C ^J A U.S. Environmental Protection Agency Industrial Environmental Research
IB • f\ Office of Research and Development
Laboratory
Research Triangle Park, North Carolina 27711
EPA-600/7-77-043
April 1977
IERL-RTP PROCEDURES MANUAL:
LEVEL 1
ENVIRONMENTAL ASSESSMENT
BIOLOGICAL TESTS FOR
PILOT STUDIES
Interagency
Energy-Environment
Research and Development
Program Report
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S.
Environmental Protection Agency, have been grouped into seven series.
These seven 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 seven series
are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Jocioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports in this series result from
the effort funded under the 17-agency Federal Energy/Environment
Research and Development Program. These studies relate to EPA's
mission to protect the public health and welfare from adverse effects
of pollutants associated with energy systems. The goal of the Program
is to assure the rapid development of domestic energy supplies in an
environmentally—compatible manner by providing the necessary
environmental data and control technology. Investigations include
analyses of the transport of energy-related pollutants and their health
and ecological effects; assessments of, and development of, control
technologies for energy systems; and integrated assessments of a wide
range of energy-related environmental issues.
This document is available to the public through the National Technical
Information Service, Springfield, Virginia 22161.
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EPA-600/7-77-043
April 1977
IERL-RTP PROCEDURES MANUAL:
LEVEL 1 ENVIRONMENTAL ASSESSMENT
BIOLOGICAL TESTS FOR PILOT STUDIES
by
K.M. Duke, M.E. Davis, and AJ. Dennis
Battelle-Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
Contract No. 68-02-2138
Program Dement No. EHE623
EPA Project Officer: Larry D. Johnson
Industrial Environmental Research Laborator
Office of Energy, Minerals, and Industry
Research Triangle Park, N.C. 27711
Prepared for
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Washington, D.C. 20460
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ACKNOWLEDGEMENT
The preparation of the biological procedures manual was conducted under
the direction of Larry Johnson, Industrial Environmental Research Laboratory,
Research Triangle Park, North Carolina (IERL-RTP), who was the task manager.
This task was part of EPA's Environmental Assessment contract number 68-02-
2138 with Battelle's Columbus Laboratory. Battelle was responsible for
editing this manual with Kenneth Duke acting as the Task Order manager and
Herman Nack as the program manager.
Special acknowledgement is given to the Environmental Assessment
Steering Committee. This committee was composed of EPA personnel in both
the Office of Energy, Minerals and Industry (OEM!) and the Office of Health
and Ecological Effects (OHEE) and had the responsibility of selecting the
procedures to implement the phased approach to environmental assessment.
Members of the subcommittee established to define the bioassays to be used
•
for Level 1 testing: Robert Botts, Environmental Research Laboratory (ERL) -
Corvallis; James Dorsey, IERL-RTP; William Horning, ERL-Duluth (Newtown); Joellen
Huisingh, Health Effects Research Laboratory (HERL) - RTF; Larry Johnson,
IERL-RTP; Jerry Stara, HERL-Cincinnati; Gerald Walsh, ERL-Gulf Breeze; and
Michael Waters, HERL-RTF.
ii
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CONTENTS
Page
INTRODUCTION ' Vli
HAPTER I. STRATEGY AND GENERAL INFORMATION 1
1-1 Definition of Strategies 1
1.1.1 The Phased Approach 2
1.1.2 Strategy of the Phased Approach 3
1.1.2.1 Definition of Level 1 Sampling 4
and Analysis
1.1.2.2 Definition of Level 2 Sampling 5
and Analysis
1.1.2.3 Definition of Level 3 Sampling 5
and Analysis
1.2 Multimedia Sampling Procedures 5
1.2.1 Classification of Streams for Sampling Purposes 6
1.2.2 Phased Approach Sampling Point Selection 6
Criteria
1.2.3 Stream Prioritization Using the Phased Approach 9
1.3 Data Requirements and Fre-Test Planning 9
1.3.1 Process Data Needs 10
1.3.2 Pre-Test Site Survey 10
1.3.3 Pre-Test Site Preparation 11
1.4 Analysis of Samples 12
CHAPTER II. SAMPLING 13
2.1 Introduction 13
2.2 Gas and Vapor Sampling 15
2.3 Sampling of Gaseous Streams Containing Particulate Matter 15
2.4 Fugitive Emissions Sampling 16
2.4.1 Airborne Fugitive Emissions 17
2.4.2 Waterborne Fugitive Emissions 17
•2.5 Liquid and Slurry Sampling 18
2.6 Solid Sampling 18
CHAPTER III. LEVEL 1 BIOASSAY TECHNIQUES 20
3.1 Introduction 20
3.2 Sample Handling 21
3.2.1 Introduction 21
itt
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CONTENTS (Continued)
Page
3.2.2 Gaseous and Farticulate Samples 24
3.2.3 Liquid Samples 26
3.2.4 Solid Samples 26
3.2.5 Range-finding and Definitive Tests 27
3.3 Health Effects Tests 27
3.3.1 Salmonella Bacterial Mutagenesis Assay (Ames1) 27
3.3.1.1 Method Description 27
3.3.1.2 Results 31
3.3.2 Cytotoxicity Assay 31
3.3.2.1 Rabit Alveolar Macrophage (RAM) Assay 32
3.3.2.2 Human Lung Embryo Fibroblast (WI-38) Assay 35
3.3.2.3 Clonal Toxicity Assay 35
3.3.3 Acute In Vivo Test in Rodents 36
