. Ind. Med.  21:  280-282.

Boyd, C. E., and E. M. Boyd.  1962.  The chronic toxicity of atropine admin-
     istered intramuscularly to rabbits.   Toxicol.  Appl.  Pharmacol.  4:  457-
     467.

Boyd, E. M.  1968.  Predictive drug toxicity.  Assessment of drug safety
     before human use.  Can Med. Ass. J.  98:  278-293.

Case, M. T., J. K. Smith, and R. A. Nelson.  1976.   Chronic oral toxicity
     studies of nefopam hydrochloride in rats and dogs.  Toxicol. Appl.
     Pharmacol.  36:  301-306.

Cattanach, B. M.  1971.  Specific locus mutations in mice.  In:  Chemical
     Mutagens:  Principles and Methods for Their Detection, Vol. 2.   A.
     Hollaender  (ed.).  Plenum Press, New York, N.Y.  pp.  535-539.

Cohen, M. M., and K. Hirschhorn.  1971.  Cytogenetic studies in animals.
     In:  Chemical Mutagens:  Principles and Methods for Their Detection,
     Vol. 2.  A. Hollaender (ed.).  Plenum Press, New York, N.Y.  pp. 515-
     534.

Collins, T. F. X., and E. V. Collins.  1976.  Current methodology in teratol-
     ogy research,  Chapter 6. In:  Advances in Modern Toxicology, Vol. 1,
     Part 1, New Concepts in Safety Evaluation.  M. A. Mehlman, R. E. Shapiro,
     and H. Blumenthal  (eds.).  John Wiley and Sons, New York, N.Y.

Embree, J. W., J. P. Lyon, and C. H. Hine.  1977.  The mutagenic potential
     of ethylene oxide using the dominant-lethal assay in rats.  Toxicol.
     Appl. Pharmacol.  40:  261-267.

Epstein, S. S., E. Arnold, J. Andrea, W. Bass, and Y. Bishop.  1972.  Detec-
     tion of chemical mutagens by the dominant-lethal assay in the mouse.
     Toxicol. Appl. Pharmacol.  23:  288-325.

Georgian, L.  1975.  The comparative cytogenetic effects of aldrin and
     phosphamidon.  Mutat. Res.  31:  103-108.

Goldstein, A.  1964.  Biostatistics - An Introductory Test, Chapter  1, p. 1-
     33.  Macmillan Publ. Co., New York, N.Y.

Goodman, L.  S.,  and A. Gilman  (eds.).  1975.  The Pharmacologic  Basis of
     Therapeutics, Chapter 1-General Principles, by E. Fingl and D.  M. Wood-
     bury, pp. 1-46.

Gove, P. B.  (chief ed.).  1976.  Webster's Third New  International Dictionary

                                     471

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     of the English Language, Unabridged.  G. and C. Merriam Co., Publ.,
     Springfield, Mass.  p.  581.

Green, S., E. Zeiger, K. A.  Palmer, J. A. Springer, and M. S. Legator.  1976.
     Protocols for the dominant lethal test, host-mediated assay and in vivo
     cytogenetic test used   in the FDA's review of substances in the GRAS
     List.  J. Toxicol. Environ. Health  1:  921-928.

Green, S., F. M. Moreland, and W. G. Flamm.  1977.  A new approach to
     dominant-lethal testing.  Toxicol. Appl. Pharmacol.  39:  549-552.

Grice, H. C., and T. Da Silva.  1973.  The Testing of Chemicals for Carcin-
     ogenic ity, Mutagenicity and Teratogenicity.  Ministry of Health and
     Welfare of Canada, Ottawa, Canada.  Section on Chemical Carcinogenesis
     Testing, p. 34-49.

Hayes, W. J.  1975.  Toxicology of Pesticides.  Chapter 2 - General principles:
     dosage and other factors influencing toxicity, p. 37-106.  Williams and
     Wilkins, Baltimore, Md.

Hinkle, D. K.  1977.  Personal communication addressing quality assurance
     and  control in animal laboratories.  U.S. Environmental Protection
     Agency, Research Triangle Park, N.C.  February 4, 1977.

ILAR.  1974a.  Guide for the Care and Use of Laboratory Animals.  Institute
     for  Laboratory Animal Resources, DHEW Publication No. NIH 74-23.  Public
     Health Service, National Institutes of Health, Bethesda, Md.

Ibid.  1960.  Standards for  the Breeding, Care and Management of Syrian
     Hamsters.  NAS-NRC, Bethesda, Md.

Ibid.  1964.  Standards for  the Breeding, Care, and Management of Guinea
     Pigs.  NAS-NRC, Bethesda, Md.

Ibid.  1964.  Standards for  the Breeding, Care and Management of Laboratory
     Cats.  NAS-NRC, Bethesda, Md.

Ibid.  1967.  Standards for  the Breeding, Care and Management of Laboratory
     Rabbits.   NAS-NRC, Bethesda, Md.

Ibid.  1969.  Procurement Specification (Contract Clause), VI.  Colony-
     Produced Cats.  NAS-NCR, Bethesda, Md.

Ibid.  1969.  Procurement Specification (Contract Clause), V.  Kennel-Pro-
     duced Dogs.   NAS-NRC, Bethesda, Md.

Ibid.  1971.  A Guide to the Infectious Diseases of Mice and Rats.  NAS-NRC,
     Bethesda, Md.

Ibid.  1973.  Procurement Specification (Contract Clause) IX.  Defined
     Laboratory Rodents and Rabbits.  NAS-NRC, Bethesda, Md.


                                    472

-------
Ibid.  1974b.  A Guide to the Infectious Diseases of Guinea Pigs, Gerbils,
     Hamsters and Rabbits.  NAS-NRC, Bethesda, Md.

Ibid.  1976.  Long-Term Holding of Laboratory Rodents.  ILAR News, 19(4);
     Ll-25.  NAS-NRC, Bethesda, Md.

Ibid.  1977.  Laboratory Animal Management:  Rodents.  ILAR News, 20(3):
     Ll-15.  NAS-NRC, Bethesda, Md.

Legator, M. S., K. A. Palmer, S. Green, and K. W. Petersen.  1969.  Cyto-
     genetic studies in rats of cyclohexylamine, a metabolite of cyclamate.
     Science  165:  1139-1140.

Litchfield, J. T., Jr., and F. Wilcoxon.  1949.  A simplified method of
     evaluating dose-effect experiments.  J. Phannacol. Exp. Ther.  96:  99-
     113.

Loomis, T. A.  1974.  Essentials of Toxicology.  Chapter 13 - Toxicologic
     testing methods, p. 177-215.  Lea and Febiger, Publ., Philadelphia, Pa.

Lubs, H. A., and J. Samuelson.  1967.  Chromosome abnormalities in lympho-
     cytes from normal human subjects.  Cytogenetics  6:  402-411.

Majumdar, S. K., H. A. Kopelman, and M. J. Schnitman.  1976.  Dieldrin-
     induced chromosome damage in mouse bone-marrow and WI-38 human lung
     cells.  J. Hered.  67:  303-307.

McLean, A. E. M., and E. K. McLean.  1969.  Diet and toxicity.  Brit. Med.
     Bull.  25:  278-281.

NCI.  1976.  National Cancer Institute Safety Standards for Research Involv-
     ing Chemical Carcinogens.  National Cancer Institute, DHEW Publication
     No. NIH 76-900.

Rand, M. C., A. E. Greenberg, and M. J. Taras (eds.).  1975.  Standard
     Methods for the Examination of Water and Wastewater.  14th Edition.
     American Public Health Association, American Water Works Association,
     and Water Pollution Control Federation.  Washington, D.C.

Robson, J. M.  1970.  Testing drugs for teratogenicity and their effect on
     fertility.  Brit. Med. Bull.  26:  212-216.

Russell, W. L.  1951.  X-ray induced mutations in mice.  Cold Spring Harbor
     Symp. Quant. Biol.  16:  327-336.

Searle, A.  1975.  The mouse-specific locus test.  Mutat. Res.  31:  298-325.

Slan.  1972.  Nutrient Requirements of Laboratory Animals.  National Academy
     of Sciences, National Research Council, Subcommittee on Laboratory
     Animal Nutrition, Washington, D.C.

Sokal, R. R., and F. J. Rohlf.  1969.  Biometry, Chapter  7 - Estimation  of

                                     473

-------
     Hypothesis Testing, p. 127-174.  W. H. Freeman and Co., San Francisco,
     Calif.

Sontag, J. M., N. P. Page, and U. Saffiotti.  1976.  Guidelines for Carcino-
     gen Bioassay in Small Rodents.  U.S. Department of Health, Education
     and Welfare, NIH 76-801.  pp. 11-29, 50.

Steinberg, M., W. A. Akers, M. Weeks, A. H. McCreesh, and H. I. Maibach.
     1975.  A comparison of test techniques based on rabbit and human skin
     responses to irritants with recommendations regarding the evaluation of
     mildly or moderately irritating compounds.  Chapter 1 in:  Animal
     Models in Dermatology.  H. Maibach  (ed.).  P. 1-11.  Churchill Living-
     stone, Publ., New York, N.Y.

U.S. EPA.  1977.  Survey and Evaluation of Techniques Used in Testing Chemi-
     cal Substances for Teratogenic Effects.  U.S. Environmental Protection
     Agency, Office of Toxic Substances, EPA-560/5-77-007.

U.S. FDA.  1959.  Appraisal of the Safety of Chemicals in Foods, Drugs and
     Cosmetics.  U.S. Food and Drug Administration, Division of Pharmacology,
     Washington, D.C.

U.S. FDA.  1966.  Guidelines for Reproduction Studies for Safety Evaluation
     of Drugs for Human Use.  U.S. Food and Drug Administration, Washington,
     D.C.

U.S. Pharmacopeia.  1975.  XIX  Edition, pp. 581-794.  U.S.Pharmacopoeial
     Convention, Inc., Rockville, Md.

Weil, C. S., and D. D. McCollister.  1963.  Relationship between short- and
     long-term feeding studies designing an effective toxicity test.  J. Agr.
     Food Chem.  11:  486-491.

Weisburger, E. K.  1975.  Critical evaluation of the methods used for deter-
     mining carcinogenicity.  J. Clin. Pharm.  15' 5-15.

Weisburger, J. H.  1976.  Bioassays and tests for chemical carcinogens,
     Chapter L In: Chemical Carcinogens, ACS Monograph No. 173, pp. 1-23.
     American Chemical Society, Washington, D.C.

Wilson, J. G.  1975.  Reproduction and teratogenesis:  current methods and
     suggested improvements.  J. Ass. Offie. Anal. Chem.  58:  657-667.

Zbinden, G.  1973.  Progress in Toxicology.  Chapter 2 - Formal toxicology,
     pp. 4-27.  Springer-Verlag, New York, N.Y.
                                    474

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                                           APPENDIX A

                          CHECK LIST  FOR PLANNING  TEST PROGRAMS

      The  following  check  list is taken from: Bicking,  C.  A.,  1954.   Some  uses
of  statistics in the planning of experiments, Industrial  Quality Control  10;
20-23.
                           CHECK LIST FOB PLANNING TEST PROGRAMS
                   A. Obtain a Clear Statement of the Problem
                       1.  Identify the new and important problem area
                       2.  Outline the specific problem within current limitations
                       3.  Define exact scope of the test program
                       4.  Determine relationship of the particular problem to the whole re-
                          search or development program
                   B. Collect Available Background Information
                       1.  Investigate all available sources of  information
                       2.  Tabulate data pertinent to planning new program
                   C. Design the  Tut Program
                       L  Hold a  conference of all parties concerned
                          a.  State the propositions to be proved
                          b. Agree on magnitude of differences considered worthwhile
                           c. Outline the possible alternative outcomes
                          d. Choose the factors to be studied
                           e. Determine the practical range of these factors and the specific
                             levels at which tests will be made
                           f. Choose the end measurements which are to be made
                          g. Consider the effect of sampling variability and of precision of
                             test methods
                          h. Consider possible inter-relationships (or "interaction!") of the
                             factors
                           j. Determine limitations  of time,  cost, T"flt*riBil», manpower, in-
                             strumentation and other  facilities and of extraneous conditions,
                             such as weather
                          k. Consider human relations angles of the program
                       2,  Design  the program in preliminary form
                           a. Prepare a systematic and inclusive schedule
                          b. Provide for step-wise performance or adaptation of  schedule if
                             necessary
                          c.  Eliminate effect of variables not under study by controlling, bal-
                             lan^yig or randomizing them
                          d. Minimize the number of experimental runs
                           e. Choose the method of statistical analysis
                           f. Arrange for orderly accumulation of data
                       3.  Review the design with all concerned
                           a. Adjust the program in line with comments
                          b. Spell out the steps to be followed in unmistakable terms
                                                 A-l

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D.  Plan and Carry Out the Experimental Work
    1.  Develop methods, materials, and equipment
    2.  Apply the methods or techniques
    3.  Attend to and check details; modify methods if necessary
    4.  Record any modifications of program design
    5.  Take precautions in collection of data
    6.  Record progress of the program
E.  Analyze the Data
    1.  Reduce recorded data, if necessary, to numerical form
    2.  Apply proper mathematical statistical techniques
F.  Interpret the Results
    1.  Consider all the observed data
    2.  Confine conclusions to strict deductions  from the evidence at hand
    3.  Test questions suggested by the data by independent experiments
    4.  Arrive at conclusions as to the technical meaning of results as well
        as thnir statistical significance
    5.  Point out implications of the findings for application and for further
        work
    6.  Account for any limitations imposed by the methods used
    7.  State results in terms  of verifiable probabilities

G.  Prepare the Report
    1.  Describe work  clearly giving background, pertinence of the prob-
        lems *»"^ tnoatitfig of results
    2.  Use tabular and graphic methods of presenting data in good form for
        future use
    3.  Supply sufficient information to permit  reader to verify results and
        draw his own conclusions
    4.  Limit conclusions to objective summary  of evidence so  that the
        work recommends itself for prompt consideration and decisive action
                                 A-2

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                                 APPENDIX B.

                     GOOD ANIMAL CARE LABORATORY PRACTICES
Designation

   CBO-1
   CBO-2
   CBO-3
   CBO-4
   CBO-5

   CBO-6
   CBO-7
   CBO-8
   CBO-9
   CBO-10
   CBO-11

   CBO-12

   CBO-13
   CBO-14

   CBO-15
   CBO-16

   CBO-17

   CBO-18

   CBO-19
   CBO-20
   CBO-21
   CBO-22
   CBO-23

   CBO-24
   CBO-25
   CBO-26
                     Subject                              Page

Laboratory Animal Care Personnel                           B-2
Provisions for Emergency Laboratory Care                   B-5
Carcinogen Bioassay Pathology Personnel                    B-6
Safety of Animal Care Personnel                            B-8
Preparation for Shipment and Transportation of
Laboratory Animals                                         B-12
Receipt and Quarantine of Laboratory Animals               B-15
Weighing of Laboratory Animals                             B-18
Examination of Laboratory Animals for General Health       B-19
Examination of Rodents for Parasites                       B-21
Randomization, Assignment, and Identification of Animals   B-22
Storage of Feed, Bedding, and Equipment for
Laboratory Animals                                         B-26
Dose Preparation and Analysis for Chemicals to be
Administered by Procedures other than Inhalation           B-29
Feeding of Laboratory Animals                              B-32
Generation and Analysis of Test Atmosphere of Chemicals
Evaluated by the Inhalation Method Program                 B-34
Watering of Laboratory Animals                             B-37
Changing of Litter or Bedding, Changing of Laboratory
Animal Cages, and Disposal of Waste                        B-40
Maintenance of Optimal Environmental Conditions for
Laboratory Animals                                         B-44
Sanitation of Equipment and Supplies for Laboratory
Animals                                                    B-51
Disinfection of Laboratory Animal Rooms                    B-54
Vermin Control in Animal Facilities                        B-56
Sacrifice of Laboratory Animals (Euthanasia)               B-58
Disposal of Dead or Sacrificed Animals and Tissues         B-60
Disposal of Radioactive Wastes Associated with Laboratory
Animal Experiments                                         B-62
Disposition of Carcinogen Bioassay Pathology Material      B-64
Required Information                                       B-67
NCI Carcinogen Bioassay Data System (CBDS)                 B-72
                                   B-l

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     Carcinogen  Bioassay  Program
Specification No.  CBO-1
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
i
Of
3
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
               LABORATORY ANIMAL CARE PERSONNEL
                                  1.  SCOPE

     This  specification covers  laboratory  animal  care personnel used  in  the
     Carcinogen Bioassay Program.
     None
     3.1   General
                           2.   APPLICABLE DOCUMENTS
                                3.   REQUIREMENTS
          3.1.1   Animal  care  program shall  be  directed by veterinarians having
     specialized training or  experience  in  laboratory animal medicine.

          3.1.2   The employment  of  a full-time staff specifically concerned
     with the animal care program is recommended.   The staff shall  include
     the professional and supporting personnel necessary to implement the
     veterinary,  animal  husbandry,  and administration aspects  of the program.

     3.2  Supervisors

          3.2.1   All personnel and  facilities  for  maintaining  laboratory
     rodents  shall be directly supervised by a professionally  qualified person
     In addition,  services of a  veterinarian trained in a laboratory animal
     medicine should be  available either on a  permanent basis  or as a part-
     time consultant.

     3.3  Technicians

          3.3.1   Technicians  employed as caretakers of laboratory animals
     shall be trained in formal  courses  designed for that specific  purpose
     or shall undergo extensive  on-the-job  training under close supervision.

          3.3.2   Caretakers shall be certified in  their job specialty by a
     nationally  recognized certification board.

          3.3.3   Caretakers shall be able to recognize symptoms  of disease
     and other abnormalities.
                                      B-2

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   Carcinogen Bioassay Program               Specification No. CBO-1
Subject:
LABORATORY ANIMAL CARE PERSONNEL
Date:
Sheet
2
Of
3
   3.4  Drivers
       3.4.1  Each driver or assistant shall be qualified to handle and
   care for laboratory animals being transported and to provide needed
   services in emergencies.  This qualification may consist of either
   completion of an approved animal care technician course or equivalent
   practical experience  in animal care.
                           4.  QUALITY CONTROL
4. 1.  Animal technicians should be divided into 2 grades.
    4. 1.1  Grade A (lower grade), minimum of 9 years of schooling, proba-
   tionary period of at  least 3 months; completion of a formal course of
   instruction; a minimum 2 year period of service before examination.
    4. 1.2  Grade B (higher grade), similar requirements for schooling and
   probationary period;  completion of an advanced formal course of instruc-
   tion; a minimum three year service period as a Grade A technician before
   examination.
4. 2.  The education of  laboratory animal technicians should be based on the
   following outline:
    4. 2.1  Introduction to animal care.
    4. 2.2  Life, living matter and biological organization.
    4. 2.3  Structure and function (skeletal and muscular system, integument;
   circulatory and respiratory systems; digestive and excretory systems;
   nervous system and sense organs; endocrine systems; reproductive system).
    4. 2.4  Genetic and  mating system.
    4. 2.5  Nutrition and metabolism.
    4. 2.6  Handling.
    4. 2.7  Animal health and disease.
    4. 2.8  Sanitation and hygiene.
    4. 2.9  Housing and  equipment design.
    4. 2.10  Administration, management, record keeping.
    4. 2.11  Shipping and receiving of animals.
    4. 2.12  Safety
    4. 2.13  Animal experimentation
 4.3.  Apart from scholastic achievements the animal technician should have
   a natural aptitude for dealing with animals and keen sense of  discipline
   and responsibility thus ensuring that the requirements of the  animal will
   always be uppermost in his mind  (6.5, p. 116).
                                     B-3

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Carcinogen Bioassay Program               Specification No. CBO-1
Subject:
LABORATORY ANIMAL CARE PERSONNEL
Date:
Sheet
3
Of
3
                             5.  PACKAGING

N/A

                        6.  REFERENCE DOCUMENTS

  6.1  Code of Federal Regulations, Title 9, Chapter 1, Subchapter A.
Animal Welfare. Parts 1, 2 and 3, May, 1972.

  6.2  Guide for the Care and Use of Laboratory Animals. 1974. U. S.
Department of Health, Education and Welfare, NIH 74-23.

  6.3  Procurement Specification VII. Rodents. 1969. Institute of
Laboratory Animal Resources, National Academy of Sciences, National
Research Council, Washington, D. C.

  6.4  Rodents. 1969. Standards and Guidelines for the Breeding, Care
and Management of Laboratory Animals.  National Academy of Sciences,
Washington, D. C.

  6.5  The UFAW Handbook on the Care and Management of Laboratory
Animals, 1972, 4th edition. UFAW Staff (eds.).  Churchill Livingstone,
Edinburgh and London.
                                 B-4

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     Carcinogen Bioassay Program
Specification No. CBO-2
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
i
Of
1
Approved:
Proj.
Q.C.
lab
Other
Date
Subject:
              PROVISIONS  FOR EMERGENCY LABORATORY CARE
                               1.   SCOPE

      This  specification  covers the steps  taken  to provide for emergency
     laboratory  animal  care.
     None
                              2.  APPLICABLE DOCUMENTS
                                 3.  REQUIREMENTS
     3.1  Provision must be made for  the  emergency care of animals.

       3.1.1  Animal  care supervisors and personnel available  for emergency
     duty shall be  identified and alerted to  the necessary emergency care
     procedures.

       3.1.2  A  list  of the animal care  supervisors and designated personnel
     shall be prominently posted at the laboratory's central  telephone center
     and in  the security department if one exists.

       3.1.3  Laboratory security personnel  and fire and police officials
     should  know  how to reach the individuals responsible for emergency care.

     3.2  Emergency animal care personnel shall be called to  duty promptly
     upon discovery of any emergency  involving animals or records.