3.3.3.1 Method Description 37
3.3.3.2 Reports 39
3.3.3.3 Discussion 39
3.4 Ecological Effects Tests 40
3.4.1 Freshwater Algal Assay Procedure: Bottle Test 40
3.4.1.1 Introduction 40
3.4.1.2 Method Description 40
3.4.1.3 Results 46
3.4.1.4 Discussion 50
3.4.2 Acute Static Bioassays With Freshwater Fish
and Daphnia 50
3.4.2.1 Introduction and Rationale 50
3.4.2.2 Method Description 52
3.4.2.3 Results 61
3.4.3 Bioassay With Unicellular Marine Algae 65
3.4.3.1 Introduction 65
3.4.3.2 Method Description 66
3.4.3.3 Procedure 67
3.4.3.4 Results 71
3.4.4 Static Bioassays With Marine Animals 71
3.4.4.1 Introduction 71
iv
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CONTENTS (Continued)
Page
3.4.4.2 Method Description 72
3.4.4.3 Procedure 74
3.4.4.4 Results 75
3.4.5 Stress Ethylene Plant Response 76
3.4.5.1 Method Description 76
3.4.5.2 Results 78
3.4.5.3 Discussion 78
3.4.6 Soil-Litter Microcosm Test . 79
3.4.6.1 Introduction 79
3.4.6.2 Method Description 80
3.4.6.3 Results 82
3.4.6.4 Discussion 83
3.5 Reporting Format 85
CHAPTER IV. LEVELS 2 AND 3 BIOASSAY TECHNIQUES 86
4.1 Introduction 86
4.2 Approach to Health Effects Testing 87
4.3 Approach to Ecological Effects Testing 87
REFERENCES 89
APPENDIX 93
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ILLUSTRATIONS
Page
1. Bioassay Protocol for Gases and Suspended Particulate 7
Hatter Streams
2. Bioassay Protocol for Liquid and Solid Streams 8
3. Biological Analysis Overview 25
4. Diagram of a Soil Mcrocosm Unit 81
5. Sample Plot of Cumulative Calcium Loss as a Function of
Time for 4 Dosage Levels of a Contaminant 84
TABLES
Page
1. Level I Sample Fractions (Bioassay) 14
2. Level 1 Minimal Test Matrix 22
3. Level 1 Health and Ecological Test Requirements 23
4. Physical Examinations in Acute Toxicity Tests in Rodents 35
5. Macronutrients Needed for Algal Nutrient Medium 41
6- Micronutrient Stock Solution 42
7. Recommended Prophylactic and Therapeutic Treatments 57
for Freshwater Fish
8. Percentage of Ammonia That is Unionized in Distilled 62
Water at Different Temperatures and pH's
9. Composition of Mixes to be Added to Algal Growth Media 6g
vi
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INTRODUCTION
This bioassay procedures manual has been prepared as a guide for
pilot studies to be conducted by the Industrial and Environmental Research
Laboratory of the Environmental Protection Agency, Research Triangle Park,
North Carolina. To assist in its preparation, a subgroup of the Environ-
mental Assessment Steering Committee was formed. The subcommittee, com-
posed of EPA experts in health and ecological effects, was given the
responsibility of recommending specific bioassays. The subcommittee
recommended an initial series of tests, which was reviewed by the committee
as a whole, various bioassay experts within EPA, and others in industry and
universities. This manual presents the agreed upon Level 1 biotests and is
the result of the efforts of the Environmental Assessment. Steering Committee.
It is written so that the sampling and analysis professional can plan
and execute the sampling and bioassay portion of an environmental source
assessment program. This manual is not intended for use by an inexperienced
professional staff or by technicians. The recommended biotests for
testing the toxicity and mutagenicity of feed and waste streams of industrial
processes is presented. Also included is a brief summary of procedures for
collecting the samples to be tested. A more detailed discussion of the
sampling program is provided in the companion procedures manual, "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment" (Ref. 1), which also
provides the procedures for chemical and physical testing of industrial
process feed and waste streams.
The bioassay procedures in this manual are designed to complement the
chemical and physical procedures and to be an integral part of the phased
environmental assessment. They apply to Level 1. The purpose of
Level 1 efforts is to obtain preliminary environmental assessment information,
identify problem areas, and provide the basis for the prioritization of streams
for further consideration in the overall assessment. A detailed discussion
of the approach along with the criteria used for method selection is given
in Chapter I.
Chapter II of this manual briefly discusses the types of Level 1
sampling activities that can be used in most industrial complexes: gas
and vapor samples, gaseous streams containing particulate matter, fugitive
emissions sampling, liquid and slurry sampling, and solids sampling. In
vii
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this way, the complex and difficult task of organizing the manpower and
equipment necessary for successful field sampling is facilitated. For
each sample type, the discussion focuses on the general problem, prepara-
tions needed for sampling, the actual sampling procedures, and packaging
of samples for shipment.
Chapter III specifies the Level 1 bioassay schemes. The schemes
identify the methods of analysis, anticipated output, and predicted level
of effort required for implementation. The format for presenting the
results of the tests is also given.
Chapter IV provides a brief discussion of a passible approach to
Levels 2 and 3 biological testing.
vlil
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CHAPTER I
STRATEGY AND GENERAL INFORMATION
The Industrial and Environmental Research Laboratory of the Environ-
mental Protection Agency, Research Triangle Park, North Carolina (IERL-RTP)
has developed a three-phased approach to performing an environmental source
assessment—the testing of feed and waste streams associated with industrial
processes in order to define control technology need. Each phase involves
distinctly different sampling and analytical activities. While all three
phases are briefly described in Section 1.1, this biological procedures manual
focuses on the Level 1 sampling and bioassay effort. A second manual provides
the chemical and physical Level 1 procedures and a detailed discussion of
sampling (Ref. 1).
This manual describes for an experienced professional a set of samp-
ling and analytical procedures for biological testing which are compatible
with the information requirements of a Level 1 environmental assessment.
An environmental assessment involves multimedia environmental source sampling.
The sampling techniques described in Chapter II will provide an adequate
sample of fugitive air and water emissions, ducted air and water emissions,
liquids and slurries, and solids for the analyses described in Chapter III.
Finally, Chapter IV briefly discusses Levels 2 and 3 of the phased approach
to environmental source assessment.
1.1 DEFINITION OF STRATEGIES
It should be stressed that the results of Level 1 tests are not
to be used for regulatory recommendations, nor are they to be used as tests
of acceptability or non-acceptability. The three-phased sampling and
analytical strategy was developed to focus available resources (both manpower
and dollars) on emissions which have a high potential for causing measurable
health or ecological effects, and to provide chemical and biological infor-
mation on all sources of industrial emissions. Discussions of this philosophy,
the information cost-benefits, and a summary of the application of the
phased approach to sampling and analysis follow.
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1.1.1 The Phased Approach
The phased approach requires three separate levels of sampling and
analytical effort. The first level (1) provides preliminary environmental
as.
assessment data, (2) identifies problem areas, and (3) generates the data
needed for the prioritization of energy and industrial processes, streams
within a process, and components within a stream for further consideration
in the overall assessment. The Level 2 sampling and analysis effort,
is designed to provide additional information that will confirm and expand
the information gathered in Level 1. Level 1 results serve to focus
Level 2 efforts. The Level 2 results provide a more detailed characteriza-
tion of biological effects of the toxic streams, define control technology
needs, and may, in some cases, give the probable or exact cause of a given
problem. Level 3, utilizes Level 2 or better sampling and analysis method-
ology in order to monitor the specific probelms identified in Level 2 so
that the toxic or inhibitory components in a stream can be determined exactly
as a function of time and process variation for control device development.
Chronic, sublethal effects are also monitored in Level 3.
To meet the environmental source assessment requirement of comprehensive-
ness, the phased approach provides for physical, chemical, and biological
tests. Physical and chemical characterization of environmental emissions
is critical to the definition of need for and design of control technology.
However, the final objective of the Industrial Environmental Research
Laboratory's environmental assessment is the control of industrial emissions
to meet environmental or ambient goals that limit the release of substances
that cause harmful biological (health and ecological) effects. Consequently,
the testing of industrial feed and waste streams for biological effects is
needed to complement the physical and chemical data and ensure that the
assessment is comprehensive. -Biological testing can provide a direct measure
of toxicity and mutagenicity of substances to organisms that the other tests
cannot. This is especially important when dealing with substances for which
there is little available data on toxicity or when assessing complex mix-
tures where synergisms and antagonisms may alter the toxicity of the indivi-
dual components. The use of biological tests in concert with the other
tests will provide a better data base for the prioritization of streams for
further study on the process of defining the need for control technology.
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1.1.2 Strategy of the Phased Approach
The phased approach recognizes that it is impossible to prepare for every
conceivable condition on the first sampling or analysis effort. In some cases,
unknown conditions and components of streams will result in unreliable informa-
tion and data gaps that will require a significant percentage of the sampling
or analysis effort to be repeated.
There is a possibility that many streams or even the entire installa-
tion may not be emitting hazardous substances in quantities of environmental
significance. Conversely, certain streams or sites may have such problems
that a control technology development program can be initiated in parallel
with a Level 2 effort. If either of these situations could be determined
by a simplified set of sampling and analysis techniques, considerable
savings in both time and money could result.
A second possibility is that budgetary limitations may require pri-
oritizing a series of installations so that the available funds can be used
in assessing only those installations most in need of control technology. Here
again, a simplified sampling and analysis methodology would be advantageous
to the overall environmental assessment effort.
The phased approach offers potential benefits in terms of the quality
of information that is obtained for a given level of effort and in terms
of the costs per unit of information. This approach has been investigated
and compared to the more traditional approaches (Ref. 2) and has been found
to offer the possibility of substantial savings in both time and funds
required for assessment.
The three sampling and analysis levels are closely linked in the
overall environmental assessment effort. Level 1 identifies the questions
that must be answered by Level 2, and Level 3 monitors the problems identified
in Level 2 to provide information on chronic effects and for control device
design and development. The following situation is an example of this
procedure.
Level 1 biological testing indicates that a small quantity of an effluent
has inhibitory effects on algal growth, adverse effects on a specified per-
centage (EC50) of the population in a static bioassay, and gives a positive
microbial mutagenicity test. Level 1 chemical testing indicates further that
polycyclic organic materials (POM) might be present in significant amounts.