                               4.  QUALITY CONTROL

     Periodically update the name, telephone  number and address of the
     responsible  individuals.   The objective  is to assure that  animals will
     be cared for should an emergency arise.
     N/A
                                5.   PACKAGING
                            6.  REFERENCE DOCUMENTS
       6.1  Guide for  the Care and  Use of Laboratory Animals.  1974.  U.  S.
     Department  of Health,  Education and Welfare,  NIH 74-23.
                                       B-5

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     Carcinogen Bioassay Program
Specification No. CBO-3
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
i
Of
2
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
         CARCINOGEN BIOASSAY PATHOLOGY PERSONNEL
                                  1.   SCOPE

        This specification covers personnel required for all pathology work
     with small rodents employed in the Carcinogen Bioassay Program.
     None
                          2.   APPLICABLE DOCUMENTS
                              3.   REQUIREMENTS
        3.1  A board-certified pathologist (veterinary or  medical)  experienced
     in laboratory animal pathology shall be responsible for all  pathology
     procedures,  evaluations,  and reporting.  Persons  not  board-certified
     may be acceptable if appropriate training and experience judged to  be
     satisfactory by program management can be demonstrated.

        3.2  Histology technician(s) shall be supervised by an HT/ASCP
     registered technician who is qualified as judged  by program  management.
     Persons not certified may be acceptable if they have  had appropriate
     training and experience that is satisfactory in the judgement  of program
     management.

        3.3  Prosectors shall be trained and experienced in laboratory animal
     dissection and must be able to recognize and describe gross  abnormalities
     "Careful performance of the necropsy for the detection of possible  tumors
     at any site is vital to carcinogenesis experiments".   Qualified and well-
     supervised personnel are absolutely essential.

        3.4  The subcontractor must have personnel available for  weekend duty
     to necropsy any dead or moribund animals.

                             4.  QUALITY CONTROL

        4.1  The credentials of all persons to be engaged  for pathology
     responsibilities or histologic duties in the National Cancer Institute
     Carcinogen Bioassay Program who are not board-certified or registered
     shall be submitted by the subcontractor for review and approval by
     program management.
                                       B-6

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 Carcinogen Bioassay Program
Specification No. CBO-3
Subject:
CARCINOGEN BIOASSAY PATHOLOGY PERSONNEL
Date:
Sheet
2
Of
2
N/A
                            5.  PACKAGING
                         6.   REFERENCE DOCUMENTS
   6.1  Sontag, J.M., N.P. Page, and U. Saffiotti. 1976. Guidelines for
Carcinogen Bioassay in Small Rodents. U.S. Department of Health,
Education and Welfare, NIH 76-801.

   6.2  Request for Proposal 76-S-12, Carcinogen Bioassay Program, Due
Date June 15, 1976, Tracer Jitco, Inc., Rockville, Maryland.
                                  B-7

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     Carcinogen Bioassay Program
Specification No. CBO-4
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
i
Of
4
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
              SAFETY OF ANIMAL CARE PERSONNEL
                                  1.   SCOPE

          This specification covers the requirements needed  to protect
     laboratory animal  care personnel from potential hazards - the
     restriction of  certain personnel into particular facilities, the
     use  of  protective  clothing and equipment,  the  personal  hygiene
     aspect  and the  enforcement of an occupational  health program.

                          2.   APPLICABLE DOCUMENTS

          2.1   NCI Safety  Standards for Research  Involving Chemical
     Carcinogens. 1975.  Department of Health, Education  and  Welfare,
     NIH  76-900.

          2.2   OSHA  Standard  for Carcinogens, Federal Register, Vol. 39,
     No.  20, Jan., 29,  1974.

          2.3   OSHA  Standard  for Carcinogens, Part  II, Federal Register,
     Vol.  41,  No. 163,  p.  35184,  August 20,  1976.

                              3.   REQUIREMENTS

          Personnel  should  receive adequate  animal  care  and  personal hygiene
     training  and instruction as  to the proper  operating procedure.

          3.1  Personnel  restrictions.

              3.1.1  Access to the animal  facilities  should  be restricted to
              those  individuals essential  to their  operation.

              3.1.2  Personnel whose  medical condition,  e.g., depressed
              immune response,  pregnancy,  and steroid or cytotoxic drug
              treatment, may  make  them unusually susceptible to the possible
             harmful effects of a test  agent should  be  excluded from any
             area where accidental exposure could  occur.

             3.1.3  Individuals who  are allergic to  laboratory animals
             should not be exposed to  them  unless  adequately protected
             and approval  has  been given  by the medical or  safety officer.

          3.2  Use of protective  clothing and equipment.
                                     B-8

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Carcinogen Bioassay Program                Specification No.  CBO-4
Subject:
SAFETY OF ANIMAL CARE PERSONNEL
Date:
Sheet
2
Of
4
        3.2.1  A complete change of clean working clothes should be
        provided daily and should include a fully fastened laboratory
        suit or jumpsuits, gloves, boots, and head cover.

        3.2.2  Clothing contaminated by chemical carcinogens shall be
        decontaminated before being sent out for laundering or it shall
        be disposed of immediately after an overt exposure.

        3.2.3  An appropriate face mask or respirator should be worn as
        protection against dust, mists, or fumes.

        3.2.4  The protective clothing should not be worn outside the work
        area.

        3.2.5  Suitable facilities should be available for storage of
        street clothing during the workday.

    3.3  Personal hygiene.

        3.3.1  There shall be no eating, drinking, smoking, application of
        cosmetics, or storage of food within animal room or in areas where
        chemical carcinogens are used.

        3.3.2  Showering or a surgical scrub to the elbows, prior to entry
        into the clean area is recommended.

        3.3.3  Face and neck skin surfaces should be hygienically cleaned.

    3.4  Occupational health program.

        3.4.1  An occupational health program is mandatory for personnel
        working in laboratory animal facilities and for other personnel
        with significant animal contact.

        3.4.2  It should include preplacement and periodic physical
        examinations.

        3.4.3  The specific occupational hazards that may exist should
        be recognized.

        3.4.4  An immunization schedule appropriate to the animal care
        program should be developed.

        3.4.5  Zoonosis surveillance should be carried out.
                                   B-9

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Carcinogen Bioassay Program               Specification No. CBO-4
Subject:
SAFETY OF ANIMAL CARE PERSONNEL
Date:
Sheet
3
Of
4
           3.4.5.1  Keep a permanent case record of individual work
           assignments.

           3.4.5.2  Retain records concerning bite wounds and  occurrence
           of any unusual illness.

           3.4.5.3  Instruct personnel to notify their supervisor of
           suspected health hazards.

           3.4.5.4  Obtain and store individual preplacement and post-
           employment sera for future diagnostic purposes.

                        4.  QUALITY CONTROL

       4.1  Periodic inspection to assure that safety regulations have
       been carried out.

                      5.  REFERENCE DOCUMENTS

       5.1  Sontag, J.M., N.P. Page, and V. Saffiotti. 1976. Guidelines
       for Carcinogen Bioassay in Small Rodents. U. S. Department of
       Health, Education and Welfare, NIH 76-801.

       5.2  Guide for the Care and Use of Laboratory Animals. 1974. U.S.
       Department of Health, Education and Welfare, NIH 74-23.

       5.3  Procurement Specification IX. Defined Laboratory Rodents
       and Rabbits. 1973. Institute of Laboratory Animal Resources,
       National Academy of Sciences, National Research Council,
       Washington, D. C.
                                               V
       5.4  Long-Term Holding of Laboratory Rodents. 1976. ILAR News
       XIX (4), L20, L21.

                             6.  NOTES

       6.1  Definitions.

          6.1.1  CHEMICAL CARCINOGEN is a chemical that has been
          demonstrated to cause tumors in mammalian species by induction
          of a tumor type not usually observed; or by induction of an
          increased incidence of tumor type normally seen, or by its
          appearance at a time earlier than would be otherwise expected.
                                 B-10

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Carcinogen Bioassay Program
Specification No. CBO-4
Subject:
SAFETY OF ANIMAL CARE PERSONNEL
Date:
Sheet
4
Of
4
          6.1.2  DECONTAMINATION is the safe removal of a chemical
          carcinogen from a contaminated item.

          6.1.3  DISPOSAL is the safe elimination of a chemical carcinogen
          from the general environment by inactivation, degradation,
          destruction, or other appropriate method.

          6.1.4  GLOVES are covers to protect the hands of a worker
          against contact with or exposure to chemical carcinogen.

          6.1.5  PROTECTIVE CLOTHING is clothes designed to protect a
          worker against contact with or exposure to a chemical carcino-
          gen.

          6.1.6  PROTECTIVE EQUIPMENT is equipment in addition to protec-
          tive clothing and gloves, such as a face mask or a respirator,
          that is designed to protect a worker against contact with or
          exposure to chemical carcinogen.
                                B-ll

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Approved:
Proj.
Q.C.
Lab
Other
Date
     Carcinogen Bioassay Program
Specification No. CBO-5
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
l
Of
3
Subject:
       PREPARATION FOR SHIPMENT AND TRANSPORTATION OF LABORATORY ANIMALS
                                   1.   SCOPE

          This  specification covers the precautions  taken during transporta-
     tion  of  experimental  animals  between facilities to minimize  contamina-
     tion  and alteration of the behavior and physiologic status of  the
     animals.
     None
                           2.   APPLICABLE  DOCUMENTS
                               3.  REQUIREMENTS
          3.1   Shipping  containers

            3.1.1   Shipping  containers must be made of new materials.  The
    materials  should be nontoxic and  impervious to moisture.

            3.1.2   All  inner surfaces of containers should be wire-screened
    when  the materials  call  for it.

            3.1.3   Twenty-five to  thirty percent of the surface areas must
    be open and covered by filter.

            3.1.4   Ventilation openings should be decreased during severe
    cold weather.

            3.1.5   The  bedding in  the shipping containers must be clean and
    adequate to assure  sanitation  and comfort.

            3.1.6   Shipping  containers should be sterilized prior to packing,

          3.2  Transportation vehicle and its environmental control

            3.2.1   Vehicles  used must be mechanically sound and equipped to
    provide fresh air without injurious draft to all animals being trans-
    ported.

            3.2.2   Exhaust from the vehicle's engine should not have ingress
    to the animal cargo  space.

            3.2.3   Animal cargo space must be kept clean.
                                     B-12

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Carcinogen Bioassay Program               Specification No. CBO-5
Subject: PREPARATION FOR SHIPMENT AND TRANSPORTATION
OF LABORATORY ANIMALS

Date:


Sheet


Of

3
        3.2.4  The shipper should be conservative in determining the
 number of animals to be placed in a shipping container.  The animals
should have sufficient space to turn freely, stand erect, and lie
naturally.

        3.2.5  During periods of high outdoor temperature, limitations
upon the number of animals per shipping container are especially impor-
tant.
        3.2.6  Vehicles must be sanitized before loading.

        3.2.7  Vehicles should maintain a temperature suitable for the
animals by air-conditioner, heater or other devices.

    3.3  Feed and water for animals during shipment.

        3.3.1  Each shipping container must have sufficient food and
water to maintain the animals for approximately double the time period
normally estimated for transit from consignor to consignee.

    3.4  Qualification of the driver of the vehicle.

        3.4.1  The driver should be qualified to handle and care for the
laboratory animals being transported and to provide needed services in
emergencies.

        3.4.2  This qualification may consist of either completion of an
approved animal care technician course or equivalent practical experience
in animal care.

    3.5  Schedule

        3.5.1  Schedule of shipments must be planned to minimize the
amount of time that animals are in transit.  For example, shipments
should be scheduled for normal working days, usually Mondays through
Thursdays, since delivery and reception are often unreliable on Fridays,
holidays and weekends.

    3.6  Shipping labels

        3.6.1  Shipping labels should contain the following information:
origin of shipment; name, address and zip code and telephone number of
the consignee; purchase order number, if available; date of shipment;
kind and total number of animals and number of containers per shipment;
instruction for special handling, feeding or watering, if required;
delivery ticket for signature of consignee acknowledging receipt of
shipment.
                                 B-13

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Carcinogen Bioassay Program               Specification No. CBO-5
Subject: PREPARATION FOR SHIPMENT AND TRANSPORTATION
OF LABORATORY ANIMALS
Date:
Sheet
3
Of
3
                        4.  QUALITY CONTROL

    4.1  The project management should check to assure that the transport
requirements have been carried but.

    4.2  Shipping containers and transportation vehicles must be inspect-
ed to see they meet the specifications set forth.

    4.3  Acceptance of animals at the purchaser's facilities depends upon
freedom from overt signs of disease and parasitism, scars, wounds,
lesions and abnormal physical and behavioral characteristics.

    4.4  Freedom from certain microbial organisms and ecto and endo
parasites should be delineated by purchasers.

                           5.  PACKAGING

N/A

                       6.   REFERENCE DOCUMENTS

    6.1  Procurement Specification IX.  Defined Laboratory Rodents and
Rabbits. 1973.  Institute of Laboratory Animal Resources, National
Academy of Sciences, National Research Council, Washington, D. C.

    6.2  Long-Term Holding of Laboratory Rodents. 1976.  ILAR News XIX
(4) L20, L21.

    6.3  Procurement Specification VII.  Rodents. 1969.  Institute of
Laboratory Animal Resources, National Academy of Sciences, National
Research Council, Washington, D. C.
                                 B-14

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    Carcinogen Bioassay Program
                                          Specification No.  CBO-6
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
1
Of
3
Subject:
Approved:
Proj.
Q.C.
Lab
Other
Date
             RECEIPT AND QUARANTINE OF LABORATORY ANIMALS
                                 1.  SCOPE

         This practice covers  examination and caging of animals upon receipt
     from supplier  through quarantine at the laboratory.
                           2.  APPLICABLE DOCUMENTS
         2.1
         2.2
         2.3

         2.4
         2.5
         2.6
          Specification No. CBM-6
          Specification No. CBO-7
          Specification No. CBO-8

          Specification No. CBO-9
          Specification No. CBO-21
          Specification No. CBO-17
                          Animal Cages and Cage Filters
                          Weighing of Laboratory Animals
                          Examination of Animals for General
                          Health
                          Examination of Animals for Parasites
                          Sacrifice of Living Animals
                          Maintenance and Optimal Environmental
                          Conditions for Laboratory Animals
                          Disinfection of Animal Laboratory
                          Rooms
Code of Federal Regulations, Title 9, Chapter 1, Subchapter A.
          2.7   Specification  No.  CBO-19
          2.8
         Animal Welfare.  Parts  1,  2  and  3, May,  1972.
                               3.   REQUIREMENTS
          3.1
          Examination upon receipt
        3.1.1  Animals shall be received, in their unopened shipping
     containers, in the designated quarantine area.
        3.1.2  Discard substandard animals on receipt for size, health
     or other reasons.
                     Minimum Acceptable Size:
                        Mice	g
                             Rats
          Examine all animals  for general  health.   Sacrifice a random sample
         	 animals and examine for parasites.   Palpate all animals
of	
and discard any with an abnormality.
                                      B-15

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Carcinogen Bioassay Program               Specification No. CBO-6
Subject:
RECEIPT AND QUARANTINE OF LABORATORY ANIMALS
Date:
Sheet
2
Of
3
      3.2   Caging before  distribution  for  test

      A  shipment may be caged  together during quarantine, acute  toxicity
 test  and  repeated dose study  according to the weight-space requirements
 in  the  following chart:

 SPECIES        .WEIGHT          FLOOR  AREA/ANIMAL              HEIGHT*
                                   (SQUARE)


 Mouse       Up to 10 g         39  cm  (6 in)            12.7 cm  (5 in)
                10-15 g         52  cm  (8 in)            12.7 cm  (5 in)
                16-25 g         77  cm  (12  in)           12.7 cm  (5 in)
              Over 25 g         97  cm  (15  in)           12.7 cm  (5 in)

 Rat          Up to 100 g       110 cm (17 in)          17.8 cm  (7 in)
               100-200 g       148 cm (23 in)          17.8 cm  (7 in)
               201-300 g       187 cm (29 in)          17.8 cm  (7 in)
             Over 300  g       258 cm (40 in)          17.8 cm  (7 in)

 * Height  means from the  resting floor to  the cage  top.

 3.3   Physical facilities of quarantine area.

   3.3.1   The quarantine area should  be located in rooms physically
 separated from existing  testing areas.  Separate rooms should be provided
 for each  species.

   3.3.2   Except for relaxed  caging requirements prior to distribution,
 physical  conditions during quarantine shall be of  the same quality as
 that  provided animals under test.

   3.3.3   If an epizootic disease  or  parasitic infection if found among
 the animals upon arrival, or  at  any time  during quarantine, the entire
 shipment  should be discarded  and the  room disinfected prior to  the
 receipt of additional animals.

 3.4   Quarantine period

    Animals should be quarantined  for a minimum of seven days.

 3.5   Reexamination of animals.
    3.5.1  At the end of the  quarantine period, the animals should be
 reexamined for health (and palpated)  and  any additional substandard ones
 discarded.

    3.5.2  If a sufficient number  of  healthy animals to satisfy test
 protocol requirements are on  hand  after reexamination, they may be
distributed for testing.   If  the number is insufficient, a new  supply of
animals may need to be obtained  and the quarantine and examination repeated
                                 B-16

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Carcinogen Bioassay Program               Specification No.  CBO-6
Subject:
RECEIPT AND QUARANTINE OF LABORATORY ANIMALS
Date:
Sheet
3
Of
3
  3.6  Disposal of animals dead on receipt or during quarantine.

       3.6.1  All procedures involved in the disposal of dead animals
shall be in conformance with Federal, State, and local laws and regula-
tions pertaining to pollution control and protection of the environment.
       3.6.2  Waste cans for use in removal of dead animals should be
equipped with leakproof disposable liners and tight-fitting lids.

                        4.  QUALITY CONTROL

  4.1  Shipments containing dead, moribund, or unsatisfactory animals
must be reported immediately to the program management and in writing to
the animal-supply house concerned, with a copy to the program management.

  4.2  Results of examination for parasites of individual animals in
the sample sacrificed, including all negative findings, shall be recorded
in a bound laboratory notebook by the clinician performing the examina-
tion and witnessed by the laboratory supervisor.  It shall be the
responsibility of the laboratory supervisor to verify that a complete
record has been made for each shipment within	days of receipt of
the shipment.

  4.3  The number of animals entering quarantine, length of quarantine,
and the number distributed for testing, with a tabulation by cause of all
discards, shall be entered in a bound laboratory notebook by the respon-
sible technician and witnessed by the laboratory supervisor.  This record
shall be available for audit and analysis.

  4.4  If occurrence of an epizootic disease has been reported, it shall
be the responsibility of the laboratory supervisor to verify in the
quarantine laboratory notebook that the quarantine area has been disin-
fected within	hours of the detection of the disease and removal of
the affected shipment.
                              5.  N/A

                       6.   REFERENCE DOCUMENTS

  6.1  Sontag, J.M., N.P. Page, and U. Saffiotti. 1976. Guidelines for
Carcinogen Bioassay in Small Rodents.  U.S. Department of Health,
Education and Welfare, NIH 76-801.

  6.2  Guide for the Care and Use of Laboratory Animals. 1974. U.S.
Department of Health, Education and Welfare, NIH 74-23.

  6.3  Guide to Infectious Diseases of Mice and Rats,  Institute of
Laboratory Animal Resources, ISBN 0-309-01914-1.
                                 B-17

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     Carcinogen Bioassay Program
      Specification No. CBO-7
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
l
Of
1
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
          WEIGHING OF LABORATORY ANIMALS
                                  1.   SCOPE

          This specification covers weighing of laboratory animals for caging,
     randomization,  recording weight  change during bioassay,  and other
     operations in the Carcinogen Bioassay Program.

                          2.  APPLICABLE DOCUMENTS
         2.1  Specification No.  CBO-6

         2.2  Specification No.  CBO-10

         2.3  Specification No.  CBP-2

         2.4  Specification No.  CBP-3

         2.5  Specification No.  CBP-4
Receipt and Quarantine of Laboratory
Animals
Randomization, Assignment, and
Identification of Animals
Repeated-Dose Test, Carcinogen
Bioassay Program
Sub-chronic Test, Carcinogen
Bioassay Program
Chronic Carcinogenicity, Carcinogen
Bioassay Program
                              3.  REQUIREMENTS

         3.1  All animals used in the Carcinogen Bioassay Program shall be
     weighed individually at times indicated: at time  of receipt, at time
     of assignment to treatment groups,  and periodically during chronic
     studies.

         3.2  Weight shall be determined to the nearest gram using an
     appropriate animal weighing scale.

                             4.  QUALITY CONTROL

         4.1  Balances employed for determining animal weight shall be
     recalibrated at least monthly and calibration data recorded in a bound
     notebook  and signed by responsible personnel.

         4.2  Supervisors shall be responsible for making certain that all
     animal weights are accurately determined and recorded to insure validity
     of the bioassay test results.
                                5.   PACKAGING
     Not applicable.
                                       B-18

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     Carcinogen Bioassay Prograa
Specification Ho. CBO-8
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
1
Of
2
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
          EXAMINATION OF LABORATORY ANIMALS  FOR GENERAL HEALTH
                                  1.   SCOPE

     This  specification covers  the observation of laboratory  animals  for  signs
   of diseases and ways of monitoring the general health of the  animals.
     None
                          2.   APPLICABLE DOCUMENTS
                                  3.  REQUIREMENTS
          3.1  Observation.   All animals should be observed  regularly  by
     properly qualified personnel for signs of diseases.   Animal  care  should
     be under direction of veterinarians with specialized training and
     experience in laboratory animal  medicine.