Considering these results, Level 2 biological sampling and analysis will be
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designed to determine such factors as toxic effect over a long time period,
bioaccumulation at low trophic levels (primary producers and consumers), and
persistance of toxicity in the receiving waters. Level 2 chemical testing
will be used to further identify and quantify the POM compounds and any other
significant materials as accurately as possible. This combination of bio-
logical and chemical testing can identify the exact nature of the toxic sub-
stance (s) and determine if a complex biological effect such as synergism,
antagonism, or bioaccumulation is occurring. Level 3 testing will be used
for long-term, continuous monitoring. Chemical testing will provide informa-
tion on seasonal or feedstock variations of the previously identified toxic
substance(s). Long-term biological testing will serve as an integrator of
such variations. In addition, Level 3 biological testing will identify
possible chronic health and ecological effects. The entire data package can
then be used to design the control technology research and development pro-
gram for the stream.
A detailed explanation of Level 1 sampling and analyses along with the
expected outputs is given in the following sections. For Level 2 and 3,
only the philosophy of the approach and some examples of the kind of tests
that might be used are given. A recommended protocol for Level 2 and 3 is
beyond the scope of this manual.
1.1.2.1 Definition of Level 1 Sampling and Analysis
The Level 1 sampling and analysis goal is to identify the pollution
potential of a source. At the initiation of an environmental assessment,
little is known about the specific sampling requirements of a source both
practically and technically, and hence the emphasis is on survey tests.
For this reason, no special procedure is employed in obtaining a statis-
tically representative sample and the accuracy of the analysis is depend-
ent on the characteristics of the sample. At this level, sampling and
analysis is designed to show within broad general limits the presence
or absence and the approximate levels of toxicity associated with a
source. Toxic effects are further divided into health effects and
ecological effects.
The results of this phase are used to establish priorities for addi-
tional testing among a series of energy and industrial sources, streams
within a given source, and components within streams. Level 1 has as its
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most important function the selection, in order of relative toxicity, of
specific streams and components for the Level 2 effort. It delineates
specific sampling, analysis, and decision-making problem areas, and directs
and establishes the methodology of the Level 2 effort so that additional
information needs can be satisfied.
1.1.2.2 Definition of Level 2 Sampling and Analysis
The Level 2 sampling and analysis goal is to provide definitive data
required in the environmental assessment of a source. The basic questions
and major problem areas to be addressed have been defined in Level 1 for
maximum cost and schedule efficiency. Consequently, Level 2 sampling and
analysis is characterized by obtaining statistically representative samples,
expanding information on the nature of the toxicity or mutagenicity, and
finally by identifying and quantifying the toxic substance(s).
Level 2 analyses are the most critical of all three levels because they
must provide a validation or confirmation of the results obtained in Level
1 and give a better characterization of the potential of the sample to
cause adverse environmental effects.
Level 2 thus provides sufficient detailed information concern-
ing the problems delineated by Level 1 that control stream prior-
ities, total environmental insult, and an initial estimate of process/
control system regions of overlap can be established.
1.1.2.3 Definition of Level 3 Sampling and Analysis
Level 3 testing is very specific to the stream components being
monitored, and it is not possible to define the exact tests that may be
necessary. The sampling and analysis are directed towards the integra-
tion of effects over time to account for seasonal or feedstock variations.
These efforts are also geared to assess the chronic health and ecological
effects of the stream components.
.1.2 MULTIMEDIA SAMPLING PROCEDURES
The Level 1 procedure described in this manual can be utilized to
acquire process samples, effluent samples, and feed stock samples. The
Level 1 environmental assessment program must, at a minimum, acquire a
sample from each process feed stock stream, from each process effluent
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stream, and of fugitive air/water emissions. The feed stream data are
necessary to establish a baseline for comparison. The effluent stream
sampling program is required to determine the mass emissions rate and
the environmental insult which will result. Sampling and analytical pro-
cedures which are required for a comprehensive environmental source assess-
ment must be multimedia in nature.
1.2.1 Classification of Streams for Sampling Purposes
The basic multimedia sampling strategy has been organized around the
five general types of sampling found in industrial and energy producing
processes rather than around the analytical procedures that are required
on the collected samples. This facilitates the complex and difficult
task of organizing the manpower and equipment necessary for successful
field sampling and establishing meaningful units of cost.
The five sample types are:
(1) Gas/Vapor - These include samples from input and output
process streams, process vents, and ambient air.
(2) Liquid/Slurry Stre*""" - Liquid streams are defined as those
containing less than 5 percent solids. Slurry streams are defined
as those containing greater than 5 percent solids.
(3) Solids - These include a broad range of material sizes
from large lumps to powders and dusts, as well as non-
flowing wet pastes. Because the distinction between solids
and slurries can become blurred, the reader should consult
Reference 1 when in doubt.
(4) Particulate or Aerosol Samples - This involves sampling in
contained streams such as ducts or stacks.
(5) Fugitive Emissions - These are gaseous, particulate, or liquid
emissions from the overall plant or various process units.
Flow diagrams which show the overall relationship of the samples to
the Level 1 analysis'scheme are presented in Figures 1 and 2.