               3.1.1  Sick or moribund animals or animals found dead should
     be removed from the colony, and  an adequate number should be examined
     by laboratory procedures (including pathology) to determine  the cause
     of the observed signs or death.
                               4.   QUALITY CONTROL
           4.1  Monitoring
               4.1.1  Routine methods.   At regularly scheduled intervals,
     water bottles and feces should be  cultured in order to determine whether
     the predominant organisms present  are similar or identical to those
     previously established and that pathogens are not present.
               4.1.2  Detailed methods.  At regularly scheduled intervals,
     normal-appearing animals should be removed from the colony for labora-
     tory tests.
                      4.1.2.1  Serum samples should be obtained and tested
     for antibodies to murine viruses.
                      4.1.2.2  Bacteria, mycoplasma, protozoa, and metazoa
     should be identified, if present.
                      4.1.2.3  Tissues  or organs should be examined histo-
     logically to determine the presence or absence of lesions.
                                      B-19

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Carcinogen Bioassay Program
Specification No. CBO-8
Subject:
EXAMINATION OF LABORATORY ANIMALS FOR GENERAL HEA
Date:
TH
Sheet
2
Of
2
          4.1.3  Record keeping.  Daily records shall be maintained on
morbidity, mortality, and laboratory findings by room, species, and
strain.  This information should be reviewed weekly.
Not applicable here.
                            5.  PACKAGING
                       6.  REFERENCE DOCUMENTS
    6.1  Procurement Specification IX.  Defined Laboratory Rodents and
Rabbits. 1973.  Institute of Laboratory Animal Resources, National
Academy of Sciences, National Research Council, Washington, D. C.
                                 B-20

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     Carcinogen  Bioassay  Program
Specification No. CBO-9
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
l
Of
l
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
           EXAMINATION OF  RODENTS  FOR PARASITES
                                   1.   SCOPE

         This  specification covers  examination of  rodents for parasites.

                          2.   APPLICABLE DOCUMENTS
     None
                                3.   REQUIREMENTS
         3.1   Parasitology.
            3.1.1  Routine methods.   Microscopic  examination of  specimens
     obtained from fresh feces  by concentration procedures  and scotch  tape
     impressions of the perianal region from representative animals  should
     be examined for the presence of parasitic  ova.

            3.1.2  Detailed methods.
               3.1.2.1  At the  time  of sacrifice,  in addition to routine
     methods described above, urine  should be examined  microscopically for
     nematode eggs, and the intestinal tract, cecum,  and bladder opened and
     examined with appropriate  magnification for  internal parasites.

               3.1.2.2  In addition,  histologic examination of the tissue
     and organs will assist in  determining whether selected protozoan  or
     metazoan parasites are present.   Special attention and selective
     strains are recommended for the lower respiratory  tract and brain
     for Pneumocystis and Nosema, respectively.

                             4.  QUALITY CONTROL

         4.1  Refer to the Diagnostic Guide (Section I) and Disease  Outlines
     (Sect 11) of "A Guide to Infectious Diseases of Mice and Rats", National
     Academy of Sciences, for descriptions of clinical  and  pathologic
     features of diseases plus  appropriate diagnostic procedures.

         4.2  Positive and negative  findings shall be reported for  each
     animal examined.  It shall be the responsibility of  laboratory
     supervision to monitor the examination to  assure its completeness
     and correctness.
                                       B-21

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     Carcinogen Bioassay Programs
Specification No.  CBO-10
Type:
GOOD
ANIMAL CARE LABORATORY PRACTICE
Sheet
i
Of
4
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:

     RANDOMIZATION,  ASSIGNMENT,  AND IDENTIFICATION OF ANIMALS
                                  1.   SCOPE

          This practice covers the age of animals  assigned  to  an experiment,
     the use of random numbers to select  animals by weight  distribution for
     assignment to experimental groups, and the unique  identification of
     individual animals.

                           2.   APPLICABLE DOCUMENTS

          2.1  Specification No.  CBO-7 Weighing of Animals

                               3.   REQUIREMENTS

          3.1  Animal  species,  strain  and sex

          Experimental groups  are composed of animals of  the same species,
     strain and sex, each group being  dealt with identically.

          3.2  Age of  animals

             3.2.1 At the start of the chronic study,  animals should be no
     older than six weeks and,  if possible,  weanlings.

             3.2.2 All animals assigned  to a study should  be within two to
     three days of the same age.   This is assured  by specifying age limits
     at  the time of animal procurement and using only animals  from the same
     shipment in an experiment.

             3.2.3 If it has  been necessary to replace animals lost from a
     shipment upon receipt or  during quarantine, the animals should be
     segregated initially by shipment.  A randomization procedure is used to
     ensure that there will be  a  proportionate number of  animals from each
     shipment and  from each weight distribution group (see  next section) in
     each  of  the experimental  groups.

          3.3  Weight  of  animals

             3.3.1 The animals should be initially segregated into equal
    weight distribution  groups  according to the following  table.
                                      B-22

-------
Carcinogen Bioassay Program               Specification No. CBO-10
Subject:
RANDOMIZATION, ASSIGNMENT, AND IDENTIFICATION OF ANI1
Date:
ALS
Sheet
2
Of
4
                    Weight Distribution Groups

                    Species            Weight
                    Mouse              Up to 10 g
                                             11-15 g
                                             16-25 g
                                       Over 25 g
                    Rat                Up to 100 g
                                             101-200 g
                                             201-300 g
                                       Over 300 g
      3.3.2 After segregation by weight, animals are to be divided into
experimental groups using a randomization procedure to assure that a
proportionate number of animals from each weight distribution group
are included in each experimental group.

    3.4  Distribution in sub-chronic and chronic studies

      3.4.1  Distribute animals from the outset of the studies as if they
were in the upper weight range in above table.

      3.4.2  No cage should contain more than five animals.
      3.4.3  As animals die or are sacrificed, surviving animals should
not be combined or redistributed among the cages.

    3.5  Randomization procedure

      3.5.1  Experimental groups must be balanced, that is, each group
must contain an equal number of animals and representation of initially
segregated weight groups (or age groups, if necessary) in each experi-
mental group must be proportional to the size of the initially segregated
groups.

      3.5.2  If segregation by age was required, first make the separa-
tion by age and then by weight within each age group. If each experimental
group is to contain 50 animals, the total number of animals in all the
initial age/weight groups together must be at least 50 times the number
of experimental groups required by the experiment design.

      3.5.3  The randomization procedure is followed separately for each
age/weight group.

     3.6  Identification  of animals

     Each animal should be uniquely identified at the time it is assigned
to a sub-chronic or chronic experimental group by toe clipping, ear
notching or other appropriate method.
                                 B-23

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Carcinogen Bioassay Program
           Specification No. CBO-10
Subject:
RANDOMIZATION, ASSIGNMENT AND IDENTIFICATION OF ANIMA
Date:
s
Sheet
3
Of
4
                         4.  QUALITY CONTROL

     A record of all shipments included in a study, weight groupings,
randomization and assignment to experimental groups and animal
identifications should be made in a bound laboratory notebook by the
technician and witnessed by the laboratory supervisor.

     4.1  Determine the number of animals available in each age/weight
group.

     4.2  Divide the number in each of these groups by the number of
experimental groups required.  This gives the proportional number of
animals for each initial group to be included in each experimental
group.  Because total available animals may be more than exactly 50
times the number of experimental groups, or because the numbers in the
initial groups may not be exactly divisible by the number of experimental
groups, it may be necessary to adjust the dividend slightly to add up
to a total of exactly 50.

     4.3  Temporarily number the animals in the first age/weight group
consecutively.  Select a random starting place in a table of random
numbers.  Read from the table, omitting 000, numbers larger than the
total number in the group, and repeats.  Arrange these numbers in
successive sets the size of the proportional number determined in the
preceding section.  These sets are assigned to the respective experi-
mental groups.

     4.4  Repeat the procedure of the preceding section for each size/
weight subgroup.
     4.5  Example of Randomization Procedure.  Suppose that the experi-
ment design calls for five experimental groups of 50 animals each, that
200 satisfactory animals are available from a replacement shipment.
Assume the following weight distribution and calculate the proportional
number as shown.
        Original Shipment

        Weight Group 1
        Weight Group 2
        Weight Group 3
        Weight Group 4
No. of Animals

    30
    80
    70
    20
        Replacement Shipment   No. of Animals
Proportional No.

     6
    16
    14
     4

Proportional No.
Weight Group 1
Weight Group 2
Weight Group 3
Weight Group 4
Grand Totals
10
20
15
5
250
2
4
3
1
50
                                 B-24

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Carcinogen Bioassay Program               Specification No. CBO-10
Subject:
RANDOMIZATION,
ASSIGNMENT AND IDENTIFICATION OF ANIM
(Date:
AIJS
Sheet
4
Of
4
     Number the animals in the first group from 1 to 30.  Enter a random
table and list numbers from 001 to 030 as they appear, omitting repeats,
until five sets of six numbers each have been obtained.  These sets are
assigned to the five experimental groups.  Repeat this procedure for
each group in the above table.
                                  B-25

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     Carcinogen Bioassay Program
Specification No. CBO-11
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
i
Of
3
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
 STORAGE OF FEED,  BEDDING,  AND EQUIPMENT FOR LABORATORY ANIMALS
                                  1.   SCOPE

          This  specification covers storage of feed,  bedding,  and  equipment
     used  in the Carcinogen Bioassay  Program.
     None
                          2.   APPLICABLE DOCUMENTS
                                3.   REQUIREMENTS
          3.1   Date  of  manufacture of  all feed  supplies  shall  be  checked upon
     receipt.   Products delivered 90 days or more after  manufacture  shall  not
     be  accepted.

          3.2   Feed  and bedding shall  be stored in a clean  area and  protected
     from  spoilage or deterioration and  infestation or contamination by
     vermin.  A continuous  pest control  program is essential.  Containers
     shall be stored off the  floor on  pallets,  racks, or carts.   The area
     shall be physically separated from  refuse  areas.

          3.3   Feed  shall be  stored in receptacles with  tightly fitting lids
     or  covers  which can be sanitized  before reuse,  or in original containers
     as  received from the supplier.  The storage area shall be cool  (10° C
     or  less),  dry,  and airy.

          3.4   Washed/sanitized equipment shall be stored in a clean area
     free  of vermin.

          3.5   All supplies of  feed and  bedding as well  as  equipment in
     storage shall be carefully protected against contamination by pesticides.
     Pesticides shall not be  used inside buildings unless specifically agreed
     to by program management.

                             4.   QUALITY CONTROL

          4.1   Date  of  manufacture and delivery date of  all feed  shipments
     shall be recorded  in a bound notebook maintained for later consideration
     and signed by personnel  receiving same.
                                     B-26

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Carcinogen Bioassay Program               Specification No. CBO-11
Subject: STORAGE OF FEED, BEDDING, AND EQUIPMENT
FOR LABORATORY ANIMALS
Date:
Sheet
2
Of
3
     4.2  The shelf life of all feed lots shall be checked as used to
avoid feeding outdated rations to test animals.

     4.3  Temperature in the feed storage area shall be recorded
continuously by an automatic recording thermometer.  Temperature
recordings shall be inspected daily and adjustments made when
necessary to maintain a temperature of 10° C or less.  All charts
shall be dated, signed, and filed for audit by program management.

     4.4  The automatic temperature recorder shall be recalibrated
at least monthly and data recorded and signed by technical personnel
performing the work.

     4.5  All storage areas shall be inspected weekly for the presence
or evidence of vermin and appropriate action taken when necessary.

     4.6  Feed in containers found open during inspections shall not
be used.

     4.7  If pesticides are used in the animal facility, supplies of
feed and bedding shall be analyzed at monthly intervals.  Results of
all analyses shall be reported immediately to program management who
will notify the bioassay Laboratory of any lots unsuitable for use.

                           5.  PACKAGING

     5.1  Feed shall not be shipped or stored in plastic containers.

     5.2  Feed containers must be sealed to prevent contamination
during transit.  Broken or repaired containers of  feed shall be
rejected.

                              6.  NOTES

     6.1  Feed that is older than 90 days may be unsatisfactory due
to loss of essential nutrients.

     6.2  Plastic materials are unsatisfactory for feed containers
since they melt during autoclaving and may, under  certain environmental
conditions, provide conditions favorable for the growth of molds.

                      7.  REFERENCE DOCUMENTS

     7.1  Long-Term Holding of Laboratory Rodents. 1976.  ILAR  News  XIX
(4), L20, L21.
                                  B-27

-------
Carcinogen Bioaseay Program               Specification No. CBO-11
Subject: STORAGE OF PEED, BEDDING, AND EQUIPMENT
FOR LABORATORY ANIMALS
Date:
Sheet
3
Of
3
     7.2  Code of Federal Regulations, Title 9, Chapter 1, Subchapter A.
Animal Welfare.  Parts 1, 2 and 3, May, 1972.

     7.3  Guide for the Care and Use of Laboratory Animals. 1974. U.S.
Department of Health, Education and Welfare, NIH 74-23.

     7.4  The UFAW Handbook on the Care and Management of Laboratory
Animals. 1972. 4th edition. UFAW Staff (eds.).  Churchill Livingstone,
Edinburgh and London.

     7.5  Tracer Jltco Subcontract for Carcinogen Bioassay with Industrie
Bio-Test Laboratories, Inc., Subcontract No. 76-33-106002, April 19,
1976.

     7.6  Tracer Jltco Subcontract for Carcinogen Bioassay with Battelle-
Columbus Laboratories, Subcontract No. 76-34-106002, April 8, 1976.
                                 B-28

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     Carcinogen  Bloassay  Program
Specification No. CBO-12
Type:
GOOD
ANIMAL
CARE
LABORATORY
PRACTICE
Sheet
1
Of
3
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject: DOSE PREPARATION AND ANALYSIS  FOR CHEMICALS  TO  BE  ADMINISTERED  BY
              PROCEDURES  OTHER THAN  INHALATION
                                  1.   SCOPE

          This  specification covers mixing  of  the  test  chemical with  feed
     or  other carrier,  storage,  and analysis of  the mixture  for concentration,
     homogeneity,  and  stability.
          None
                           2.   APPLICABLE DOCUMENTS
                              3.   REQUIREMENTS
          3.1   A procedure for mixing test  chemical with  feed  or vehicle
     which will Insure homogeneity of dose  preparations shall  be developed
     by  the analytical subcontractor  prior  to  bloassay.

          3.2   The  stability  and storage parameters of each  test chemical
     also  shall be  determined by the  analytical  subcontractor  prior  to  Its
     bloassay.

          3.3   The  bloassay laboratory shall follow the mixing procedure,
     storage conditions,  and  frequency of dose preparation recommended  by
     the analytical subcontractor.  Any difficulties with or deviations
     from  these procedures shall be reported promptly  to  program management.

          3.4   The  bloassay laboratory shall analyze all  dosage mixtures by
     procedures developed by  the analytical subcontractor.

               3.4.1  A sample of each dose-feed mixture  and stock liquid
     mixture (highest level only for  the latter) during  the  chronic  study
     at  time of mixing shall  be stored In Individual labelled  and  sealed
     containers at  5° C or lower.

               3.4.2  One-eighth of the chronic  test samples,  selected
     randomly  and blind to dosage preparation  personnel,  shall be  analyzed
     by  the bloassay laboratory immediately after mixing  or  no more than  one
     week  later.

               3.4.3  During the sub-chronic  study,  a  single sample at  each
     level will be  analyzed to demonstrate  efficiency  of  the mixing procedure
     and of the analytical method.
                                      B-29

-------
 Carcinogen  Bioassay  Program                Specification  No. CBO-12
Subject: DOSE PREPARATION AND ANALYSIS FOR CHEMICALS |Da**:
TO BE ADMINISTERED BY PROCEDURES OTHER THAN INHALATIOl
Sheet
2
Of
3
           3.4.4   Analysis  shall  consist  of determination of  the
 concentration  of  test  chemical in dose mixture  to  Insure accuracy
 of weighing and mixing processes as well as stability of the chemical.

           3.4.5   Analytical  methodology  normally will consist of
 re-Isolation of test chemical from the dose mixture and spectroscoplc
 or chromatographic  analysis  by a procedure developed for each test
 chemical Individually.

           3.4.6   Analytical  values which differ from that of the expected
 concentration  by  more  than 10% shall be  considered out of tolerance and
 shall  not  be given  to  test animals (cause of deviation will  be discussed
 in the analytical report).   Replacement  preparations shall be analyzed
 immediately.   Unanalyzed samples shall be discarded 90 days  after mixing.

           3.4.7   Analytical  results will be reported to the  Principal
 Investigator immediately.  Copies of results will be submitted to
 program management  as  Indicated  on section Reports.

     3.5   An Inventory of  each dosage mixture shall be maintained on
 a current  basis.  Preparation date, amount prepared, usage dates,
 amounts used,  and names of responsible personnel shall be Included.

     3.6   Any  Instability  of chemical In dose mixture shall  be reported
 immediately to program management.

                           4.  QUALITY CONTROL

     4.1   Prior to  the  bioassay, the analytical subcontractor shall
 document:

           4.1.1   Homogeneity of  dose preparation of test chemical
 according  to the  mixing procedure developed.

           4.1.2   Stability of the test chemical under conditions of
mixture with feed or vehicle and storage.

     4.2  All analytical Instruments used by the analytical  subcontractor
and bioassay laboratory shall be re-calibrated monthly.  All recalibra-
 tlon data  shall be  recorded  in a bound notebook maintained for the
purpose and signed  by personnel  and supervisor Involved.

     4.3  All temperature charts of refrigerated storage for dose
preparations throughout the course of study shall be dated,  signed,
and filed  for audit.  Thermometers shall be re-calibrated monthly and
data filed as in 4.2 above.
                                 B-30

-------
Carcinogen Bioassay Programs              Specification No. CBO-12
Subject: DOSE PREPARATION AND ANALYSIS FOR CHEMICALS (Date:
TO BE ADMINISTERED BY PROCEDURES OTHER THAN INHALATIOB
Sheet
3
Of
3
                             5.  PACKAGING

     Not Applicable

                      6.  REFERENCE DOCUMENTS

     6.1  Sontag, J. M., N. P. Page, and U. Safflottl. 1976. Guidelines
for Carcinogen Bioassay In Small Rodents.  U. S. Department of Health,
Education and Welfare, NIH 76-801.
                                 B-31

-------
     Carcinogen Bioassay Program
Specification No. CBO-13
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
1
Of
2
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
             FEEDING OF LABORATORY ANIMALS
                                  1.   SCOPE
          This specification covers the feeding procedures and requirements
     for feeders.
                           2.   APPLICABLE DOCUMENTS
          None
                               3.   REQUIREMENTS
          3.1  FEEDING PROCEDURES
               3.1.1  Provide feed as  often as necessary, but not  less  than
     once weekly,  to assure an adequate supply of  fresh rations.
               3.1.2  Supply sanitized feeder at least  once weekly.
               3.1.3  Analyze the  feed for  pesticide, mycotoxin, and
     industrial contaminants periodically.
               3.1.4  Sterilize feed whenever practical and consistent  with
     the disease control program.
               3.1.5  Care should  be taken  that nutrients are not  degraded or
     the palatability of the feed  altered.
          3.2  REQUIREMENTS FOR FEEDER
               3.2.1  Feeder shall be  accessible to  all animals.
               3.2.2  Feeder shall be  located so as  to  minimize contamination
     by excreta.
               3.2.3  Feeder shall be  durable and  kept  clean.
               3.2.4  Sanitize feeder  at least once  every two weeks.
               3.2.5  Discard disposable feeder after each feeding.
                               4.   QUALITY  CONTROL
          4.1  Nutrient Analysis.   Collect  random  feed  samples  quarterly and
     analyze in accordance with the AOAC methods of  analysis  (Association of
     Official Analytical Chemists, 1975).
                                      B-32

-------
 Carcinogen Bioassay Program               Specification No. CB0-1 '
Subject:
FEEDING OF
LABORATORY ANIMALS
Date:
Sheet
2
      4.2   Retention of Feed Samples.                                       <
           4.2.1  Retain a 500 to 800 g sample from each production ha  •   j
.of  feed used.                                                              j
           4.2.2  Store in freezer or in sealed containers placed in a     |
 cool,  dry, area,  for the duration of the experiment invovled.              j
      4.3   Microbiologic Monitoring
           It is recommended that periodic sterilizer runs be moniu :  1  *..•
 assure that vegetative forms of microorganisms have been killed.  'i'-ij..,
 may be most easily accomplished by placing a filter paper strip inn   g     s
 nated with Escherichia coli in the center of load.  The strip is t'- i     \
 incubated in a suitable medium and examined for growth.  Food nay '   nel i '
 in  a clean storage area until culture results are available.

      4.4   A program of periodic assay for the chemical contaminant'  than
 may interfere with results of a particular study is recommended.
           4.4.1  If unacceptable concentrations are detected,  a c»v" ue
 in  ration or source may be in order.

      4.5   A continuing pest control program is essential in the fond
 storage area.

                            5.  PACKAGING

      N/A

                       6.  REFERENCE DOCUMENTS

      6.1   Sontag, J. M., N. P. Page,  and U. Saffiotti. 1976.  Gulch '
 for Carcinogen Bioassay in Small Rodents.  U. S. Department of Heal Mi.
 Education and Welfare, NIH 76-801.

      6.2   Long-Term Holding of Laboratory Rodents. 1976.  ILAR Hews VT
 (4), L20, L21.

      6.3   Code of Federal Regulations, Title 9, Chapter 1, Subchapt
 A.  Animal Welfare. Parts 1, 2 and 3,  May, 1972.
                                  B-33

-------
     Carcinogen Bioassay Program
Specification No.  CBO-14
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
l
Of
3
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:  GENERATION AND ANALYSIS OF TEST ATMOSPHERES  OF CHEMICALS
              EVALUATED BY THE INHALATION METHOD PROGRAM
                                  1.   SCOPE

          This specification covers generation of test atmosphere of
     chemicals studied by the inhalation method in the Carcinogen
     Bioassay Program together with analytical and control procedures
     employed.
          None
                          2.   APPLICABLE DOCUMENTS
                               3.   REQUIREMENTS
          3.1  Gases shall be introduced into the main chamber air supply
     by means of a pressure regulator in combination with a flowmeter and
     mixed with the air supply by turbulence in the mixing chamber prior
     to actual introduction into the exposure chamber.

          3.2  Inhalation test preparations of liquids shall be generated by
     bubbling clean (charcoal and HEPA-filtered), dry air (-40° C dewpoint)
     through all-glass impingers containing the test chemical.

          3.3  The concentration of test chemical in exposure chamber shall
     be monitored continuously by means of an automated sampling system.
     Sampling from a single port will suffice if preliminary data demon-
     strated uniform concentration of test chemical throughout the chamber.

          Chamber concentration of the agent shall be calculated also
     from data on mass transfer from generator and flow rate through
     chamber as a backup method.

          3.4  The exposure chamber atmosphere shall be maintained at a
     temperature of 23.3° + 1.1° C (74° + 2° F) and 50 + 5% relative
     humidity.  Chamber pressure shall be negative (approximately 0.5-1.0
     cm H20) in relation to the room pressure.  Air flow rate, temperature,
     and humidity shall be monitored continuously and recorded.  Air
     pressure shall be recorded at least daily.

          3.5  Chamber temperature shall be determined at two locations at
     least by remote sensors.  Air flow rates shall be controlled by
                                      B-34

-------
Carcinogen Bioassay Program               Specification No. CBO-14
Subject: GENERATION AND ANALYSIS OF TEST ATMOSPHERES
OF CHEMICALS EVALUATED BY THE INHALATION METHOD PROG&
Date:
M
Sheet
2
Of
3
precision rotameters, calibrated pressure-drop orifices, and mass
flowmeters.

     3.6  The exposure chamber shall be equipped with an emergency
alarm system for detection of all significant deviations from test
limits for air flow, chamber pressure, temperature, and test chemical
concentration.  Laboratory personnel also shall carefully observe all
chamber instruments throughout the study.

     3.7  Uniformity of test chemical throughout the exposure chamber
shall be documented during development of the exposure technique and
again at beginning of the bioassay with animals in the chamber.

     3.8  The exposure chamber atmosphere shall be checked for the
absence of test chemical during non-exposure periods prior to the
bioassay and at intervals during the study.  Appropriate action shall
be taken to insure the absence of the agent during non-exposure
periods if necessary.

     3.9  If stability of test chemical is questionable, the chamber
atmosphere shall be tested for known or suspected degradation
products at intervals during the study.

     3.10  When liquid chemicals are tested in the form of a molecular
vapor rather than as an aerosol, the test atmosphere shall be tested
by photometric or other appropriate means to insure the absence of
significant concentration of particulates of the agent.

     3.11  Exhaust test atmospheres from the chamber shall first be
passed through an appropriate scrubber for the test chemical and
then through filters in the common exhaust vent to an outside stack,
and finally through a second air scrubber.

     Effluent air stacks shall be sampled daily for air concentration
of the test chemical.

                         4.  QUALITY CONTROL

     4.1  The following equipment shall be calibrated at least monthly
by qualified technicians:  pressure regulators, flowmeters, temperature
sensors, rotameters, photometers, pressure-drop orifices, and automated
sampling system for determination of test chemical concentration.  All
calibration data shall be dated and recorded in a bound notebook main-
tained for the purpose and signed by personnel and supervisors involved.
                                 B-35

-------
Carcinogen Bioassay Program
Specification No.  CBO-14
Subject: GENERATION AND ANALYSIS OF TEST ATMOSPHERES
OF CHEMICALS EVALUATED BY THE INHALATION METHOD PROG
Date:
AM
Sheet
3
Of
3
     4.2  The emergency alarm system for indicating significant
deviation from test parameters shall be tested weekly and results
recorded and signed as in 4.1 above.

     4.3  Bioassay supervisors shall carefully supervise all steps in
the bioassay study to make certain that all procedures are in
compliance with Subcontract regulations and "NCI Guidelines for
Carcinogen Bioassay in Small Rodents" including Appendix C - Safety
Standards for Research Involving Chemical Carcinogens.

     4.4  All pertinent data in "NCI Guidelines" - Appendix F -
Carcinogen Bioassay Information - shall be collected and reported in
accordance with the Carcinogenesis Bioassay Data System (CBDS)
procedures.
Not Applicable
                           5.  PACKAGING
                       6.   REFERENCE DOCUMENTS
     6.1  Sontag, J. M., N. P. Page, and U. Saffiotti. 1976. Guidelines
for Carcinogen Bioassay in Small Rodents. U. S. Department of Health,
Education and Welfare, NIH 76-801.
                                 B-36

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     Carcinogen Bioassay Program
Specification No.  CBO-15
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
i
Of
3
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
             WATERING  OF LABORATORY ANIMALS
                                  1.   SCOPE

          This  specification  covers the  operations  in the watering of
     laboratory animals  and the  product  requirements for water bottles,
     bottle  stoppers,  and  sipper-tubes.
     None
                           2.  APPLICABLE DOCUMENTS
                               3.   REQUIREMENTS
          3.1   Procedure requirements  for watering of laboratory animals.
    Watering  bottles  may be used  although  an  automatic watering system
    is  preferred.

               3.1.1  Provide the  animals with an adequate supply of fresh
    and treated water ad libitum.

               3.1.2  Check to ensure  that  the water bottles are accessible
    to  all  animals.

               3.1.3  Supply sanitized water bottles, stoppers, and sipper-
    tubes at  least  twice weekly.

               3.1.4  Wash dirty water bottles promptly in a washer contain-
    ing at  least  one  cycle of water at  180°F  or higher.

               3.1.5  Sanitize bottle  stoppers by a germicide  treatment prior
    to  washing, by  boiling  after washing, or by autoclaving.

               3.1.6  Sterilize sipper tubes by a germicide  treatment prior
    to  washing, or  by boiling after washing.

               3.1.7  Fill the bottles and  insert the stoppers and sipper
    tubes into the  bottles only outside of the animal rooms.

               3.1.8  Replace empty  or partially full water  bottles
    instead of refilling them.

               3.1.9  Locate water bottles  in  a position  to  prevent the
    stoppers  from being chewed by the animals.

               3.1.10   Routinely examine watering device  to  assure  their
    patency and use by the animals.
                                       B-37

-------
Carcinogen Bioassay Program               Specification No. CBO-15
Subject:
WATERING OF LABORATORY ANIMALS
Date:
Sheet
2
Of
3
          3.1.11 Kill potential pathogens carried in the water or
remove them through appropriate treatment, such as sterilization,
pasteurization and filtration.

          3.1.12  Periodically assay drinking water for compounds
that may influence experimental data (see American Public Health
Association, Inc. 1971).

          3.1.13  If automatic watering system is used, overall labor
involved with changing, washing and filling water will be reduced.
However, nozzles shall be inspected daily in order to ensure that they
are functioning properly.

          3.1.14  Water supply for the automatic watering device shall
be treated.

     3.2  Product requirements for watering equipment.

          3.2.1  Water bottles.  It should be made of glass or plastic
with large openings and smooth surface.  500-ml capacity size is
practical.

          3.2.2  Sipper tubes should be made of stainless steel.  The
internal diameter of the sipper-tube should be 6 to 9mm and that of the
terminal aperture about 3mm.
          3.2.3  Stopper.  One-hole-rubber-stopper is practical.  It
shall be protected by a suitable device to prevent gnawing by the
animals.

          3.2.4  Nozzles of the automatic watering system shall be
capable of being rapidly disassembled for cleaning.
          3.2.5  Valves of the automatic watering system shall be
capable of 100 percent operative efficiency at all times.

                        4.  QUALITY CONTROL

     4.1  Microbiologic monitoring of water.

                      5.  REFERENCE DOCUMENTS

     5.1  Guide for the Care and Use of Laboratory Animals. 1974. U.S.
Department of Health, Education and Welfare, NIH 74-23.

     5.2  Sontag, J.M., N.P. Page, and U. Saffiotti. 1976.  Guidelines
for Carcinogen Bioassay in Small Rodents. U.S. Department of Health,
Education and Welfare, NIH 76-801.
     5.3  Long-Term Holding of Laboratory Rodents. 1976. ILAR News XIX
(4), L20, L21.
                                 B-38

-------
Carcinogen Bioassay Program
Specification No. CBO-15
Subject:
WATERING OF LABORATORY ANIMALS

Date:


Sheet

3
Of

3
     5.4  The UFAW Handbook on the Care and Management of Laboratory
Animals. 1972. 4th edition.  UFAW Staff (eds.).  Churchill Livingstone,
Edinburgh and London.

     5.5  Workshop on Criteria for Successful Rodent Chronic Studies.
National Cancer Institute, National Institutes of Health, Bethesda,
Maryland, April 4-5, 1973.
                                  B-39

-------
     Carcinogen Bioassay Program
      Specification No. CBO-16
Approved:
Proj.
Q.C.
Lab
Other
Date
Type:
GOOD
ANIMAL CARE LABORATORY PRACTICE
Sheet
l
Of
4
Subject:  CHANGING OF LITTER OR BEDDING,  CHANGING OF LABORATORY ANIMAL
          CAGES AND DISPOSAL OF WASTE
                                   1.   SCOPE

          This specification covers changing of  litter  or bedding and
     laboratory animal cages and  disposal  of wastes  from small  rodents
     used  in the Carcinogen Bioassay Program

                           2.   APPLICABLE  DOCUMENTS

          2.1  Code  of Federal  Regulations,  Title  9,  Chapter  1,  Subchapter
          A.  Animal  Welfare.  Parts  1,  2, and 3,  May,  1972.

          2.2  Specification No.  CBM-6  Laboratory  Animal Cages and Cage
                                        Filters
          2.3   Specification No. CBM-7
          2.4   Specification No. CBO-4
Feeders for Laboratory Animals

Safety Standards for Research
Involving Chemical Carcinogens
                              3.  REQUIREMENTS

         3.1  Provision  shall be made  for prompt removal and disposal of
         all  food wastes from laboratory animal cages so as to minimize
         vermin  infestation,  odors, and disease hazards.

         3.2  Measures must be taken to prevent molding, contamination,
         deterioration,  or caking of feed.  Uneaten fruit or vegetable
         supplements must not be allowed to accumulate in animal cages.
         3.3  Litter or  bedding shall  be removed from cages as necessary
         to keep the animals  clean and dry, and to minimize offensive
         odors.  One to  three changes  per week will suffice for small
         rodents.  Cages shall be emptied in an area set aside for  the
         purpose away from the animal  rooms.
         3.4  Catch-pans for  animals caged in exposure chambers shall be
         cleaned and relined  with new  absorbent paper daily.

         3.5  Individually caged animals in exposure chambers shall be
         changed to a sanitized stainless steel wire mesh cage weekly.

         3.6  Animals housed  in polycarbonate cages shall be changed to
         a sanitized cage with fresh bedding at least twice weekly
         excepting when  the cage population falls to one or two animals
         when one weekly cage change is permissible.
                                     B-40

-------
Carcinogen Bioassay Program               Specification No. CBO-16
Subject: CHANGING OF LITTER OR BEDDING, CHANGING OF
LABORATORY CAGES AND DISPOSAL OF WASTE
Date:
Sheet
2
Of
4
     3.7  Cages shall be chemical specific.  They shall be returned to
     the same chemical group and dose level to prevent test chemical
     contamination.

     3.8  All waste shall be collected and removed in a safe, sanitary
     manner.  Cage waste may be removed to storage by vacuum.  If waste
     cans are used, they should be made of metal or plastic and shall
     be leakproof and equipped with tight-fitting lids.

     3.9  Waste material should be removed regularly and frequently.
     Waste which must be stored before final disposal shall be kept
     in an area maintained at a temperature of 7° C (45° F) or less.
     The storage area shall be separated from any other cold storage
     and shall be used exclusively for refuse storage.  The area must
     be kept clean and free of vermin.

     3.10  Wastes which are contaminated with chemical carcinogens shall
     be disposed of in accordance with applicable NCI and other Federal
     safety regulations (see 5.1).

     3.11  Infectious wastes should be autoclaved or rendered noninfec-
     tious by other effective measures before removal from the animal
     facility.

     3.12  Wastes which are not contaminated with carcinogens or
     infectious agents may be disposed of at a public incinerator or
     burned at the facility.  Incineration shall comply with Environ-
     mental Protection Agency regulations.

          3.12.1  The incinerator shall be located in such a position
          that stack vapors, fumes, and particulate matter will not be
          drawn into air-handling intake vents.
          3.12.2  Stacks shall be of design which prevents emission of
          fly ash.
     3.13  Waste disposal shall comply with all Federal, state, and
     municipal laws, statutes, or ordinances.

                        4.  QUALITY CONTROL

     4.1  Animal cages shall be inspected daily and litter or bedding
     changed as frequently as necessary, but not less than once per
     week, to comply with requirements set forth in Section 3 above.

     4.2  Animal care personnel shall be responsible  for changing
     animals to sanitized cages with fresh bedding as indicated in
     3.5 and 3.6.
                                 B-41

-------
Carcinogen Bioassay Program               Specification No. CBO-16
Subject: CHANGING OF LITTER OR BEDDING, CHANGING OF
LABORATORY ANIMAL CAGES AND DISPOSAL OF WASTE
Date:
Sheet
3
Of
4
     4.3  Supervisors shall monitor removal and disposal of all wastes
     containing chemical carcinogens or infectious agents to make
     certain that all procedures are in compliance with applicable
     Federal, state, and local laws and regulations of the NCI Office
     of Research Safety.

     4.4  Commercially available spore strips shall be included in
     all autoclave loads of infectious waste and subsequently cultured
     to monitor the efficacy of the sterilization procedure.

     4.5  Data pertaining to the disposal of infectious wastes or
     wastes containing chemical carcinogens shall be entered in a
     bound notebook, dated, and signed by personnel involved and the
     supervisor.

                           5.   PACKAGING

     5.1  Food and other wastes contaminated with chemical carcinogens
     shall be placed into separate plastic bags or other suitable
     impermeable containers for each carcinogen, closed,  sealed,  and
     labelled with both name of carcinogen and "DANGER 	 CHEMICAL
     CARCINOGEN",  before being transported to storage or disposal
     area.   Final  disposal shall be in conformance with Federal,
     state,  and local laws and with the Office of Research Safety
     Regulations.

                        6.   REFERENCE DOCUMENTS

     6.1  Sontag,  J.  M., N.  P.  Page,  and U.  Saffiotti.  1976.   Guidelines
     for Carcinogen Bioassay in Small Rodents.  U.  S.  Department of
     Health,  Education  and Welfare,  NIH 76-801.

     6.2  Request  for Proposal  76-S-12, Carcinogen Bioassay,  NCI
     Program,  Due  date  June 15,  1976,  Tracer Jitco,  Inc.,  Rockville,
     Maryland.

     6.3  Carcinogen Bioassay  Subcontract with Industrial  Bio-Test
     Laboratories,  Inc.,  Subcontract  No.  76-33-106002,  Apr.  19, 1976.

     6.4  Guide for  the Care and Use  of Laboratory Animals.  1974.  U.  S.
     Department  of Health,  Education  and Welfare,  NIH 74-23.

     6.5 The UFAW Handbook  on  the  Care and  Management  of  Laboratory
    Animals. 1972.  4th  edition.  UFAW  Staff  (eds.).  Churchill Living-
     stone, Edinburgh and  London.

     6.6  Procurement Specification IX.   Defined Laboratory  Rodents and
     Rabbits. 1973.  Institute of  Laboratory  Animal Resources,  National
    Academy of  Sciences,  National Research  Council,  Washington, D. C.
                                 B-42

-------
    Carcinogen Bioassay Program
Specification No.  CBO-16
Subject:  CHANGING OF LITTER OR BEDDING,  CHANGING OF
LABORATORY ANIMAL CAGES AND DISPOSAL OF WASTE
        Date:
Sheet
Of
         6.7  Long-Term Holding of Laboratory Rodents.  1976.  ILAR News XIX
         (4), L20, L21.
                                     B-43

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il 3 '.
.•inogen Bioassay Program
                                          Specification No.  CBO-17
•I' ANIMAL CARE LABORATORY PRACTICE
                                                          Sheet
                                                             l
                                                                Of
 'TENANCE OF OPTIMAL ENVIRONMENTAL CONDITIONS FOR LABORATORY ANIMALS
                  Q.C.
                                  Lab
Other
Date
                           1.  SCOPE

  This specification covers temperature, ventilation, lighting,
 ti control, and maintenance of animal facilities used in the
 inogen Bioassay Program.

                    2.  APPLICABLE DOCUMENTS

  2.1  Code of Federal Regulations, Title 9, Chapter 1, Subchapter
  A.  Animal Welfare.  Parts 1, 2, and 3, May, 1972.
  2.2  Specification No. CBM-4

  2.3  Specification No. CBO-4

  2.4  Specification No. CBM-1
                                      Air Filters for Carcinogen
                                      Bioassay Facilities
                                      Safety Standards for Work
                                      Involving Chemical Carcinogens
                                      Construction of the Physical  Plant
                        3.  REQUIREMENTS

  3.1  Temperature and humidity

       3.1.1  Each animal room or group of rooms with a common purpose
       shall have individual temperature and humidity controls.

       3.1.2  The heating-cooling-ventilation system of the animal
       facility shall be sensitive to permit adjustments within +
       1°C for any temperature within the range of 18° to 29° C
       (65° -85°F).

       3.1.3  A temperature of 23.3° C ± 1.1°C (74°F + 2°F) shall
       be maintained in all mouse and rat rooms.

       3.1.4  The optimum temperature for hamsters is 20-24°C.
       According to Federal regulations, the ambient air temperature
       in rooms where these rodents are quartered shall not be less
       than 15.6°C (60°F) or greater than 29.4°C (85°F).

       3.1.5  A relative humidity of 40% + 5% shall be maintained
       in all mouse and rat rooms.

       3.1.6  The relative humidity for hamsters shall be 40-45%.

       3.1.7  An automatic recording and alert system shall be used
       to monitor the ambient temperature and relative humidity in
       each animal room.
                              B-44

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Carcinogen Bioassay Program               Specification No. CBO-17
Subject: MAINTENANCE OF OPTIMAL ENVIRONMENTAL
CONDITIONS FOR LABORATORY ANIMALS
Date:
Sheet
2
Of
7
          3.1.8  An emergency power source shall be available with a
          capacity sufficient for the air conditioning and light
          systems of the animal facility.

     3.2  Ventilation

          3.2.1  Each animal room shall have 10-15 fresh-air changes per
          hour without drafts.

          3.2.2  All air shall be adequately filtered (Specification No.
          CBM-4) before entering and before discharge from the animal
          facility.

          3.2.3  The general exhaust air from areas where chemical
          carcinogens are used is subject to Federal regulations
          (Specification No. CBO-4).

          3.2.4  Recirculation of exhaust air from rooms where chemical
          carcinogens are used is not permitted (Specification No. CBO-
          4).

          3.2.5  Air pressure shall be adjusted so that all animal rooms
          are slightly positive to the "dirty" corridor and "negative"
          to the "clean" corridor.  Rooms bordering a single access
          corridor shall be kept under negative pressure with respect
          to the corridor.

          3.2.6  The animal facility and human occupancy areas shall have
          separate ventilation systems.

     3.3  Lighting

          3.3.1  Housing quarters for laboratory animals shall have ample
          light of good quality which is uniformly diffused throughout
          the area.

          3.3.2  Light intensity at the cage level shall be a minimum of
          100 foot-candles.

          3.3.3  Examination and animal treatment areas shall have a
          minimum light intensity of 125 foot-candles at the work
          surface.

          3.3.4  Continuous strip fluorescent lighting mounted flush
          in the ceiling is recommended.  Fixtures shall be properly
          sealed to prevent the harboring of vermin.

          3.3.5  Convenience outlets should be waterproof, recessed  in
          walls and partitions, and located a minimum of 0.6m  (2  ft)
          above the floor.
                                   B-45

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Carcinogen Bioassay Program               Specification No. CBO-17
Subject: MAINTENANCE OF OPTIMAL ENVIRONMENTAL
CONDITIONS FOR LABORATORY ANIMALS
Date:
Sheet
3
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7
          3.3.6  Animal cages and other primary enclosures shall be
          positioned so as to protect the animals from excessive
          illumination.

          3.3.7  A time-controlled system to provide regular diurnal
          lighting cycle shall be provided.  Controls shall be located
          in the main control room.

          3.3.8  Provisions must be made to provide hamsters with a
          lighting period of approximately 12 hours which is somewhat
          less than the optimum for other small rodents.

          3.3.9  Light switches should be located outside each room in
          both clean service and evacuation corridors.

          3.3.10  Lights should be serviced via a crawl space or other
          method which does not necessitate entering the room.

     3.4  Noise control

          3.4.1  Laboratory rodents, particularly mice, shall be
          protected from noise, especially high pitch noise (upper
          limits of human auditory range and beyond).  Audiogenic
          strains must be maintained at very low noise levels.

          3.4.2  All noisy operations in the animal facility, such as
          cage and rack cleaning and washing, etc., shall be carried
          out in an area separate from rooms where laboratory animals
          are housed.

          3.4.3  Animals shall not be caged near incompatible species
          which disturb or distress them.

          3.4.4  Carts, trucks, racks, and other moveable equipment!
          used in animal quarters should have rubber-tired casters
          and rubber bumpers.

          3.4.5  Concrete walls are preferred over metal or plaster
          construction to contain noise in animal quarters.  Acoustical
          tile and similar materials should be used wherever possible
          to reduce the effect of "noise pollution" in animal rooms.

     3.5  Facility maintenance

          3.5.1  The operation of all animal facilities shall conform
          with the requirements of PL 91-579 (Animal Welfare Act, 1970),
          the amendment to PL 89-544.

          3.5.2  Sanitation

               3.5.2.1  Premises (building and grounds) shall be kept
               clean.
                                  B-46

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    Carcinogen Bioassay Program               Specification No.   CBO-17
Subject: MAINTENANCE OF OPTIMAL ENVIRONMENTAL
           CONDITIONS FOR LABORATORY ANIMALS
Date:
Sheet
   4
Of
  7
                        3.5.2.1.1   Sanitization  and  sterilization  of  rooms
                        and  corridors.

                          3.5.2.1.1.1   Room and corridor  floors,  sinks,
                          and  pipes  shall be washed with  a microbicidal
                          solution weekly.   Ceilings, walls, and  partitions
                          shall be treated  in a like manner at regular
                          intervals.

                          3.5.2.1.1.2   After a  room has been emptied of
                          animals, all  surfaces and fixed equipment  shall
                          be washed  with a  microbicidal solution.  Portable
                          equipment  for the room shall be sanitized  and
                          sterilized, returned  to the room, and the  room
                          equipment  fumigated.   Paraformaldehyde  is  recom-
                          mended  for this purpose.