1.2.2 Phased Approach Sampling Point Selection Criteria
The selection of sampling points in processes where phased level
sampling techniques are employed relies cin the concept previously stated:
that Level 1 sampling is oriented towards obtaining data with relaxed
accuracy requirements for determination of the pollution potential
of a sxmrce, whereas Level 2 sampling is intended to acquire more accurate
data necessary for a definitive environmental assessment on prior-
itized streams. Stream parameters such as flow rates, temperature, pressure
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GASES AND SUSPENDED
PARTICULATE MATTER
(DUCTED AND FUGITIVE)
Gaseous Grab
Sample
Particulate Matter
Extract from Sorbent
Plant Stress
Ethylene
Soil Microcosm
Microbial
Mutagenicity
Cytotoxicity
Rodent
Acute Toxicity
Microbial
Mutagenicity
Cytotoxicity
FIGURE 1. BIOASSAY PROTOCOL FOR GASES AND SUSPENDED PARTICULATE MATTER STREAMS
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LIQUIDS
AND
SOLIDS
Soil Microcosm
Microbial
Mutagenlcity
Cytotoxicity
Rodent
Acute ToxicIty
Marine or Freshwater
Ecology, as appropriate
oo
Freshwater -
Algal Bottle Test
Fathead Minnow Toxicity
Daphnia Toxicity
Marine -
Algal Test
Sheepahead Minnow
Toxicity
Grass Shrimp Toxicity
FIGURE 2. BIOASSAY PROTOCOL FOR LIQUID AND SOLID STREAMS
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and other physical characteristics will be obtained on both levels
within the objectives of a given level of sampling. The recommendations
in this manual are restricted to Level 1 sampling and analysis criteria.
In most cases, Level 1 sampling methods encompass approved standard
Environmental Protection Agency (EPA), American Society for Testing and
Materials (ASTM), and American Planning Institute (API) techniques.
Modifications are made to these techniques to adapt them to the time and
cost constraints consistent with the Level 1 sampling philosophy. These
modifications include: (1) reducing port selection criteria; (2) eliminating
the requirements for traversing, continuous isokinetic sampling, and
replicate sampling in the collection of particulate matter; and (3) using
grab samples for ambient, water, and solid samples.
1.2.3 Stream Prioritization Using the Phased Approach
Industrial and energy producing processes are highly complex systems
consisting of a wide variety of interrelated components. Level 1 sampling
will show that many influent and effluent streams have no environmentally
significant impact. These data can be used to reduce the number of samples
required for Level 2 substantially, and can permit reallocation of resources.
Thus, comprehensive stream prioritization based on the Level 1 sampling and
analysis effort will identify streams with widely varying environmental
priorities. In many cases, the Level 1 information will be sufficient to
eliminate certain streams entirely from the Level 2 effort. In other cases,
limited resources may require the omission of certain low priority streams.
.1.3 DATA REQUIREMENTS AND PRE-TEST PLANNING (References 1-3)
The final decision to test a particular plant will depend on the results of
the prioritization studies, on the preliminary selection process based on the
site selection criteria of a given program, and on the data requirements of
the overall program or general EPA objective.
Before the actual sampling and analysis effort is initiated, the data
requirements must be established and used to help identify test require-
ments as well as any anticipated problems. The following paragraphs pre-
sent a general summary of these requirements and planning functions which
must be applied or expanded to meet the needs of the individual tests to
be performed. Specific recommendations concerning data requirements asso-
ciated with each of the process streams are discussed in the appropriate
chapters of this manual.
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1.3.1 Process Data Needs
Before traveling to a plant for a pre-test site survey, it is necessary
to become familiar with the process used at the site. This involves
understanding the chemistry and operational characteristics of the various
unit operations as well as any pollution control processes. It is partic-
ularly important to know that detailed relevant process data are necessary
for the sampling and analysis effort as well as for the overall environ-
mental assessment in order to:
(1) Know where to look for waste streams, including fugitive
emissions.
(2) Know how plant operating conditions are likely to affect
waste stream flow rates and compositions.
(3) Permit design of a proper sampling program.
(4) Draw conclusions about pollutants likely to be found in
waste streams.
(5) Extrapolate to conditions in other sizes of the system being
assessed from thorough knowledge of the interrelationships
among process variables.
(6) Develop a checklist of the requisite data Including tempera-
tures, pressures, flow rates, and variations of conditions
with time for the pre-test site survey.
For any given sampling and analysis task, the data collected must
be consistent with the overall Level 1 objectives. Thus, the minimum
amount of data for a given stream is flow rate per unit time at a given
temperature and pressure. Additional data that may be necessary are
average flow per unit time, the effect of process variations on stream flow
and composition, and normal variations in flow and compositions with
variations in process cycling. It is expected that professional
sampling and analysis personnel in conjunction with the EPA Project
Officer and the Process Measurements Branch—Industrial Environmental
Research Laboratory—Research Triangle Park (PMB-IERL-RIP) will select
the appropriate data requirements for a given Industry.
1.3.2 Pre-test Site Survey
After establishing the necessary process data needs and selecting a
tentative set of sampling points, a pre-test site survey should be per-
formed. At the test site, the survey team should meet with the plant
engineer to verify the accuracy of the existing information and arrange
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for the addition of any missing data. Using this information, the survey
team will then proceed to select the actual sampling sites with the follow-
ing criteria in mind:
(1) The sampling points should provide an adequate data base
for characterizing the environmental impact of the source.
(2) When possible, each sampling point should provide a
representative sample of the effluent streams.