               3.5.2.2   Primary enclosure shall  be cleaned and sanitized often
               enough to prevent an accumulation of  excreta, debris,  dirt and
               harmful  contamination.

                        3.5.2.2.1   It shall  be sanitized by washing with hot
                        water  (180°F) and soap or detergent, or by washing
                        all  soiled  surfaces  with a detergent solution follow-
                        ed by a safe  and effective disinfectant, or by clean-
                        ing  all soiled surfaces  with live steam.

             3.5.2.3   All  wastes  should be collected and removed  regularly
                        and  frequently in a  safe sanitary manner.  For
                        example:  highly infectious  waste should be rendered
                        noninfectious, by autoclaving or other effective
                        means,  before removing it from the animal  facility.

               3.5.2.4   Most states or municipalities have statutes or
               ordinances controlling disposal of wastes.  Compliance with
               these  requirements is an  institutional responsibility.
             3.5.3   Inspection and repair

               3.5.3.1   Inspection  of automatic  watering system.

                  3.5.3.1.1 Nozzles shall  be inspected daily in  order to
                  assure that  they are  functioning  properly.

                  3.5.3.1.2 All pipings between filters and house supply
                  lines shall  be dismounted quarterly, thoroughly cleaned,
                  and  sterilized.

                  3.5.3.1.3 The pressure control and supply tank for each
                  rack or group of racks shall  be cleansed and sterilized
                  semiannually.
                                     B-47

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Carcinogen Bioassay Program               Specification No.  CBO-17
Subject: MAINTENANCE OF OPTIMAL ENVIRONMENTAL
CONDITIONS FOR LABORATORY ANIMALS
Date:
Sheet
5
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7
          3.5.3.2  Maintain facility in good repair,  to protect the
          animals from injury, to contain the animals,  and to restrict
          the entrance of other animals.

                   3.5.3.2.1  Check for sharp corners and edges, broken
                   wires, etc.

                   3.5.3.2.2  Check the walls, doors, ceilings and
                   corners of cracks.

          3.5.3.3  Check drainpipe, electric power and water supply.

          3.5.3.4  Check machines such as washing machines,  autoclave,
          etc.

                        4.  QUALITY CONTROL

     4.1  Temperature and humidity

          4.1.1  The temperature and relative humidity record charts  for
          each 24-hour period throughout each bioassay test shall  be
          dated, signed, and filed for audit.

          4.1.2  The automatic devices for recording temperature and
          relative humidity shall be recalibrated monthly.  All pertinent
          data shall be entered in a bound notebook and signed by
          technical personnel who performed the work, and by the
          supervisor.

          4.1.3  The alert and emergency power systems shall be tested
          at monthly intervals and results recorded as in 4.1.2 above.

     4.2  Ventilation

          4.2.1  A maintenance check on all mechanical ventilation
          equipment (air conditioner, blowers, fan motors, etc.) shall
          be made monthly.
          4.2.2  Air intake and discharge filters shall be inspected  at
          least monthly and replaced when necessary.

          4.2.3  Air pressure of animal rooms with regard to entrance
          and egress corridors shall be checked, and adjusted if necessary
          each day.

          4.2.4  The number of fresh-air changes per hour in animal
          rooms shall be monitored at least weekly and appropriate
          adjustments made when indicated.

          4.2.5  The concentration of chemical carcinogens in discharge
          air must be determined as indicated in Specification No. CBO-4.
                                 B-48

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Carcinogen Bioassay Program               Specification No. CBO-17
Subject: MAINTENANCE OF OPTIMAL ENVIRONMENTAL
CONDITIONS FOR LABORATORY ANIMALS
Date:
Sheet
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          4.2.6  All data pertaining to the above shall be entered in a
          bound notebook and signed by personnel involved.

     4.3  Lighting

          4.3.1  Light intensity at cage level and at the work surface
          in animal examination and treatment areas shall be determined
          weekly and adjusted if necessary.

          4.3.2  Instruments for determining light intensity shall be
          calibrated monthly.

          4.3.3  The light cycle shall be monitored regularly and
          adjusted if necessary  to provide diurnal cycle for the
          species in question.

          4.3.4  The position of animal cages with respect to the light
          source shall be checked regularly to make certain that  animals
          are not subjected to excessive illumination.

          4.3.5  All test results and observations above shall be entered
          in a bound notebook and signed by personnel involved.

     4.4  Noise control

          4.4.1  Evaluation of noise control practices  shall be included
          in all Inspections of the laboratory animal facility and
          remedial measures instituted where necessary.

     4.5  Facility maintenance

          4.5.1  Periodic inspection of facilities.

          4.5.2  Microbiologic monitoring of room surfaces,  including
          benches,  walls, and ceilings,  should be done  on a routine
          basis,  but frequency depends on the desired level of protec-
          tion.

          4.5.3  Monitor for radiologic,  toxicologic and infectious
          agents.

                           5.   PACKAGING

     Not applicable

                              6.   NOTES

     6.1  It should be noted that the relative humidity in the immediate
     vicinity of  an animal in a cage (microenvironment)  may be much high-
     er  than that of the animal room itself.
                                 B-49

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Carcinogen Bioassay Program               Specification No. CBO-17
Subject: MAINTENANCE OF OPTIMAL ENVIRONMENTAL
CONDITIONS FOR LABORATORY ANIMALS
Date:
Sheet
7
Of
7
      6.2  Humidification may be necessary in cold months to maintain
      the humidity optimal for small laboratory animals.

      6.3  DBA mice and certain other stocks are very susceptible to
      audiogenic seizures.
      6.4  Convulsions have been produced even in audiogenic seizure-
      resistant stocks of mice by a single explosion of intense sound.

      6.5  Congenital malformations have been induced in one or more
      animal species by audiovisual stimulation.  Certain types of
      noise pollution could possibly alter other types of experimental
      results as well.

                      7.  REFERENCE DOCUMENTS

      7.1  Sontag, J. M., N. P. Page, and U. Saffiotti. 1976.  Guidelines
      for Carcinogen Bioassay in Small Rodents.  U.S. Department of
      Health, Education and Welfare, NIH 76-801.
      7.2  Guide for the Care and Use of Laboratory Animals. 1974.  U.S.
      Department of Health, Education and Welfare, NIH 74-23.

      7.3  Long-Term Holding of Laboratory Rodents. 1976. ILAR News XIX
      (4), L20, L21.
      7.4  The UFAW Handbook on the Care and Management of Laboratory
      Animals. 1972. 4th edition. UFAW Staff (eds.).  Churchill Living-
      stone, Edinburgh and London.
      7.5  Procurement Specification VII. Rodents. 1969.  Institute of
      Laboratory Animal Resources, National Academy of Sciences, National
      Research Council, Washington, D.C.
      7.6  Procurement Specification IX.  Defined Laboratory Rodents and
      Rabbits. 1973.  Institute of Laboratory Animal Resources, National
      Academy of Sciences, National Research Council, Washington, D.C.

      7.7  Whitney, R. A., Jr. Physical Environment. In:  Workshop on
      Criteria for Successful Rodent Chronic Studies, National Cancer
      Institute, National Institutes of Health, Bethesda, Maryland,
      April 4-5, 1973.
      7.8  Request for Proposal 76-S-12, Carcinogen Bioassay, NCI Program
      Due date June 15, 1976, Tracer Jitco, Inc., Rockville, Maryland.
                                 B-50

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     Carcinogen  Bioassay Program
Specification No. CBO-18
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
l
Of
3
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
  SANITATION OF EQUIPMENT AND SUPPLIES  FOR LABORATORY ANIMALS
                                  1.   SCOPE

          This specification covers sanitization of  cages,  feeders, water
     bottles,  and certain ancillary equipment  for laboratory animals used
     in the Carcinogen Bioassay Program.

                          2.  APPLICABLE  DOCUMENTS

          2.1   Specification No. CBM-6     Laboratory Animal Cages and  Cage
                                           Filters
          2.2   Specification No. CBM-8     Watering  Devices for  Laboratory
                                           Animals

          2.3   Specification No. CBM-7     Feeders for Laboratory Animals

          2.4   Specification No. CBM-9     Exposure  Chambers for Inhalation
                                           Tests
          2.5   Specification No. CBM-5     Racks for Laboratory  Animal  Cages

                              3.  REQUIREMENTS

          3.1   Cages,  racks, feeders,  water bottles, catch-pans, exposure
          chambers,  and certain ancillary equipment  must  be sanitized at
          specified  intervals and before  reuse.

          3.2   Cages shall be washed at least  weekly in a machine which
          provides at least one cycle of  82°C  (180°F) water.

          3.3   Racks shall be either run  through a rack washer (which has
          one  cycle  of 82°C water) every  two weeks,  or washed, in  the saniti-
          zation area, with a suitable detergent and hosed  down under high
          pressure.

          3.4   Soiled feeders should be soaked,  if necessary,  and  then
          washed in  a system that uses at least  one  cycle of 82° C water.

          3.5   If water bottles are used, bottles, bottle stoppers,  and
          sipper tubes must be washed in  water of at least  82° C tempera-
          ture.  Stoppers and sipper tubes must  be sterilized  either by
          germicide  treatment prior to washing or by boiling after wash-
          ing.
                                      B-51

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Carcinogen Bioassay Program               Specification No. CBO-18
Subject: SANITATION OF EQUIPMENT AND SUPPLIES FOR
LABORATORY ANIMALS

Date:


Sheet

2
Of

3
     3.6  Inhalation chambers shall be hosed down dally and sanitized
     weekly.

     3.7  Catch-pans shall be cleaned and relined with fresh absorbent
     paper  each day.

     3.8  Mechanical washing machines with flexible time settings are
     highly  recommended for all Items where practicable.

          3.8.1  Washing phase should be at least 1 1/2 minutes.

          3.8.2  Recirculation rinse should be at least 1 minute.

          3.8.3  Final fresh water rinse should be a minimum of 1/2
          minute.
          3.8.4  Weekly preventive maintenance shall be routinely
          practiced for washing machines.  Strainers shall be cleaned
          at least once dally, or oftener, depending upon the work load.

     3.9  Portable cleaners which dispense detergent and hot water
     or steam under pressure should be used, if possible, for cage
     racks  and other pieces of equipment which are too large for the
     washing machine.

     3.10  The pH of the detergent solution should be within the range
     of 10  to 12.  An automatic detergent dispenser is recommended.

                        4.  QUALITY CONTROL

     4.1  Washing machine operators shall make certain that all nozzles
     and manifolds are constantly operative.
     4.2  The pH sensing devices and heating colls shall be maintained
     free of any accumulation of foreign material that would impair
     their accuracy.
     4.3  All sanitized cages, feeders, watering devices, racks, catch-
     pans, and exposure chambers shall be inspected for physical
     cleanliness prior to reuse.   Unsatisfactory items shall be resani-
     tlzed.
     4.4  Frequency of sampling of cages and other items for microbio-
     logical monitoring of the sanitlzatlon procedure will depend upon
     type of decontamination and level of protection desired.  No Gram-
     negative organisms should be detected, especially Pseudomonas spp.,
     but spore-formers and heat-resistant non-pathogens will be found
     occasionally.
     Detection of Gram-negative organisms should result in an immediate
shutdown of  washing equipment and correction of the defect or institu-
tion of better room sanitlzatlon procedures, depending on probable
source.
                                 B-52

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Carcinogen Bioassay Program               Specification No. CBO-18
Subject: SANITATION OP EQUIPMENT AND SUPPLIES FOR
LABORATORY ANIMALS
Date:
Sheet
3
Of
3
      4.5  Cages are to be Program and Chemical specific.  They ahall be
      returned to the same chemical group and dose level to avoid
      possible contamination.

                           5.  PACKAGING

      Not applicable

                             6.  NOTES

      6.1  The term "sanitize" is defined as "making physically clean
      and removing and destroying to the maximum degree that is
      practical, agents injurious to health."

                       7.  REFERENCE DOCUMENTS

      7.1  Code of Federal Regulations, Title 9, Chapter 1, Subchapter
      A.  Animal Welfare. Parts 1, 2, and 3, May, 1972.

      7.2  Sontag, J. M., N. P. Page, and U. Saffiotti. 1976.  Guidelines
      for Carcinogen Bioassay in Small Rodents.  U.S. Department of
      Health, Education and Welfare, NIH 76-801.

      7.3  Guide for the Care and Use of Laboratory Animals. 1974. U.S.
      Department of Health, Education and Welfare, NIH 74-23.

      7.4  Procurement Specification IX.  Defined Laboratory Rodents
      and Rabbits. 1973. Institute of Laboratory Animal Resources,
      National Academy of Sciences, National Research Council,
      Washington, D.C.

      7.5  Request for Proposal 76-S-12, Carcinogen Bioassay, NCI
      Program, Due date June 15, 1976, Tracer Jitco, Inc., Rockville,
      Maryland.
                                 B-53

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    Carcinogen Bioassay Program
Specification No.  CBO-19
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
i
Of
2
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
          DISINFECTION OF LABORATORY ANIMAL ROOMS
                                  1.   SCOPE

          This  specification covers procedures  for disinfecting laboratory
     animal  rooms  in  the Carcinogen Bioassay Program.
                          2.  APPLICABLE DOCUMENTS
      None
                               3.  REQUIREMENTS

           3.1   If an  epizootic disease occurs among animals in quarantine or
           on test,  the area  shall be disinfected before use for new animals.
           3.2   Disinfectants for use in any part of the bioassay facility
           shall be  approved  by program management.

           3.3   If formaldehyde gas  is used, the room shall be sealed and
           then  treated by evaporating 500 ml of formalin  (37% solution of
           formaldehyde in water and stabilized with methyl alcohol) for
           each  27 m3  of space.  The temperature shall be  at least 21° C
           (70°  F) and the relative  humidity 75-80% during fumigation.
           The exposure period  shall be 24 hours.

           3.4   Disinfected animal rooms shall not be reused until results
           of microbiological analyses indicate the absence of microorganisms
           pathogenic  for humans and domestic animals.

                              4.  QUALITY CONTROL

           4.1   Effectiveness of the disinfection procedure shall be
           evaluated by sample  swabbing of tables, benches, racks, walls
           and floor (at least  one swab per area) and culturing or suscep-
           tible-host  inoculation (cell cultures, embryonated eggs, or
           laboratory  animals).  Acceptable diagnostic practices of the
           American Society of  Clinical Pathologists or an equivalent
           organization shall be used.
                                5.  PACKAGING
          Not applicable
                                      B-54

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Carcinogen Bioassay Program
Specification No. CBO-19
Subject: DISINFECTION OF LABORATORY ANIMAL ROOMS
Date:
Sheet
2
Of
2
                      6.  REFERENCE DOCUMENTS

     6.1  Sontag, J. M., N. P. Page, and U. Saffiotti. 1976. Guidelines
     for Carcinogen Bioassay in Small Rodents. U. S. Department of
     Health, Education and Welfare, NIH 76-801.

     6.2  The UFAW Handbook on the Care and Management of Laboratory
     Animals. 1972. 4th edition. UFAW Staff (eds.). Churchill Living-
     stone, Edinburgh and London.

     6.3  Guide for the Care and Use of Laboratory Animals.  1974. U. S.
     Department of Health, Education and Welfare, NIH 74-23.

     6.4  Long-Term Holding of Laboratory Rodents. 1976.  ILAR News XIX
     (4), L20, L21.                                      	
                                 B-55

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     Carcinogen Bioassay Program
Specification No.  CBO-20
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
i
Of
2
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
           VERMIN CONTROL IN ANIMAL FACILITIES
                                  1.   SCOPE

          This specification covers the control of flies,  cockroaches,
     rodents,  and like pests in quarters for animals used  in the
     Carcinogen Bioassay Program.

                          2.  APPLICABLE DOCUMENTS

          2.1  Code of Federal Regulations, Title 9, Chapter 1, Subchapter
          A.  Animal Welfare. Parts 1,  2, and 3, May, 1972.

                              3.  REQUIREMENTS

          3.1  A safe and effective program for the control of insects,
          ectoparasites, avian, and mammalian pests in and around the
          animal facility shall be established and maintained under the
          supervision of a veterinarian or other qualified person.

          3.2  The building structure shall be vermin-proof with  rodent
          barriers at all doorways.

          3.3  Waste shall not be allowed to accumulate in outdoor  storage
          areas in the vicinity of animal quarters.

          3.4  Breeding sites of insects and other pests shall be eliminated
          or sealed with suitable materials resistant to detergents and
          disinfectants.

          3.5  Drains in animal rooms shall be plugged.

          3.6  Insulation on cage and rack washer pipes must not  be exposed.

          3.7  Pesticides shall not be allowed to contaminate any test
          animals or stored material.   Pesticides ("bait") may be used
          inside the building only in raceways and hallways but not in
          animal rooms.
          3.8  Strict sanitary practices on a regular basis shall be an
          integral part of the vermin control program.

               3.8.1  All animal rooms and other areas where food and
               bedding particles may accumulate shall be wet-mopped daily.

               3.8.2  Storage items shall be moved once each week and the
               floor beneath thoroughly cleaned and mopped.
                                       B-56

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Carcinogen Bioassay Program               Specification No. CBO-20
Subject:
VERMIN CONTROL IN ANIMAL FACILITIES
Date:
Sheet
2
Of
2
     3.9  A system for controlling escaped rodents by means of
     suitable traps shall be maintained at all times.

                        4.  QUALITY CONTROL

     4.1  The animal facility shall be inspected weekly for the
     presence or evidence of vermin and remedial measures instituted
     if necessary.  Results of inspections and remedial action taken
     shall be recorded in a bound notebook, dated, and signed by
     inspector and supervisor.

                            5.  PACKAGING

     Not applicable

                             6.  NOTES

     6.1  Wild rodents and other vermin carry a variety of bacteria,
     viruses, and parasites which may be transmitted to experimental
     animals should they gain entrance to the facility.  The popula-
     tion of wild rodents in the vicinity of animal buildings should
     be reduced or eliminated.

                       7.  REFERENCE DOCUMENTS

     7.1  Guide for the Care and Use of Laboratory Animals. 1974.  U.S.
     Department of Health, Education and Welfare, NIH 74-23.

     7.2  The UFAW Handbook on the Care and Management of Laboratory
     Animals. 1972. 4th edition. UFAW Staff (eds.). Churchill Living-
     stone, Edinburgh and London.

     7.3  Rodents.  Standards and Guidelines for the Breeding, Care,
     and Management of Laboratory Animals.  National Research Council,
     National Academy of Sciences, Washington, D.C., 1969.

     7.4  Long-Term Holding of Laboratory Rodents. 1976.  ILAR NEWS XIX
     (4), L20, L21.
                                 B-57

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     Carcinogen Bioassay Program
                Specification No.  CBO-21
Type:
GOOD ANIMAL
CARE LABORATORY PRACTICE
Sheet
i
Of
2
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
         SACRIFICE OF LABORATORY ANIMALS (EUTHANASIA)
                                   1.   SCOPE

          This specification covers the requirements of euthanasia and  the
     operational steps of euthanasia by physical and chemical methods.
          None
                           2.   APPLICABLE DOCUMENTS
                              3.   REQUIREMENTS
          3.1  Procedure requirements for euthanasia.

               3.1.1  Euthanasia should be performed by trained persons  in
               accordance with institutional policies and applicable laws.

               3.1.2  The choice of method should depend on the species
               of animal and the project for which the animal was used.

               3.1.3  The method of euthanasia should not interfere with
               any postmortem examinations or determinations to be perform-
               ed.

          3.2  Operational steps of euthanasia.  Mice and rats can be killed
          by two main methods: the physical method and the chemical method.

               3.2.1  Physical methods.  Some manual dexterity is essential
               for this method.  When done by skilled operator, apprehen-
               sion on the part of the animal is minimal, death is quick
               and suffering slight.

                      3.2.1.1  Cervical dislocation.  The animal is held by
                      its tail and placed on a surface that it can grip,
                      when it will stretch itself out so that a pencil or
                      similar object can be placed firmly across the neck.
                      A sharp pull on the tail will then dislocate the neck
                      and kill at once.
                      3.2.1.2
                      here.
Stunning.  Concussion is the cause of death
3.2.1.2.1  Hold the animal's head downwards
and strike very hard behind the ears with a
stout wooden stick or stunner.
                                      B-58

-------
Carcinogen Bioassay Program               Specification No. CBO-21
Subject:
SACRIFICE OF LABORATORY ANIMALS (EUTHANASIA)
Date:
Sheet
2
Of
2
                          3.2.1.2.2  Hold the animal firmly, belly
                          upwards, and strike the back of the head
                          very hard against a hard horizontal surface
                          such as a sink or a table.

         3.2.2  Chemical Methods.

                3.2.2.1  Carbon dioxide euthanasia, using a specially
                designed cabinet is the recommended method.

                3.2.2.2  Sodium pentobarbital, injected intra-
                peritoneally at three times the anesthetic dose can
                also be used.

                3.2.2.3  The use of ether in an uncrowded chamber has
                been done before.

                3.2.2.4  The use of nitrogen is not recommended.

                        4.  QUALITY CONTROL

    Not applicable

                          5.  PACKAGING

    Not applicable

                            6.  NOTES

    6.1  EUTHANASIA means the humane destruction of an animal accom-
    plished by a method which produces instantaneous unconsciousness
    and immediate death without visible evidence of pain or distress,
    or a method that utilizes anesthesia produced by an agent which
    causes painless loss of consciousness, and death following such
    loss of consciousness.

                       7.   REFERENCE DOCUMENTS

     7.1  Guide for the Care and Use of Laboratory Animals.  1974.  U.S.
     Department of Health,  Education and Welfare,  NIH 74-23.

     7.2  The UFAW Handbook on the Care and Management of Laboratory
     Animals.  1972.  4th edition.  UFAW Staff (eds.).  Churchill Living-
     stone,  Edinburgh  and  London.
                                 B-59

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     Carcinogen Bioassay Program
     Specification No. CBO-22
Approved:
Proj.
Q.C.
Lab
Other
Date
Type:
GOOD ANIMAL CARE LABORATORY PRACTICE
Sheet
i
Of
2
Subject:
           DISPOSAL OF DEAD OR SACRIFICED ANIMALS AND TISSUES
                                  1.   SCOPE

          This  specification covers disposal of  dead or sacrificed
     animals and tissues involved in the Carcinogen Bioassay  Program.