(3) The sampling site must have a reasonably favorable
working environment. The survey personnel must consider
the temperature and noise level in the sampling areas,
if protection from rain or strong winds exists, and
whether safe scaffolding, ladders, pulleys, etc. are
present.
Identification of support facilities and services is an important
aspect of the site survey. Generally, the sampling team will arrive in
a mobile lab unit carrying (1) sufficient electric generating capacity
to operate all testing and support equipment and (2) a water tank for
essential services. In an effort to minimize (1) the requests made upon
the operators for support services and (2) to minimize scheduling problems,
it is desirable but not mandatory that the mobile lab unit operate inde-
pendently of external support facilities. Where available, electrical
power and water services may be connected for auxiliary service.
The results of the pre-test site survey must be sufficiently detailed
to enable complete definition of the field-test problem of sampling the
correct process stream at the proper sampling location and using the appro-
priate methodology prior to arrival of the field-test team at the source
site.
1.3.3 Pre-test Site Preparation
Since in most cases the manpower requirements for site preparation are
low to moderate, a relatively low effort is assumed for site preparation.
Major modifications required in extreme cases are out of the scope of this
manual.
Thus, it is assumed that the erection of scaffolding and the provision
of power will be a major part of site preparation; a further assumption is
that the required manpower will be associated to a large extent with stack
sampling, the most complex sampling procedure. Preparation of other sites
is assumed to be minimal and/or part of the actual sampling procedure. The
installation of special samplers, valves, fittings, etc. is considered beyond
the scope of a Level 1 sampling effort.
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1.4 ANALYSIS OF SAMPLES
Chapter III discusses biological analysis procedures that will provide
the data requirements specified above for Level 1 environmental assessment.
These discussions identify the proposed methods of analysis, the type and
format of data generated, and the estimated time required to implement
each analysis.
There are two categories of analysis:
(1) Health Effects Bioassays - These tests are used to detect
toxic and/or mutagenic effects in bacterial and mammalian
systems. These include a mutagenesis test using
Salmonella (Ames test), an acute toxicity test using
rodents, and a cellular toxicity test using rabbit
alveolar macrophages and other mammalian cells.
(2) Ecological Effects Bioassays - These tests are used to
detect toxic or inhibitory effects on the soil-litter
community and on selected species of fish, macro-
invertebrates, algae, and higher plants.
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CHAPTER II
SAMPLING
2.1 INTRODUCTION (References 1, 3, 4)
Level 1 sampling stresses the concept of completeness by presuming
that all streams leaving the process will be sampled unless empirical
data equivalent to Level 1 programmatic outputs already exists. Further,
Level 1 sampling is not predicated on a priori judgements as to the
composition of streams. The techniques prescribed presume that whatever
prior knowledge is available is at best incomplete. Predictive and
extrapolation techniques employed during source assessments serve as
a cheek on the empirical data and not as a replacement for it (Ref. 3).
Level 1 sampling programs are designed to make maximum use of
existing samples and stream access sites. While some care must be
exercised to ensure that the samples are not biased, the commonly
applied concepts of multiple point, isokinetic, or flow proportional
sampling are not rigidly adhered to. Normally, a single sample of
each stream should be collected under average process operating conditions
or, alternatively, under each condition of interest. These samples
should be time-integrated over one or more process cycles. When a
series of discreet samples results, they are combined to produce a
single "average" for analysis.
This chapter discusses briefly the general methodology for obtaining
gaseous, particulate, liquid, and solid feedstock and waste-stream samples
for the biological analyses. Only an overview of the sampling procedures
is presented. A more detailed description of procedures is found in
the IERL-RTP Procedures Manual: Level 1 Environmental Assessment (Ref.l).
The sampling methodology is designed to collect, prepare, and ship the
samples in a manner compatible with the sample requirements for the
biological tests. Table 1 characterizes the samples that will be
available for the bioassays.
13
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TABLE 1. LEVEL I SAMPLE FRACTIONS (BIOASSAY)
Source Sample
Air
Process Gases SASS - 2-C
+SASS - 3
SASS - 4
Process Fugitive SASS - 6
Emissions
SASS - 7-C
GRAB
HV - 1
Fugitive Gases HV - 2-C
GRAB
Description
Solids > 3p,m
Solids l-3p.ro
Solids < l|im
XAD-2 extract
Gas
Solids
XAD-2 extract
Gas
Comments
-
May be inorganic, organic or both.
Approximately 50 grams maximum
Same as above except 20 grams maximum
On fiberglass mat. Combine with
SASS -4 if possible
Organics in pentane. If solids
present, may have additional solvent
present. 5 grams maximum
Organic, inorganic, or both.
Unlimited sample
Organic, inorganic, or both.
Less than 0.5 grams
Same as SASS - 7-C.
Less than 0.5 grams
Same as GRAB above
Water
All Sources W - 1
Solids
Piles, Conveyors, S - 1
Bins, etc.
Untreated
Untreated solids
Aqueous, organic, solids; unlimited
sample except for fugitive run-off
Coal, ash, residues, products;
organic and inorganic; unlimited
sample
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2.2 GAS AND VAPOR SAMPLING
A single grab sample of each stream to be tested is sufficient for
Level 1 needs. The grab sample may be taken in one of three ways
depending on the pressure of the stream in question. The three grab
sampler types are high pressure line, grab purge, and evacuated grab.