                          2.  APPLICABLE DOCUMENTS

          2.1  Code of Federal Regulations,  Title 9, Chapter  1,  Subchapter
          A. Animal Welfare.  Parts 1,  2,  and 3,  May,  1972.
          2.2  Specification No CBT-2


          2.3  Specification No.  CBT-5


          2.4  Specification No.  CBO-4
Gross Necropsy of Carcinogen
Bioassay Animals

Histopathologic Examination of
Carcinogen Bioassay Animals

NCI Safety Regulations for Research
Involving Chemical Carcinogens
                              3.   REQUIREMENTS

          3.1   All  procedures involved  in disposal  of  dead  animals and
          tissues shall  be in canformance with  Federal,  State, and local
          laws  and  regulations pertaining to  pollution control and protec -
         tion of the environment.

          3.2   All  animals which  die or are sacrificed in repeated-dose,
          subchronic, and  chronic studies shall be  subjected  to  gross
          necropsy  (unless cannibalism  or autolysis make the  animal
          unfit for all  or part of the  examination).
          3.3   Carcasses of animals may be discarded Immediately after
          necropsy  and fixation of all  tissues  required  for histopathologic
          examination.
          3.4   Contaminated wastes, cleaning  devices,  and animal carcasses
          shall be  transported to the disposal  area in a closed  impermeable
          container and  disposed  of by  methods  approved  by  the Office of
          Research  Safety.
          3.5   Refrigerated storage shall be  available for  holding dead
          animals until  necropsy.   The  area shall be separate from all
          other cold storage and  shall  be used  exlusively for refuse storage.
          The temperature  shall be kept below 7° C  (45°F).  The animals shall
          not be frozen.
                                     B-60

-------
Carcinogen Bioassay Program               Specification No. CBO-22
Subject:
DISPOSAL OF DEAD OR SACRIFICED ANIMALS AND Tli
Date:
SUES
Sheet
2
Of
2
     3.6  All dead animals shall be subjected to full gross and
     histologic examination in accordance with CBT-2 Gross Necropsy
     Examination and CBT-5 Histopathologic Examination.  Carcasses
     may be discarded immediately following necropsy and fixation
     of all required tissues.

                        4.  QUALITY CONTROL

     4.1  Supervisory personnel shall monitor the disposal of all dead
     and sacrificed animals and tissues to make certain that all
     procedures are in accord with Federal, State, and local laws as
     well as with regulations of the Office of Research Safety.

     4.2  Containers, liners, covers, etc., used in storage and
     disposal of sacrificed animals and tissues shall be inspected
     during operations to maintain conformance with safety regulations.

                           5.  PACKAGING

     Not Applicable.

                       6.  REFERENCE DOCUMENTS

     6.1  Sontag, J. M., N. P. Page, and U. Saffiotti. 1976. Guidelines
     for Carcinogen Bioassay in Small Rodents.  U. S. Department of
     Health, Education and Welfare, NIH 76-801.

     6.2  Request for Proposal 76-S-12, Carcinogen Bioassay, NCI Program,
     Due date June 15, 1976, Tracor Jitco, Inc., Rockville, Maryland.
                                 B-61

-------
Harffnoffen RlnaRf
Type:


GOOD ANIMAL
sav Program Snerif leation Nn

CARE LABORATORY PRACTICE
. PRO— y\
Sheet
i
Of
2
Subject: DISPOSAL OF RADIOACTIVE WASTES ASSOCIATED  WITH LABORATORY ANIMAL
                                  EXPERIMENTS
Approved:
Proj.
Q.C.
Lab
Other
Date
                                  1.   SCOPE

          This specification covers the operations  of  the disposal  of  radio-
     active wastes associated with laboratory animal experiments  and the
     product requirements involved in these procedures.
          Not applicable here.
                               2.   DEFINITIONS
                               3.   REQUIREMENTS
          3.1  Procedure requirements.

               3.1.1  Radioactive waste must be disposed of  in accordance
               with applicable regulations and license.
               3.1.2  Set up regular schedule for the elimination of radio-
               active waste.
               3.1.3  Use leakproof disposable liners in waste cans  for
               disposal of radioactive waste.

          3.2  Product requirement.

               3.2.1  Facilities must be provided for holding radioactive
               waste.
                      3.2.1.1  The storage area for radioactive waste should
                      be physically separated from other storage facilities
                      and animals.
                      3.2.1.2  In some instances, the ordinary storage
                      facilities may be used for holding the waste,  if
                      properly monitored, until all radioactivity has
                      decayed.
               3.2.2  Special shielding of the storage area  may be required.
               3.2.3  Cage washing equipment should be of type capable of
               decontaminating cage and accessories without  accumulating
               radioactive waste.  Machines should not recirculate the
               wash solution.
                                      B-62

-------
    Carcinogen Bioassay Program
Specification No.  CBO-23
Subject: DISPOSAL OF RADIOACTIVE WASTES ASSOCIATED
WITH LABORATORY ANIMAL EXPERIMENTS
        Date:
Sheet

   2
Of

  2
                            4.  QUALITY CONTROL


         4.1  Institute a system of equipment monitoring.


                          5.  REFERENCE DOCUMENTS


         5.1  Guide for the Care and Use of Laboratory Animals.  1974. U. S
         Department of Health, Education and Welfare,  NIH  74-23.
                                     B-63

-------
     Carcinogen Bioassay Program
Specification No. CBO-24
Type:
STANDARD OPERATING PROCEDURE
Sheet
l
Of
3
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
         DISPOSITION OF CARCINOGEN BIOASSAY PATHOLOGY MATERIAL
                                  1.  SCOPE

              This specification covers pathology materials to be submitted
         to program management as well as materials to be retained by the
         bioassay laboratory.
                          2.  APPLICABLE DOCUMENTS
         None
                              3.  REQUIREMENTS

              3.1  The following material shall be submitted to program
              management:

                   3.1.1  One hematoxylin and eosin (H & E) stained slide and
                   the tissue block representative of each different neoplasm
                   or treatment-related lesion from each chemical test group
                   shall be sent to program management as soon as the test
                   is completed or earlier, if possible, or if consultation
                   is desired.  These samples shall then be submitted to the
                   Tumor Pathology Section for record file.  There should
                   be no more than 10 slides and blocks per compound.

                   3.1.2  All pathologic specimens are the property of the
                   sponsor and must be submitted to the program management
                   upon request and automatically at end of the subcontract.

              3.2  The subcontractor shall retain all wet tissues, blocks,
              and slides of all animals (test, vehicle controls, untreated
              controls) in a vermin-proof, temperature-controlled area
              until termination of the bioassay investigation.

              3.3  All animal tissues shall be retained in formalin until
              program management directs their disposal.  However, tissues
              of repeated-dose animals may be discarded after the subchronic
              test has been started.  Subchronic and chronic residual
              material shall be retained and shipped to the repository
              when directed.
                                      B-64

-------
    Carcinogen Bioassay Program                Specification No.  C
Subject:
DISPOSITION OF CARCINOGEN BIOASSAY PATHOLOGY  MATERIAL
Date:
Of
                            4.  QUALITY CONTROL

         4.1  Pathologist-in-Charge and Histology  Supervisor .sha?"  re
         responsible for making certain that all required  slide-  and
         tissue specimens as indicated in  Sections 3  and 5 are submitted
         for storage and shipment.

         4.2  Responsible bioassay personnel shall see  to  it t» ,;. a>
         materials to be retained by the laboratory are packaged  , • .:
         stored as indicated in Sections 3 and 5.

         4.3  The Shipping Department Supervisor shall  make certain that  ?.'..'.  j
         tissue specimens, blocks, and slides to be sent to pr-!r,'t- r<-z:.-!'>   i
         ment are packaged and shipped in accordance  with  Sections  <  .r:      ;
         5 requirements.  All clearance to ship and shipment p.-'pers  . -.1 '     •
         be dated, signed, and filed for audit.

                               5.  PACKAGING                                  j

         5.1  At termination of the bioassay investigation,  re: • !ue of  a.11    i
         chronic animals, and those of sub-chronic  animals  which ^K-re         •
         subjected to histopathologic diagnosis, shall  be organic !,  pa-:  P-   j
         marked, and shipped to program management, after obta'-ii:  cleT-    (
         ance.                                                                '
         5.2  Wet tissues (residue from harvested tissues, not
         shall be stored in two plastic bags (one inside the o1' • >.; a. d       •'
         organized by histology number.  A label (permanent ink-  cental njr.g   \
         name of subcontractor and histology number shall be piac> •'. Wt:  _ ; a
         the two bags.  Bags shall then be packed in double-wall  cardboard    j
         boxes (350 Ib-test) and labelled on one end as follow.".:
                                                                              i
                            (a) Name of subcontractor                         i
                            (b) Subcontract number
                            (c) Chemical number                               ;
                            (d) Animal Group number(s)                        ;
                            (e) Histology numbers in that box                 i
              Boxes shall be sealed shut with shipping tape, IM-.:M,,;   . r-       !
         filament tape, and shipped to program management upon  '~^e.', t or     j
         clearance to ship.

         5.3  Blocks shall  be  resealed with paraffin,  permanently label'
         with name of subcontractor and histology number and or:, "i c>.
         according to the histo number.  For shipment, blocks sir;1.1. >.-
         placed in single-wall cardboard boxes (approximately <"><> hi >  '
         size) which shall then be packed into double-wall card: • &V
         containers (350 Ib-test) approximately 16" x 18" x 7 1/2" ?•. <
                                     B-65

-------
Carcinogen Bioassay Program               Specification No. CBO-24
Subject:
DISPOSITION OF CARCINOGEN BIOASSAY PATHOLOGY MATERIAL
Date:
Sheet
3
Of
3
     sealed with pressure tape and bound with filament tape.  One end
     shall show the information given in 5.2 above.

     5.4  Slides shall be organized by histology number and placed in
     plastic boxes which shall then be packaged in a 350 Ib-test
     cardboard box, separated by abundant packing material, for ship-
     ment to program management.

          5.4.1  Each plastic slide box shall be labelled with name of
          subcontractor and range of histology numbers.

          5.4.2  Each cardboard shipping box shall contain a packing
          list with name of subcontractor, number of slide boxes, and
          cross-reference information (animal number, histology number,
          chemical number) for complete identification of the contents.

          5.4.3  A master log (reproduction acceptable) of histology
          number assignments shall be sent to program management along
          with the first shipment of slides.  This log shall be updated
          as required.

          5.4.4  Plastic slide boxes will be sent to the bioassay
          laboratory upon request.  All other supplies for shipment
          of residual material to the repository shall be obtained
          by the subcontractor.

                      6.  REFERENCE DOCUMENTS

     6.1  Sontag, J. M., N. P. Page, and U. Saffiotti. 1976. Guidelines
     for Carcinogen Bioassay in Small Rodents. U. S. Department of
     Health, Education  and Welfare, NIH 76-801.

     6.2  Request for Proposal 76-S-12, NCI Carcinogen Bioassay Program,
     Due Date June 15,  1976, Tracor Jitco, Inc., Rockville, Maryland.
                                 B-66

-------
     Carcinogen Bioassay Program
          Specification No.  CBO-25
Type:
DATA RECORDS AND REPORTS
SPECIFICATION
Sheet Of
1 5
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
           REQUIRED INFORMATION
                                  1.   SCOPE

         This  specification lists the minimum required  information  for  the
     report  on  bioassay  study.
                           2.   APPLICABLE DOCUMENTS
    None
                               3.   REQUIREMENTS

          3.1  Devise  a plan  for the  collection  of  required  information
          before  the start of the bioassay  study.

          3.2  All  information and  data pertaining  to  the bioassay shall
          be completely and accurately recorded  on  a current basis.

          3.3  Minimum required information includes:

              GENERAL:  1.   Outline  of the bioassay study
                        2.   Bioassay study number  in  the investigator's file
                        3.   Names  of the investigators responsible for the
                             bioassay study,  including histopathological
                             diagnoses
                        4.   Name of  the bioassay laboratory
              CHEMICAL  (TEST AGENT)
 1.  Name, chemical abstract number,
     NCI number
 2.  Name, synonyms
 3.  Formula
 4.  Source (generic)
 5.  Manufacturer
 6.  Batch number
 7.  Date(s) when received
 8.  Storage (before its reception)
 9.  Physical state and other
     characteristics
10.  Melting point
11.  Solubility
12.  Criteria of purity
13.  Impurities (generic)
                                     B-67

-------
    Carcinogen Bioassay Program
                         Specification No. CBO-25
Subject:
         REQUIRED INFORMATION
                                 Date:
                                       Sheet
                                          2
Of
 5
           PREPARATION:
           ANIMALS:
1.
2.
3.
4.
5.
             14.  Methods of synthesis
             15.  Storage conditions and dates
             16.  Other

 1.  Chemical(s): name, data sheet number
 2.  Vehicle(s): name, data sheet number
 3.  Preparation and concentration
 4.  Methods of preparation
 5.  Amount prepared each time
 6.  Frequency of preparation
 7.  Physical state
 8.  pH
 9.  Stability and decomposition
10.  Storage
11.  Date(s) prepared
12.  Other

 Species
 Strain and subline
 Initial number by sex (male and female)
 Date(s) of birth (male and female)
 ^Source
   a.  Own colony (give reference)
   b.  Other
 Breeding
   a.  Inbred
   b.  Random
   c.  Outbred
   d.  Other
 Disease control
   a.  Specific pathogen-free
   b.  Germ-free
   c.  Conventional
   d.  Vaccinated
 Distribution in groups
   a.  Pooled at weaning
   b.  Random
   c.  Random tables
   d.  Littermates
   e.  Other
 Other experimental groups
                                                   included in the same
                    10.
                    11.
                    12.
    distribution
    Initial number per cage
    Divided by sex
    Age when obtained from Animal House
                                     B-68

-------
                        Pff>pi*am
                                     Snecification No  CBO-25
Subject:
          REQUIRED INFORMATION
                                             Date:
Sheet
   3
Of
                     13.   Maintenance (general conditions)
                            a.   Own standard (give reference)
                            b.   Special
                     14.   Cages
                     15.   Bedding
                     16.   Room temperature (range)
                     17.   Light
                            a.   Source
                            b.   Time cycle
                     18.   Diet
                            a.   Type
                            b.   Source
                     19.   Amount of diet
                            a.   Ad libitum
                            b.   Measured
                     20.   Water
                            a.   Tap
                            b.   Other
                     21.   Amount of water
                            a.   Ad libitum
                            b.   Measured
                     22.   Other or special conditions
         TREATMENT:
            1.  Special pretreatment conditions
            2.  Treatment multiplicity
                  a.  Single type
                  b.  Combined
Item 3-14 should apply for each treatment
            3.  Preparation administered
            4.  Dose per administration
                  a.  Volume
                  b.  Weight
                  c.  How measured
            5.  Route
            6.  Site
            7.  Methods and instruments used
                  a.  Surgical procedures
                  b.  Anesthesia
                  c.  Sterility
            8.  Frequency of administration
            9.  Total number of doses
           10.  Total dose given
           11.  Total time of treatment
           12.  Date treatment started
           13.  Date treatment ended
           14.  Others
                                     B-69

-------
    Carcinogen Bioassay Program
                       Specification No. CBO-25
Subject:
        REQUIRED INFORMATION
                               Date:
Sheet
   4
Of
         PLAN OF OBSERVATIONS:
         1.  Age of animals at start of experiment
         2.  Weight of animals at start of experiment
         3.  Duration of experiment
               a.  Lifespan
               b.  Interruptions (from when to where
                   and why)
         A.  Frequency of checking
         5.  Frequency of weighing
         6.  Frequency of charting
         7.  Frequency of measuring consumption of:
               a.  Feed
               b.  Water
         8.  Other observations
         9.  Autopsies
               a.  On all animals
               b.  With the exception of:
                     (1) decomposed animals
                     (2) lost animals
                     (3) other
        10.  Autopsy examinations
               a.  Complete
               b.  Except cranial cavity
               c.  Other exceptions
        11.  Histology
               a.  All tumors (note exceptions)
               b.  Other tissues
        12.  Other pathological observations
         CONTROLS:   1.
 List:
   a.  Each group
   b.  Selection
         VARIATIONS:   1.   List protocol additions or change
         REPORTS:   1.

                   2.
Animal groups
  a.  Body weight curves
Individual animals
  a.  Identification number
  b.  Mode of death (died or sacrificed)
  c.  Time of death (in days or weeks of age, or time
      from start of bioassay study)
  d.  Diagnosis of tumors found at necropsy and other
      pertinent pathology
  e.  Indication if necropsy not done (as in de-
      composition) and animal is considered lost from
      the study
                                     B-70

-------
Carcinogen Bioassay Program
Specification No. CBO-25
Subject:
REQUIRED INFORMATION
Date:
Sheet
5
Of
5
                        4.  QUALITY CONTROL

     Proof-read the report to ensure that no required information is
missing.
Not applicable here.
                           5.  PACKAGING
                      6.  REFERENCE DOCUMENTS
     6.1  Sontag, J. M., N. P. Page, and U. Saffiotti. 1976. Guidelines
     for Carcinogen Bioassay in Small Rodents. U. S. Department of
     Health, Education and Welfare, NIH 76-801.
                                 B-71

-------
     Carcinogen Bioassay Program
Specification No.  CBO-26
Type:
DATA RECORDS
AND REPORTS SPECIFICATIONS
Sheet
i
Of
2
Approved:
Proj.
Q.C.
Lab
Other
Date
Subject:
         NCI CARCINOGEN BIOASSAY DATA SYSTEM (CBDS)
                                  1.   SCOPE

          This specification covers the purpose and requirements  of  a  log
     book.  Carcinogenesis Bioassay Data System (CBDS)  by The National
     Cancer Institute is described, including its quality control processes.
                          2.   APPLICABLE DOCUMENTS
     None
                               3.   REQUIREMENTS

          3.1  The purpose of a log book is to enable ease,  accuracy,  and
          completeness in recording and retrieving data,  since the prepa-
          ration of reports require the extraction, review,  consolidation,
          and tabulation of data from the log books.

          3.2  The National Cancer Institute has develdped a computerized
          system to collect, retrieve, tabulate, and analyze bioassay  test
          data.

          3.1  This CBDS also serves to manage and monitor the status  and
          progress of individual bioassay studies as well as to summarize
          the total effort of the CBP.

          3.2  Data input is through a series of forms (see specification
          on "CBDS Data Forms") submitted to the CBP where they are
          processed for entry into a computer.

          3.3  The Systemalized Nomenclature of Pathology (SNOP) is used
          to code the pathology results collected on individual animals.

          3.4  Data output is available as a series of standard or special
          reports and tables presenting the data in the following way:
               a.  Bioassay studies underway within the total program or
               within a particular contract effort
               b.  An individual bioassay study
               c.  Special pathology reports
         	d.  Survival and weight curves
                                      B-72

-------
Carcinogen Bioassay Program               Specification No. CBO-26
Subject:
DATA RECORDS AND REPORTS SPECIFICATIONS
Date:
Sheet
2
Of
2
           e.  Selected analysis of the test data

                         4.  QUALITY CONTROL

     4.1  Closely monitor data before their entrance into the CBDS.

     4.2  Periodically hold special training classes for data
     technicians.

     4.3  Data forms must be checked before being translated into
     machine-readable format and entered into the system, microfilmed,
     and stored as microfiche for future reference.

     4.4  Data are only available for selective or complete recall
     after errors have been edited by computer.

                           5.  PACKAGING

     Not applicable here.

                      6.   REFERENCE DOCUMENTS

     6.T  Sontag, J. M., N. P. Page, and U. Saffiotti. 1976.  Guidelines
     for Carcinogen Bioassay in Small Rodents. U. S. Department of
     Health, Education and Welfare, NIH 76-801.
                                 B-73

-------
                                         APPENDIX C

          QUALITY CONTROL SURVEILLANCE CHECK LIST  FOR MICROBIOLOGY


    The  following appendices, 1,  LA,  IB,  1C,  and  2,  are reprinted  with
permission  from the publisher.   They are from "Functional Quality Control" by
R.  C. Bartlett,  in  Quality  Control  in Microbiology  edited by J. E. Prier,
J.  T. Bartola and H. Friedman,  and  published by  the University Park Press,
Baltimore,  Maryland 21202,  in  1973.
               APPENDIX 1

               Surveillance Report

               Personnel responsible for conducting surveillance activities are defined in the
               surveillance schedule. If any item is not monitored as scheduled, an explana-
               tion must be provided on a Surveillance Report supplement, sheet A.  Any
               error or deficiency found must be reported.
                  Verification of the recording of error in the Surveillance Report will be
               made by circling unacceptable observations on laboratory work sheets along
               with the insertion of the letters SR, the date, and the recorder's initials. A
               complete description of  the error is made  on sheet  B. This must include a
               description of the action taken to resolve  the problem, whether a solution
               was found or the matter is pending. A and B sheets must be submitted to the
               supervisor for compilation into the monthly surveillance report to the direc-
               tor. The supervisor will review  all pending deficiencies reported during the
               previous month. The ones that continue in pending status are submitted  with
               the next surveillance report.
                  The following symbols  have been appended to each item to assist other
               laboratories in attaching  priorities to the progressive development of surveil-
               lance programs: *, low priority; **, medium priority; and ***,high priority.
                  Abbreviations:  BAP, blood  agar plates; CTA, cystine Trypticase agar;
               DNase, desoxyribonuclease; G-N, gram-negative; MR-VP,  methyl red/Voges-
               Proskaver; ONPG, orthonitrophenylgalactosidase; PAD,  phenylalanine de-
               aminase; PSE, Pfizer selective Enterococcus; RA, rheumatoid arthritis  (RA
               factor); SAE, Society of Automative Engineers; SIM, sulfide  indole motility;
               TSA, tryptic soy agar; TSN, tryptic sulfite neomysin; XL, xylose lysine; XLD,
               xylose lysine decarboxylase.
                  Suppliers:  a,  BBL, Division of BioQuest, Becton,  Dickinson and Co.
               (Cockeysville, MdJ; *, Clay Adams (New York, N.Y.); c, Difco Laboratories
               (Detroit, Mich.):  , Ortho  Diagnostics (Raritan, N.J.); ', Pfizer, Diagnostics
               Division (New  York, N.Y.); '   Roche Diagnostics,  Division of Hoffman-
               LaRoche Inc.  (Nutley, N.J.); *, Statens Seruminstitut  (Copenhagen,  Den-
               mark); ", Wampole Laboratories (Stamford, Conn.).
                                              C-l

-------
                                                                                                                           i ht, Mo • •>L'. • a-•.
                       METHODS (responsibility for iurveilUnce
                       b indicated under each item)

                       Procedure Book •••
3 Months
O
                       Evening Shift Personnel Review
                       Night Shift Personnel Review
                       Proficiency Testing Specimens *
1-6 Months
1-6 Months
Monthly
All sections of the procedure book are indexed by author.
Each section will be monitored every 3 months by the
author. The supervisor will reassign sections when
employees terminate.