Only the lower molecular weight organic and inorganic gaseous effluents
are sampled using these procedures. The particulate content along with
high molecular weight hydrocarbons are obtained via the Source Assessment
Sampling System (SASS) discussed below.
For chemical analysis on-site gas chromatography, a 3-liter, glass-
bulb sample container is used. For biological testing, a much larger
quantity of gas is required and the sample must be shipped for analysis;
therefore, the sample must be collected in a larger container. Various
containers can be used including large Teflon or Tedlar bags, and
rigid glass or stainless steel containers.
2.3 SAMPLING OF GASEOUS STREAMS CONTAINING PARTICULATE MATTER
Streams, vents, and effluents containing particulate matter are
sampled using the Source Assessment Sampling System* (SASS) developed
by the Industrial Environmental Research Laboratory of the Environmental
Protection Agency, Research Triangle Park (IERL-RTP). (Sampling of
fugitive gaseous emissions containing particulate matter is accomplished
using the high-volume sampler discussed below.) The SASS sampling train
consists of a stainless steel probe which enters an oven module containing
three cyclones and a filter. Size fractionation is accomplished in the
series to provide large quantities of particulate matter size-classified
into three ranges: (1) >10pan, (2) 3pm to 10M
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Whether or not quantities sufficient for analysis have been
acquired cannot be determined until subsequent gravimetric analyses have
been performed; therefore, some sampling guidelines should be followed.
3
At least one process cycle and 30 standard m of the process effluent
are to be sampled. If the process is not cyclic in nature, the 30
3
standard m must still be satisfied over a period of time conducive to
obtaining a sample representative of process conditions. A sampling
duration of 5 hours generally satisfies this requirement and provides
enough material for chemical analysis. A longer period may be necessary
to obtain adequate samples for biological analysis.
At the conclusion of the sampling run, the train is disassembled
and transported to the mobile lab unit or prepared work area. The
three cyclones and the filter chamber must each be tapped and brushed to
remove all contents on the walls. Liquids should not be used in removing
particulates for biological analysis. The IQim and 3y,m cyclone partic-
ulates are combined as are the liun and filter chamber particulates. The
filter should also be tapped to remove particulate matter, which is
added to the smaller fraction. Since it may be very difficult to remove
all particulates, this fraction may be tested separately. The filter
should not be brushed or scraped as this process leaves glass fibers in
the sample. For shipping, the two particulate fractions are each trans-
ferred to a tared nalgene container. The filter is also shipped in a
tared petri dish. The XAD-2 cartridge is removed from its container and
placed into a widemouth, amber-glass jar. All containers are shipped to
the environmental assessment laboratory for further preparation, splitting,
and shipping to appropriate labs for biotesting.
2.4 FUGITIVE EMISSIONS SAMPLING
Fugitive emissions are those air and water pollutants generated by
any activity at an industrial site that are transmitted from their,
source directly into the ambient air or receiving surface and ground
waters without first passing through a stack, duct, pipe, or channel
designed to direct or control their flow. Airborne emissions consisting
of particulate matter and gaseous pollutants may be generated by sources
16
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enclosed in buildings and transmitted to the atmosphere through structural
openings or vents, or generated by sources in open areas and transmitted
directly into the atmosphere. Waterborne fugitive emissions, which
consist primarily of suspended and dissolved solids, may be generated
by process leaks and spills, runoff from a wide variety of material storage
piles, and fallout from emissions initially airborne. They are trans-
mitted to surface waters by runoff and to ground waters by infiltration.
2.4.1 Airborne Fugitive Emissions
Airborne fugitive emissions may be of three types each requiring
different sampling procedures: (1) a site source in which no specific
source of emissions can be identified, (2) a specific source which
generates a highly diffuse cloud over an extensive area, or (3) a specific
source which generates an emission which could be generally classified
as a plume. In case 1, samples must be taken at both an upwind and a
downwind location. Particulates are sampled with a high volume sampler
equipped with a 3. Sum filter and a XAD-2 cartridge. Gases are sampled
with an evacuated grab sampler. In case 2, samples are taken at a
downwind location only. The high volume sampler is used to collect
the particulate fraction and an evacuated grab sampler is used to collect
a gaseous sample. In case 3, the plume is sampled using the SASS train
described in the previous section and an evacuated grab sampler.
After sampling, particulates are tapped and brushed from the 3.5p-m
fractionating head and tapped from the filter into a tared nalgene container
for shipping. The XAD-2 cartridges are placed in amber-glass jars.
2.4.2 Waterborne Fugitive Emissions
Waterborne fugitive emissions are sampled using plug collectors.*
The plugs are driven into the ground at selected locations where runoff
will occur. Water runoff samples are similar to airborne fugitive
emissions in that both specific source and site source samples are
obtainable. Collectors placed for general plant runoff are analagous
to site source upwind-downwind samples, and collectors placed for specific
* Plugs are designed and built by Kahl Scientific Instrument Corp.,
P.O. Box 1166, El Cajon, California 92022.
17
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problem areas are analagous to specific source downwind samples. Runoff
samples from specific sources are combined in order to perform
a single analysis to characterize a potential problem area. Waterborne
fugitive emission samples are handled and analyzed as are other liquid
samples, as described in the following section.