Corrections will be submitted to the division secretary
for retyping, copying, and distribution to established
procedure book locations. Deficient procedures will be
listed as pending until corrections are distributed.

Review of procedures with evening personnel by area
assistant supervisor. Procedures to be reviewed and
frequency will be established by supervisor.

Review of procedures with night shift personnel by
assistant supervisor. Procedures to be reviewed and
frequency will be established by supervisor.

Supervisor receives samples, submits them to area assistant
supervisors, and later checks all forms before mailing
by the deadline. Assistant supervisor reports results
at weekly meeting. Supervisor receives critiques and
discusses any discrepancies with area assistant supervisors.
Surveillance is applied to completion of analyses and
mailing of reports before the deadline, review of
antiques, maintenance of specimen viability, and
resolution of discrepancies. To be conducted by area
assistant supervisor.

-------
Appendix  Ik-Continued
                                                    Frequency of
                                                    SurveilUncc
                                          Standuds to be Monitored
Culture* Submitted to Reference Laboratory •*•     Monthly
Blind Unknown! *•
  Bacteriology
  Clinical microscopy
  Serotogy
  Mycology
  Mycobacttrium
  Florescence microscopy
BACTERIOLOGY (surveillance conducted
by bacteriology assistant nrpervuor)

General
  Antimicrobial susceptibility
  testing disc method
    Materials •*•
Monthly
Daily
Monthly
2 Months
Monthly
Monthly
Each batch of
medium
                       Area assistant supervisor is responsible for preparing and
                       sending samples to reference laboratory, copying lab slips
                       and putting them in proper book, maintaining samples for
                       retesting in case of discrepancies, resolving any discrepancies,
                      .and discussing results with supervisor. Surveillance is
                       applied to resolution of discrepancies, and maintenance
                       of specimen viability.
 In duplicate by supervisor.
 Submitted by area assistant supervisor.
 Submitted and results reviewed by supervisor.
Blind unknowns are submitted by nondiagnostic supervisor.
Result* on all of the above are correlated and discussed
with diagnostic supervisor and area assistant supervisors.
Diagnostic supervisor  then discusses results.
Control strains tested with each batch of media:
(a) coordinate media preparation for testing,
(b) coordinate stock and use of antibiotics in test,
(c) maintain control strains, and
(d) calculate precision and error. Control reporting
of results on drugs yielding unacceptable results.

-------
                      Appendix Ik—Continued
                                                                         Frequency of
                                                                         Surveillance
                                                                                         Standards to be Monitored
O
   Personnel *•

  Antimicrobial susceptibility
  testing-tube dilution-penicillin **

Frequently Used Reagents •
  Antisera lor bacteriological identi-
  fication
   k'scherichia coli, A3, and C
   HemophUut influenzac. A and B
   Micella AD
   Salmonella A--I, vi, polyvalent
   Alkalcscens dispar
   Bcthcsda Ballerup
   Arizona

  Stains
   Gram stain reagents
                                  stain
                        Hydrogen peroxide
                                                                        3 Months

                                                                        Monthly or as
                                                                        requested
                                                                        6 Months
Monthly


Monthly



Monthly
                      Test each worker for technique, precision, and accuracy.

                      Monitor technique and procedure with stock strain of
                      Enterococcus of known minimum inhibitory concentration.
                      Results must be 3-12 units/ml.

                      Culture antisera in use for 6 months. Rotate all antisera in
                      use, according to inventory. Test new antisera with known
                      cultures.
Fresh stock dated. Discard remaining portions every 6
months.

Fresh stock dated. Discard remaining portions yearly.
Combined stain made fresh with each use and discarded
after use.

Replace with fresh stock every 2 months.
                       Equipment (surveillance to be conducted by
                       assistant supervisor in each area)

                         Refrigerators •*•
                           MB 4338 Foster 2 door                        Weekly
                           MB 4348 Jewett
                           MB 4347 Foster 6 door
                                   Foster S door
                           Automatic alarms                              6 Months
                                                                       2-8°C

-------
                        Appendix Ik—Continued
                                                                       Frequency of
                                                                        Surveillance
                                                                                Standards to be Monitored
?
                          Freezer ***
                           MB 4307 DiUon Lilly

                          Incubators •**
                           MB4339Ublineld
                           MB 4371 LabHne 2d

                             Temperatures
                             Water
                             Fan motors
  Vents


MB4380LablineCO,
  Temperature
  Water

  Fans
  Fan motor
  CO, tanks
      surge tank

      flow tank

  CO, concentration
                                           Weekly
Weekly
Weekly

Weekly
Weekly
Weekly
6 Months

6 Months
                                                                      Weekly
                                                                      Weekly

                                                                      Weekly
                                                                      6 Months
                                                                      Weekly
                                                                      Weekly

                                                                      Weekly

                                                                      Yearly
                    2-%'C
35 t 1°C
Pan full
Running smoothly
MB 4371 oil with
20SAE
Change glasswool
filter
                    35 ± 1°C
                    Bubbler system
                    full
                    Running smoothly
                    Oil with 20 SAE
                    Not empty
                    12 pounds during
                    surge
                    12 pounds at 0.3
                    liters/min
                    5-10%
                    Monitor for 48 hr
                    with medical gas
                    analyzer (pulmon-
                    ary lab)

-------
                 Appendix Ik—Continued
o

Humidity
Water baths ind healing bloc* •••
MB Hospital control
MB 4319 variable
MB 4369 TSN bloc
MB 4368 variable
Inoculating loops •
Inoculating wire *
Safety hood •••
Air conditioner *
Grinding motor *
Microscopes*
Anaerobic Jars •••
Frequency or
Surveillance
Monthly
Weekly
Weekly
Monthly
Monthly
6 Months
Monthly
3 Months
Monthly
6 Months
6 Months
Monthly
Daily
Daily
Daily
Standards to be Monitored
Submit syringe full of gas
60-80%
54-56"C
45-47'C
3-mm diameter ; re-
place as needed
5-8 cm; replace
as needed
Face velocity, SO
feet/min
Filter pressure,
0.5-1. 5 cm
Wash interior with
germicidal cleaner
Change filter (engi-
neering department)
Oiled
Fan belt, wear,
tension
General inspection
Ctottridium novyi control
Change catalyst
Anaerobic indicator

-------
                      Appendix 1A—Continued
?
                                                                          Frequency of
                                                                          Surveillance
                                                                                      Standards to be Monitored
IMMUNOLOGY (surveillance to be conducted by
area assistant supervisor)

Procedures •*•
  Antislreptolysis "O" titer                        As performed or
  Brucvlla                                       once a month
  Rheumatoid factor
  Thyroglobulin antibody
  Typhoid O
  VDRL
  Antinuclear factor
  E.coli
  Neisseria gonorrhoeas
  Streptococcus
  Rhodamine-auramine staining
  of mycobacterb

  VDRL (Veneral Disease Research Laboratory) •**  Weekly
                         Rhodamine-auramine stain •*

                        Refrigerators •••
                          MG 4357
                        Waterbaths ***
                          MB 4384 VDRL
                          MB 4390 miscellaneous
                                                                                            Prepare and distribute positive and negative
                                                                                            controls, approve results before reporting.
                                                                                            Record control data grapliically.
                                                Monthly
                                               Weekly; automatic
                                               alarms every 6 months.
                                               Weekly
                                               Weekly
Monitor proper use and recording of controls.
Graphically record comparison of state laboratory
and Hartford Hospital results. Resolve results
differing by more than two tubes.

Fresh stock dated. Discard remaining portion
every 2 months.
55-57°C
36-38°C

-------
                     Appendix Ik-Continued
n
00
                                                                      Frequency of
                                                                       Surveillance
                          VDRL Rotators *
                            MB 4335 FA
                            MB 4387 VDRL

                          VDRL rotators *
                          Inoculating loops *

                          Safety hood •**
                          Microscope* *
                          Fluorolume illuminator *
                         CLINICAL MICROSCOPY (surveillance to be
                         conducted by area assistant supervisor)

                         Biological* **

                          Occult blood lest

                            (Bcnzidine dihydrochloride: H,O,)
                            Hydrogen  peroxide

                          Wright stain
                          White and red blood cell diluting
                          fluids and stains
                          Trichome stain
                          Sudan
Weekly
Weekly

Weekly
Replace monthly
or as needed
6 Months
6 Months
As bulb change
needed
Daily
Monthly
                                     Standards to be Monitored
Lubricate bearings
180 rpm (VDRL only)
5-mm diameter loop

Face velocity. 50-200 fpm
General inspection
Bulb should be
changed every ISO
hr, with mainte-
nance as follows:
Check reflector ad-
justments and
clean; check bulb
adjustments and
fan motor; Clean
exciter filter and
window assembly.
Affirm 4+ positive reaction to 1:1,000
aqueous solution blood

Replace with fresh stock every 2 months.

Replace with fresh stock every 2 months.


Discard remainder and prepare fresh yearly.

-------
Appendix I A—Continued

Equipment
Refrigerator •*•
MB 4372
Refractomer •••
MB 4388
MB 4390
Glassware •
Fune hood *
o Microscopes *
• 1
^ MEDIA ROOMfcurvdOance to be conducted
by media room personnel)
Equipment
Refrigerator ***
MB 4394
Hot air oven •*•

Autoclaves •••
Frequency of
Surveillance

Weekly
6 Month*
3 Months
Monthly
3 Months
Monthly
Weekly
Daily
3 Months
3 Months
Weekly
Standards to be Monitored

2-88C
Test automatic
alarms.
Calibrate with
H,O to 1.000
Discard chipped
glassware
Wash with gerrnid-
dal cleaner
General inspection
2-8°C
Record each run;
must be 1 55-1 65eC
Sterility check
(spore strips)
Sterility check
(spore solution)
Check gaskets

-------
                    Appendix I ^-Continued
o
H»
O

Glassware *
Balance *
MYCOLOGY (surveillance to be conducted
by area assistant supervisor)
Equipment
Water haths •*•
Incubator •*•
MB 4350
Safety hood ***
Microscope •
Frequency of
Surveillance
Monthly
3 Months
6 Months
Weekly
Weekly
6 Months
3 Months
Monthly
Standards to be Monitored
Discard chipped
glassware
Calibrate
Clean and general
maintenance (de-
scribed in instruc-
tion manual)

35 t 1°C
Face velocity
50-200 fpm
Wash with germi-
ddal cleaner
General inspection
                       MISCELLANEOUS (surveillance to be conducted
                       by delegated supervisor)

                       Equipment

                         Vacuum pump (for ryophilizaUon)*
                                                                     3 Months

                                                                     3 Months
                                                                     6 Months
                                                                     Yearly
Before use and
when In continual
use the following
arc performed:
0.5-inch belt ten-
sion
Oil level
Oil parts
Change oil, order
new oil

-------
 Appendix I A- Continued
                                                 Frequency of
                                                 Surveillance
                                   SUndards to be Monitond
   Vacuum pump (for air sampler)*

   Pipettes •
6Monthi

As received
Oil as indicated

Check calibration
   Thermometers *
As received
Calibrate with
National Bureau
of Standards cali-
brated ther-
mometer
• MEDIA (surveillance functidns to be delegated by supervisor)

 Autoclave Cycles***                             Each batch

   Test tubes, 18 X  ISO mm with 10 ml of agar
   1,000-ml volume in flask
   2,000-mI volume in flask

 pH***                                         Each batch
 Storage***                                     Weekly
   Pbtes
    Not bagged: 2-week duration
    Bagged: 16-week duration
                     Recording of min of exposure to
                      121*C (minimum/maximum)
                                   12/34
                                   20/54
                                   30/72

                     Must be t 0.2 from recommended
                     pH of manufacturer (except
                     Mueuer-Huiton, 7.2-7.4)
                     2-8*C

-------
                   Appendix I A—Continued
                                                                    Frequency of
                                                                     Surveillance
                                                                                    Standards to be Monitored
O

M
NJ
Tubes, sponge-plugged
  Not bagged: 2-week duration
  Bagged: 2-month duration
Tubes, screw-capped
  Not bagged: 2-month duration
  Bagged: 4-month duration
Exceptions
  Cystcine Trypticase agar (CTA) sugars: 2-week duration
  Indote nitrate: bagged, 2-week duration
  Thkjglycolate: stored in darkness, 4 months
                      Depth of Plates**
                       Plates
                       Tubes
                         Agar
                         Broths

                      Sterility Check***
                       Broth (blood)
                       Plates (BAP and chocolate only)
                     Testing with Stock Culture****
                                             Each batch
                                             Each batch
                                             Each batch
                                             Each batch*
                                                or each tot •f
                                                                                                              2-8°C
                                                                                                              2-8°C
Room temperature
Room temperature
Room temperature
3 Mm (except Mueller-Hinton, 4-6 mm)

Length of slant equal to length of butt
Specified in reagents and media section
3 Tubes, incubated 48 hr
5% Of plates incubated 48 hr
(chocolate agar, 72 hr)

Proper hemotysis
Correct color reactions
Inhibitory or selective properties

-------
APPENDIX IB: Testing with Stock Cultures
Item
Plates BAP*
Bipbtes*
Brain heart infusion agar*
Brain heart infusion agar and
chloramphenicol
Casein pbtes*
0
£J Chocobte*
Control Organisms
G^oup A 0-hemolytic Streptococcus
Streptococcus viridani
Blank
See BAP and MaoConkey
Oyptococcui sp.
Nocardia sp.
Escherichla coU
Streptomyces sp.
Nocardia aiteroldes
HemophUus tnfluemae
Acceptable Results
Good /Miemolyab
Good a-hemorysis
Sterile
See BAP and MacConkey
' Growth
Growth
No growth
Hydrolysis
No hydrolysis
Good growth
J"»^_J «A«^L fAO m._\
Martin-Lester*
MacConkey-f
Mycobiotic agar*
Ncisseria gonorrhoeae
Staphylococcta aureta
Eschertchia coll

Proteus mirablla
Candida alblcam

Etchertchla coU
Shigella flexneri (B)
Enterococau

Candida alblcant
Atpergttlut
Etchertchla colt
Good growth (48 hi)
Inhibition
Inhibition

Inhibition of swarming
Inhibition

Lactose-positive; correct morphology
Colorless colonies
Inhibition

Growth
No growth
No growth

-------
o
Appendix It-Contlmied
Item
Mueller-Minion*
Phenyfcthyl alcohol agar (PEA)**
Potato dextrose agar*
Rice agar*
Rodac*"
Sabouraud's dextrose*
*Sabouraud's dextrose*
agar and chloramphenicol
Trypticase soy agar+
Tyrosine pbtes*
Xanthine plates*
XLD
Control Organisms
See surveillance of disk susceptibilty
(Appendix 14. Bacteriology, General)
Staphylococcus aureut
Etcherichia coll
Pteudomonat aeruglnota
Trichophyton rubrum
Candida alhicant
Candida knuei
rlavobacterium
Candida alblcant
AspergSlus sp.
t'scherichia coli
Staphylococcus aurcus
Streplococcut virtdans
Streptomyces sp.
Nocardla asteroidet
Streptomyces sp.
Nocardia asteroldet
Salmonella typhimurium
ShigeUaflexneriW
Exherichia coli
Acceptable Results
Disc
Growth
Inhibition (tiny colonies)
Inhibition (tiny colonies)
Growth
Chlamydospores (72 hr)
No chlamydospores (72 hr)
Growth
Growth
Growth
No growth
Growth
Growth
Positive
Negative
Positive
Negative
Black colonies
Red (colorless) colonies
Yellow colonies
                           Abbreviations: *, each batch; *, each lot; +, positive; 0, negative; D, delayed; A, acid; ALK, alkaline; AC, add and gas; NC, no change.

-------
                  Appendix IB-Continued
                                          Item
                                                                 Control Organisms
                                                Acceptable Results
Ul
                  Tubes

                    Christensen's urea*
                    Christensen's urea* slants
                    (mycology version)

                    DNase*
Middlebrook 7H-10*


Pseudosel*



SIM*




Simmon's dtrate*


Stuart's transport*
Proteus mirabSts
Klebsiella pneumonlae
Escherichla coll

Torulopsis sp.
Candida sp.

Serrotia
Staphylococcus epidermidis

Nocardla
Mycnbacterium tuberculosis
                                                                             Pseudomonas aeruginosa
                                                                             Escherlchlo coll
Proteus mirabilis
Klebsiella pneumonlae
Escharichla coll

Klebsiella pneumonlae
Escherichla coll

Heisenia gonorrhoea*
22°C, 6-hr transfer
2-8°C, 6-hr transfer
                                                                                                D
                                                                                                0

                                                                                                Positive
                                                                                                Negative
Positive
Positive

Growth and pigment

0           0

H,S  Indote  motility
*     0        *
0     +        *
0     *        *

*
0
                                                                                                                    Growth (48 hr)
                                                                                                                    Growth (48 hr)

-------
Appendix I B-Conf/nucd
                       Item
Control Organisms
                                     Acceptable Results
           TSA+


           Triple sugar iron (TSI)+




           TSN+


           Indole nitrate*
           Gelatin* (mycology version)
           Rke grains*
           Bile* (sodium detoxycholate)
           Blood broth*
Cloiirtdium novyl
22°C, 6-hr transfer
2-8°C, 6-hr transfer

Staphylococcus aureus
Streptococcus virtdans

Escherichta coll
Salmonella typhimurium
Proteus rettgeri
Pseudomonat aerugtnosa

Clostrlditim perfrlngeni
Clottridium novyi

Indole
  Eschertchla colt
  Klebtiella pneumonlae
Nitrate
  Staphylococcus aureut
  Herellea tp.
  .Pseudomonat acruginosa

dadotporium
P.pedrosot

Mlcroiporum canis
Microsporum audouinl

Streptococcus pneumonlae
Streptococcus viridam

Hemophitus influenue
fietiserta mentngittdis
                                       Growth (24 hr)
                                       Growth (24 hr)

                                       Growth
                                       Growth

                                       A/AG
                                       ALK/AGH,S+
                                       ALK/A
                                       ALK/NC
                                                                                                       0

                                                                                                       •»•
                                                                                                       0
                                       Positive
                                       Negative

                                       Positive
                                       Negative
                                      Growth (24 hr)
                                      Growth (24 hr)

-------
              Appendix IB—Continued
o
Item
Brain heart infusion agar'
Brain heart infusion agar
and chloramphenicol*
Decarboxybse*
Control*
With ornithine*
With lysine*
Control Organisms
Cryptococcus albidus
Cryptococcus albidus
Proteus mirahilis
Klebsiella pneumoniae
Pseudomonas aemginosa
Proteus mirabilis
Klebsiella pneumoniae
Pseudomonas aeruginosa
t'sclierichia coll
t'nterobacter cloacae
Pseudomonas aeruginosa
Acceptable Results
Growth
Growth
0
0
NC
•f
0
NC
0
NC
Gelatin*


Gluconate+


G-N Broth*
                                                                          Pesiidomonas aeruginosa
                                                                          Herellea

                                                                          Pseudomonas aeruginosa
                                                                          Herellea
E. coH:Shigella
6-hr transfer
18-hr transfer
                                                                          E.co1i:Shigetla
                                                                          6-hr transfer
                                                                          18-hr transfer
                                                                                           1:1
                                                                                                                   Recovery of Shigella
                                                                                                                   Recovery of Shigella
                                                                                                                   Inhibition off. coli
                                        Recovery of Shigella
                                        Recovery of Shigella

-------
o

M
00
Appendix IB-Continued
Item
Inositol*
Malonate*
MR-VP+
ONPG+
PAD*
PSE**
Rabbit plasma*
6J% Salt broth*
Sucrose assimilation*
TWamine*
TMogtycobte*
Control Organisms
Trichosporon verrucosum
Klebsiella pneumonlae
Eschericliia coli
Eschtrichia coli
Klebsiella pneumoniae
Eacherichia coll
Proteus mirabilis
Proteus mirabilis
Eschericliia coll
Enterococcus
Group D not Enterococcus
Llsterla (48 hr)
Group A 0-hemolytic Streptococcus
Staphylococcus aureus
Staphylococcus epidemldis
Enterococcus
Group D not Enterococcus
Staphylococcus aureus
Group A 0-hemolytic Streptococcus
Candida albtcans
Trtchophyton tonsurani
Trtchophyton menta
Ctostridlum novyl
Streptococcus viridins
Acceptable Results
Positive
0
+/0
0/+
0
0
•f
0
0
0
0
+
0
Growth
Growth

-------
                 Appendix IB-Continued
                                            Item
                                                         Control Organisms
                                                                   Acceptable Results
                   Tryptic soy broth*

                   Urea broth* (Rustigian
                   and Stewart)
                                           Streptococcus vMdant
                                           Nocardla broSten*
                                                          Growth (24 hr)
                                Oxidation-fermentation  Proteut rcttgeri       Proteut morganU  Pteudomonet    Pteudomonat   Arizona
                                (OF) sugars*            (lactose-fermentative)                   aerugfnom      maltophBia
V£>
Glucose
Sucrose
Maltose
Xylose
Arabinose
                                CTA sugars' "

                                Glucose
                                Maltose
                                Sucrose
                                Lactose
A
A
0
0
Semtia
Enterobacter

Netaeria
gonorrhotae

A
0
0
0
A
0
0
0
0
A

Neiaeria
menlngitidit

A
A
0
0
A/0
0
0
A
                                                             Neiaeria ticca

                                                             A
                                                             A
                                                             A
                                                             0
0
0
A
0
A
0
A
A
                              Netaeria
                              catarrhaKs

                              0
                              0
                              0
                              0

-------
APPENDIX: 1C

Inventory of reagents and biological***

Storage code: A, room temperature; B, 2-8°C; C, cool; D. dry; E, protect from light; F, dangerous to handle for one or more of the following:  1,
poison, 2, caustic, 3, corrosive, 4. avoid  contact (absorbed  through skin, strong oxidizing agent, etc.), 5, should not be inhaled, 6, volatile,  7,
extremely toxic, or 8, carcinogen; G, freeze; H, dedicated; NS, none stated or none found in reference.

Expiration code: M, month; W, week; Y, year; S, slated on product; D, does not apply; NS, none stated or none found in reference.

References
Merck Index of Chemicals and Drags. 1960. Merck A Co., Railway, N J.
BBL Manual of Products and Laboratory Procedures. 1968. 5th Ed. BBL-BioQuest, Division of Becton, Dickinson and Co., Cockeysville, Md.
Personal communication, David A. Power, Ph. D., Manager of Marketing Communications, BioQuest, Division of Becton, Dickinson and Co.,
Cockeysville, Md.

BIOLOGICALS-IMMUNOLOGY (surveillance to be conducted  by area assistant supervisor)
1
to
O
Item
Albumin, bovine
ANF conjugate
ASO buffer
ASO reagent
ASO standard
Brucclla
t'. coli A(conjupate)
(Difoo)r
K. coli A(scrological)
E. coli B(conjugate)
(Difoo)f
K. coll Bfserological)
Expiration

Opened
2M
6M
1M
lOmin
2M
2M

2M
6M

2M
6M

Closed
3Y
1Y
2YorS
2YorS
2YorS
S

1Y
2Y

1Y
2Y
Storage and/or Precautions

Opened
B
G
B
B
G
B

B
B

B
B

Closed
B
B
B
B
B
B

B
B

B
B
Surveillance
Interval
Weekly
Weekly
Weekly
Weekly
Weekly
Weekly

Weekly
Weekly

Weekly
Weekly

-------
              Appendix 1C—Continued
?
to
Expiration
Item
t'. oo/i C(conjugate)
£'. co/iC(Jcrological)
Hemagglutination buffet
Monospol kit 4
Neiaeria gonorrhoeae
conjugate
Phosphate-buffered
saline (PBS)
Pregnoslicon (sHde)-
50 test/
Pregnosticon (sHde)-
10 test/
RA buffer
RA latex 0.81
RA plasma fraction II
RA test kit
Rabbit globulin;
fluorescent antibody
Rabbit plasma, discard
remainder after use
Streptococcui conjugate
Stnptococcia controls
Streptozyme Ut *
Thyroid kit
Typhoid "O"
Typhoid "O" and
Bntcdla control
(MS.)
Opened
2M
6M
1M
1M

2M

1M

2W

1M
1M
1M
1M
1M

2M

2W
2M
2M
1M
1M
2W


3M
Closed
1Y
2Y
2Y
S

1Y

MS

S

S
S
1Y
1Y
S

1Y

1Y
4M
1Y
S
S
S


NS
Storage
Opened
B
B
B
B

B

B

B

B
B
B
B
B

B

B

A
B
B
B


G
Closed
B
B
B
B

B

A

B

B
B
B
B
B

B

B

B
B
B
B


B
Surveillance
Interval
Weekly
Weekly
Weekly
Weekly

Weekly

Weekly

Weekly

Weekly
Weekly
Weekly
Weekly
Weekly

Weekly

Weekly
Weekly
weekly
Weekly
Weekly
Weekly


Weekly

-------
                           1C-
                                                                   Expiration                          Storafe
                                                                                                                              SurvciHance
                 	Item	Opened	Cloud	Opened	doted	inl«*val

                 Typhoid "O" and
                 AVNrrflf control
                 (DIM.)                                        3M             S                G               B               Weekly
                 VDRL antfcjten
                 end buffer                                    2W             1Y              A               E               Weekly
                 VDRL control eera                             3M             S                G               B               Weekly



                 BIOLOGICALS-BACTERIOLOGY (	IBun to be cowtneted fry era
M
4rfrww (open
wpjen needed)
dljieiiiiii
•MMUpnesk
CVftiniliiBni
OepMotMn
«T*t>A*UAMkMft*AMl«MhB.
vMOffnipVBBiBwIN
Coatfe

yinromycMi
GeflrteinkinMlCite
HeeMpkHM type A
HeMopMtos type B
lloiM sefiun
Kanunycin
unconiycM
nydfocMonde
MetkicflHn
NnMixteKid

OnMieenwfor

Oxytetracydine
6M
IY
IY

•M

-------
               Appendix 1C—Continued
JsJ
OJ
Expiration
Item
Penicillin (powder,
standard)
Penidllinase
Rabbit phana,
normal-coagulase
Salmonella O group A
Salmonella O group B
Salmonella O
group Cl
Salmonella O
group C2
Salmonella O group D
Salmonella O group E
Salmonella O group F
Salmonella O group G
Salmonella 0 group H
Salmonella O group 1
Salmonella O
poly a'tent
Salmonella vi
.r/i/.L''7.'i7 |7rGUp A
Sliifclla inoup b
thizrlta proupC
Shi fella f.toup D
i'/itfr/Az -Alkalesccas
dispar
Sueploinycin
Opened

6M
1Y

2W
6M
6M

6M

6M
6M
6M
6M
6M
6M
6M

iJM
6M
fiM
GM
yM
6M

6M
»jM
Closed
-
S
5Y

3Y
2Y
2Y

2Y

2Y
2Y
2Y
2Y
2Y
2Y
2Y

1Y.
?Y
2Y
2Y
2Y
2Y

2Y
S
Storage and/or Precautions
Opened

G.H
B

G
B
B

B

B
B
B
B
B '
B
B

B
B
B
B
13
B

E
G,H
Closed

G41
B

B
B
B

B

B
B
B
B «
B '
B
B

B
B
n
H
b
B

R
u.ii
Surveillance
Interval

Weekly
Weekly

Weekly
Weekly
Weekly

Weekly

Weekly
Weekly
Weekly .
Weekly
Weekly
Weekly
Weekly

^Vckiy
Weekly
Y.Vwkly
\Vcckly
Wcck'y
"..-^kly

We«klv
-, Al>

-------
               Appendix IC-Continued
o
to
Expiration
Item
Tetracydine
Vancomycin
hydrochloride
Opened
6M

6M
Closed
S

S
Storage and/or Precautions
Opened
GJi

BJI
Closed
BJi

BJI
a? *ll
Surveillance
Interval
Weekly

Weekly
STOCK CARBOHYDRATES-SURVEILLANCE
Frequently used stored in crystal form
Arabinose
Dextrose
Lactose
Maltose
Sucrose
D-Xylose
Infrequently used stored in disc form
Adonitol
Dulcitol
Galactose
Inositol
Inulin
Levulose
Mannitol
Mannose
Rafilnosc
Rhamnose
Salidn
Sorbitol
Trehalose

6M
6M
6M
6M
6M
6M

6M
6M
6M
6M
6M
6M
6M
6M
6M
6M
6M
6M
6M

2Y
2Y
2Y
2Y
2Y
2Y

2Y
2Y
2Y
2Y
2Y
2Y
2Y
2Y
2Y
2Y
2Y
2Y
2Y

CJ)
CJ)
CJ)
C,D
CJ)
C,D

CJ)
CJ)
CJ)
C,D
CJ)
BJI
BJi
BJi
BJi
BJi
BJi
BJi
BJi

CJ)
CJ)
CJ)
CJ)
CJ)
CJ)

CJ)
CJ)
CJ)
CJ)
CJ)
B.A
B,A
B.A
B,A
B.A
B.A
B.A
B.A

Weekly
Weekly
Weekly
Weekly
Weekly
Weekly

Weskly
Weakly
Weekly
Weskly
Weekly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Moithly
Monthly

-------
              Appendix 1C—Continued
o

to
in
STOCK CHEMICALS
Item
Acetic acid, glacial
N-AcctyW-cystein
p-Aminodimethyhnlline ozalate
Ammonium sulfate powder
Aniline
Auramine O
Barium chloride
Barium sulfate
Benzidine dihydrochloride
Bromthymol blue
Calcium chloride
China blue powder (Poirier's blue)
Chlorazol black E
Crystal violet
Cystcine hydrochloride
p-Dimethylaminobenzaldehyde
Dimelhylxulfoxide
LorinY
Ether
Ethyl alcohol

Ferric ammonium citrate
Ferric chloride
Fuchsin, acid
Fuchsin, basic
Glycerine
Gramercy indicator
Hemin
Expiration
NS
NS
NS
NS
NS
5Y
NS
3M prepared
6M prepared
5Y
NS
NS
5Y
5Y
NS
NS
NS
5Y

3M opened
NS sealed
NS
NS
5Y
5Y
NS
NS
NS
Storage and/or
Precautions
F, 4
B,
D

F,,E
D
F,
NS
NS.F4.8

C.D
D
D
D
D
E
NS
D
F5.6
D

E
E
D
D
F4
NS
NS
Surveillance
Interval
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly


Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly

-------
o
to
Item
Hydrogen chloride, cone, (chemistry)
Hydrogen peroxide 3%
Iodine crystals
Lactic acid
Magnesium sulfate
Malachite green
Menadione
Mercuric chloride
Merthiolate
Methyl red
Methylene blue
Naphthol
Naphthylamine
Orthonitrophenyl-0ri-galactopyranoiide
OxgaU
Periodic acid
Phenol (crystals)
Phenolphthalein diphospliate
Phenyl red
Phenylalanine
Potassium alum
Potassium ferrocyanide

Potassium hydroxide
Potassium iodide
Potassium permanganate
Potassium phosphate dibasic
Potassium phosphate monobasic
Rhodamine O
Safranin
Sedi stain *
Sodium m-bisulfate
Sodium carbonate
Sodium chloride
Expiration
NS
NS
NS
NS
NS
5Y
NS
NS
NS
SY
5Y
NS
NS
NS
1Y
NS
NS
NS
5Y
NS
NS
working
soln.-immediatdy
NS
NS
5Y
NS
NS
5Y
5Y
5Y
NS
NS
NS
Storage and/of
Precaution*
F4.S
EAR
NS
NS
D
D
E
Fl

D
D.F1
E
F4.8
NS
C4>
NS
D.E.F4
F4
D
NS
NS
NS

F2
NS
D
NS
NS
D.F1
D
D
NS
D.E.FM
NS
Surveillance
Interval
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly

Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
, Monthly
Monthly

-------
              Appendix 1C- Continued
to
Item
Sodium citrate
Sodium desoxychobte
Sodium hydroxide
Sodium phosphate monobasic
Sodium phosphate tribasic
Sodium succinate
Sodium thiosulfate
Sudan 111
Sulfanilic acid
SulfosalicyKc acid
Tannic acid
N.N.N.N.-Tetramethytp-phenylene
diamine monohydrocttoride
Thiamine
Thymol (Merck)
Toluidine blue
Trkhrome
TriphenyltetrazoUum chloride
Trisodium citrate
Trypan blue
/-Tryptophane
Tyros! nc
Xanthine
Zinc dust metal
Zinc sulfate
Expiration
NS
NS
NS
NS
NS
NS
NS
SY
NS
NS
NS
NS

NS
NS
SY
SY
NS

SY
NS
NS
NS
NS

Storage and/or
Precautions
NS
NS
F2
NS
NS - .
NS
NS
D
NS
DJB
E
FM

NS
NS
D.F1
D
NS

D
NS
NS
Fl
F5

Surveillance
Interval
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly

Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly
Monthly


-------
Appendix 1C- Continued
MEDIA (surveillance to be conducted by media room personnel under direction of super*!
Expiration Date
Product
Agar-agar
Anaerobic agar
Brain heart infusion agar
Brain heart infusion broth
Brilliant green agar
Bordet-Oengon
Chloramphenicol
Clostrisel agar"
Columbia agar base
Cooked meat medium
Cystine Trypticase agar (CTA) medium *
DNase test medium
Entamoeba medium
Fletcher medium base
OC medium base
Gelatin
GN broth
Indole nitrate broth
Litmus milk
Liftman OxgaO (prepared)
LoeiTIer blood serum
Loeffler (prepared)
Lysine iron agar
MacConkey
Malonate
MoeOer decarboxylase
MR-VP
Mueller-Hinton agar
Mycobactoset agar0
Mycobactosd L-l agar*
Mycobk>ticc
Neopeptone c
Oxidation-fermentation basal medium
Pfizer selective Enterococcus
Phenol red broth base
Phenylethyl alcohol agar (PEA)
Phytone peptone*
Plate count agar
Pseudoset*
Purple mflk
Resazurin
Rice extract agar
Sabonraud's agar (modified)
SIM
Simmon's citrate
Skim milk powder
Spirit blue agar
Stuart's transport
Thiosulfate citrate bile salts (TCBS) medium
Tellurile glycine agar base
Thiogel medium *
Thiogylcobte with dextrose and Eh indicator
Thioglycolate without dextrose and Eh indicator
Opened
1Y
1Y
1Y
1Y
1Y
1Y
6M
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
D
1Y
D
1Y
1Y
1Y
1Y
1Y
1Y
D
D
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
1Y
Closed
3Y
3Y
3Y
3Y
3Y
3Y
S
2Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
2Y
S
2Y
1Y
2Y
3Y
3Y
3Y
3Y
3Y
1Y
1Y
2Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
3Y
Storage
CJ)
C4>
CJ)
CJ)
CO)
CJ)
BJ)
CJ)
CJ)
CJ)
CJ)
CJ)
CJ)
CJ)
co>
CJ)
CJ)
CJ>
CJ)
B
CJ)
B
CJ)
CJ)
CJ)
B
CJ>
CJ)
B
B
CJ)
CJ)
CJ)
CJ)
CJ)
B
CJ)
C.D
CJ)
CJ)
CJ)
C4)
C4>
CJD
C4>
CJ)
CJ>
B
C4)
CJ)
CJ)
CJ)
CJ)
                             C-28

-------
Appendix 1C—Continued
                                                 Expiration Pate
                 Product                       Opened     Closed    Storage

Thioglycolate fluid                                 1Y       3Y       CJD
Todd-Hewitt                                      1Y       3Y       CJD
Triple sugar iron (TSI)agar                          1Y       3Y       CJD
Tripticase soy agar with tethicin and polysorbate SO"    1Y       3 Y       B
Trypticase soy agar «                                1Y       3Y       B
Trypticaie soy broth «                              1Y       3Y       C4>
TSN «                                           1Y       2Y       CJD
Tuberculosis (TB) niacta tert                         1Y       1Y       CJD
Urea agar base                                     1Y       2Y       B
Urea broth                                        1Y
XLagarbase                                      1Y       2Y       CJ>
Yeast extract                                      1Y       3Y       CJD
                                       C-29

-------
APPENDIX 2:

         QUALITY CONTROL PROGRAM MONTHLY REPORT*

                       Division of Microbiology
                       Department of Pathology
                          Hartford Hospital
                            Date	

           .#.                                             Deficiency
     £    '*   -                                 Conducted   observed
                                               as scheduled   (if YES
                                                (if NO give  give expla-
                                               explanation   nation
                                                 sheet A)     sheet B)
                                                Yes 'No f Yes1  4*o  -
Methods
  Procedure book
  Evening shift review
  Night shift review
  Referee samples CAP
                CD€
  Check samples CAP
                ASCP
  Evaluation (Conn. State
  Department of Health)
  Reference cultures
  Blind unknowns
    Clinical microscopy
    Bacteriology
    Serology
    Mycology
    Parasitology
    Mycobacterium
    Fluorescent miscroscopy
  Susceptibility (disc control)
  Antimicrobial susceptibility
  Tube dilution

Biological materials
  Antisera (bacteriological)
  Antigens (serology)
  Fluorescent controls
  Reagents

Equipment
  Refrigerator
  Freezers
  Incubators
                                  C-30

-------
Appendix 2-Continued
Conducted
as scheduled
(if NO give
explanation
sheet A)
Deficiency
observed
(if YES
give expla-
nation
sheet B)
                                                Yes  No    Yes   No
 Water baths and heating blocks
 Hot air oven
 Autoclaves
 VDRL rotator
 Inoculating loops
 Inoculating wires
 Safety hood (small)
 Safety hood (large)
 Vacuum pump (lybphile)
 Vacuum pump
 Refractometer
 Glassware
 Detergent
 Grinding motor
 Media room balance
 Microscopes
 Pipettes
 Thermometer

Inventory
 Biologicals-serology
   ASO buffer
   ASO reagent
   ASO standard
   Brucetta
   Cold agglutinin cells
   Fever control (negative)
   Fever control (positive)
   Hemagglutination buffer
   Monospot kit
   Pregnosticon (slide)
   Pregnosticon (tube)
   RA buffer
   RA latex 0.81
   RA plasma fraction II
   RA test kit
   Syphilitic serum (4+)
   Thyroid  kit
   Typhoid "O"
   VDRL antigen and buffer
                                      C-31

-------
Appendix 2-Continued

Conducted
as scheduled
(if NO give
explanation
sheet A)
Deficiency
observed
(if YES
give expla-
nation
sheet B)
                                                 Yes  No    Yes  No
Biologicals
  Ampicillin
  ANF conjugate
  Arizona diphasic
  Arizona monophasic
  Carbenicillin
  Cephalothin
  Chloramphenicol
  Colistin
  Erythromycin
  E. coli A (conjugate) (Difco)
  E. coli A (serological)
  E. coli B (conjugate) ODifco)
  E. coli B (serological)
  E. coli C (conjugate)
  E. coli C (serological)
  Gentamicin sulfate
  Hemophilus type A
  Hemophilus type B
  Kanamycin
  Lincomycin hydrochloride
  MethiciUin
  Naladixic acid
  Neisseria gonorrhoeae conjugate
  Nitrofurantoin
  Omni serum for Pneumococctu
  Oxytetracycline
  Penicillin (disc)
"Abbreviations: CAP, College of American Pathologist*; CDC. Center for Disease Con-
trol; ASCP, American Society of Clinical Pathologists; ASO, antistreptolysin 0; RA,
rheumatoid arthritis; ANF, antinuclear factor.
                                C-32

-------
 Sheet A
Form used to report itemi not monitored as scheduled. Reason must be given along with
corrective action.
Sheet A:  Use for  each surveillance item  not  conducted  as scheduled. Use additional
sheets if necessary.
Item
Reason not conducted:
Item.
 Reason not conducted:
Item.
Reason not conducted:
                                          C-33

-------
•j.Vei Bl                                 :                 •                      i
    Form used to record detection of deficiency through monitoring. Description most
be  thorough and  include corrective action. Ultimate resolution of problem must be
                                       '
Sheet Bl: Use for items when surveillance reveals deficiency.
Item	
Deficient/ observed: Date
Describe:
Date corrective action taken.
Nature of corrective action:
V a.-, deficiency corrected?	Ye-        No Date.
If not corrected, Explain:
Further surveillance needed O
                                       C-34

-------
Sheet 12:
Item

Date
observed

•nd action
taken

lUeatUof
action taken and
OMCjMie*

Procedural

Was
Deficiency
Corrected
Yet No

Further
ramUbnce
needed

                                                     •ft US. VO:l«T».7»VOMAXM
                                      C-35

-------
                                    TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
 1. REPORT NO.

  EPA-600/4-78-043
             3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE

 QUALITY ASSURANCE GUIDELINES FOR BIOLOGICAL TESTING
             5. REPORT DATE
                August 1978
                                                            6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
 Tracer Jitco, Inc.
 Rockville,  Maryland 20852
             B. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
 Tracer Jitco, Inc.
 Rockville,  Maryland 20852
              10. PROGRAM ELEMENT NO.

                IHD621
              11. CONTRACT/GRANT NO.
                                                               68-03-2462
 12. SPONSORING AGENCY NAME AND ADDRESS
 U.S. Environmental Protection Agency-Las Vegas, NV
 Office of Research and Development
 Environmental Monitoring and Support Laboratory
 Las Vegas, Nevada 89114	
              13. TYPE OF REPORT AND PERIOD COVERED
              14. SPONSORING AGENCY CODE
                EPA/600/07
 15. SUPPLEMENTARY NOTES
 16. ABSTRACT
 This guideline document was prepared  to address the need for  a manual of quality
 assurance practices aimed specifically at biological testing.  These guidelines
 draw from the  good practices published for analytical and clinical laboratories,
 and incorporate observations made  in  a number of U.S. EPA laboratories, contractor
 laboratories,  and biological research laboratories in general.  As quality assurance
 aspects of biological testing depend  on the particular test systems being used,
 these guidelines cover the general aspects of quality assurance, aquatic bibassay,
 microblologic  assay,  and mammalian bioassay.  Hopefully, attention to the principles
 presented in this document will assist in Improving the validity and integrity of
 the data generated by biological testing.
 7.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
b.lOENTIFIERS/OPEN ENDED TERMS  C.  COSATI Fkld/GrOUp
Quality Assurance
Microorganisms
Plankton
Fishes
Birds
Mammals
Plants
 Biological Testing
 Methods Standardization
 Biological Sampling
 Good Laboratory Practicee
 Macroinvertebrates
14B
06C.M
 8. DISTRIBUTION STATEMENT

 RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
   Unclassified	
                                                                          21. NO. OF PAGES
    600
                                               20. SECURITY CLASS (This page/
                                                  Unclassified
                           22. PRICi
EPA Form 2220-1 (R«w. 4-77)   PREVIOUS EDITION i» OBSOLETE

-------