2.5 LIQUID AND SLURRY SAMPLING
Many diverse influent and effluent streams exist in liquid or
slurry form. Three sampling methods are used for sampling liquid
streams: heat exchange, tap sampling, and dipper sampling. The heat
exchange system is used for high-temperature lines and involves the
use of a water-cooled condenser system. The condensate from the stream
is collected in a reservoir for later analysis. Tap sampling is
used to sample both moving streams (lines) and non-moving streams
(tanks or drums). It involves taking a sample at a tap from a line or •
tank wall. Dipper sampling is applicable to sluices or open
discharge streams of thick slurry or stratified composition. The dipper
is made with a flared bowl, coated with teflon, and attached handle. It
is inserted into the free-flowing stream so that a sample is collected
from the full cross-section of the stream.
Crude liquid effluent containing aqueous, organic, and solid
material should be tested for health and ecological effects. The material
should generally be transported in tightly sealed, amber-glass containers.
Aqueous samples may be shipped in high-density polyethylene containers.
All samples should be shipped and stored at 4 C and no stabilizers
should be added to samples to be used in bioassays. In addition,
special care should be taken not to agitate the samples or to open
the containers before analysis.
2.6 SOLID SAMPLING
Solid samples range in size from large lumps to fine powders and
dusts and in consistency from anhydrous solids to thick, nonflowing
pastes. Level 1 solid sampling procedures use manual grab sampling
techniques such as shovel or grab sampling, boring techniques including
pipe or thief sampling, and auger sampling.
18
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Samples should be stored in air-tight, high-density polyethylene
containers until ready for analysis. Large samples should be placed
in metal containers lined with polyethylene bags.
19
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CHAPTER III
LEVEL 1 BIOASSAY TECHNIQUES
3.1 INTRODUCTION
Level 1 environmental source sampling procedures provide a set of
samples which represent the "average" composition of solid, liquid, and
gaseous feed or waste streams of industrial processes. Each fraction or
stream will be evaluated with survey techniques to define its basic
physical, chemical, and biological characteristics. The survey methods
or tests selected are compatible with a very broad spectrum of materials
and have sufficient sensitivity to ensure a high probability of detecting
potential environmental problems. The methods and instrumentation have
been kept as simple as possible to minimize cost but still provide the
information required by Level 1 objectives. Each individual piece of
data should add a relevant point to the overall evaluation. Conversely,
since the information from a given test is limited, all the tests must
be accomplished to permit a valid assessment of the sample. This is
particularly true for the bioassays because physical and chemical infor-
mation alone cannot provide a reliable measure of potential biological
response. In addition, only bioassays can detect complex biological
effects such as synergism and antagonism. On the other hand, bioassays
cannot identify the cause of toxicity or mutagenicity or suggest means
of controlling it".~ Thus, physical, chemical, and biological analyses
must be used to complement one another at all three levels of the phased
approach to environmental assessment.
Level 1 biological testing is limited to whole sample testing which
is consistent with the survey nature of this level. The testing of
fractionated samples or specific components of a given sample involves
a degree of specificity appropriate to levels 2 and 3 testing. This
chapter includes a brief description of how the various samples should
be handled in preparation for biological analysis as well as detailed
descriptions of the bioassays recommended for Level 1.
20
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The bioassays include both assessments of health and ecological effects,
Health effects tests estimate the potential mutagenicity, potential or
presumptive carcinogenicity, and potential toxicity of the samples to
mammalian organisms. The ecological effects tests focus on the potential
toxicity of the samples to vertebrates (fish), invertebrates, and plants
in freshwater, marine, and terrestrial ecosystems. The species chosen for
the tests have been used for many documented environmental assessments
in most cases. A total of nine tests comprise Level 1 testing (there are
two tests - marine and freshwater - for the algal and static bioassays).
Table 2 shows which bioassays are to be used with each sample type and
constitutes the minimal bioassay protocol. The approximate time and
amount of sample required for each of the tests is shown in Table 3.
The minimal test matrix (see Table 2) shows several tests to be used
as tests of secondary priority for various sample types. These tests can
give useful Level 1 data and should be used if time, money, and the amount
of sample available for testing permit. This is an example of the flexibi-
lity that is possible in implementing the Level 1 bioassays. The minimal
test procedures should be performed first. If conditions permit, additional
testing may be performed at this Level. This testing would serve to
clarify confusing or inconclusive results or provide additional data that
would permit a better prioritization of the various influent and effluent
streams. Such additional testing, if done, should be designed to meet the
objective of Level 1.
Following the bioassay descriptions is a brief section on the
suggested format for reporting results of the bioassays. Results of all
Level 1 bioassays plus information from chemical and physical Level 1
testing will permit the identification and relative ranking of the feed-
stock or waste streams so that Level 2 testing can be initiated on the
most environmentally significant streams with the testing of lower priority
streams to follow.
3.2 SAMPLE HANDLING
3.2.1 Introduction
Due to the variation in samples which could be received for analysis,
it is impossible to provide explicit instructions on how every sample
should be handled and administered to the test organisms. This ultimately
21
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TABLE 2. LEVEL 1 MINIMAL TEST MATRIX
Sample Type
Water and Liquids
Solids- (Aqueous Extract
Feed, Product, Waste
Gases- (Grab Sample)
Farticulates
Sorb ent- (Extract)
Health Effects Tests
Microbial
Mut agenesis
Microbial
Mut agenesis
(Microbial ',.
Mutagenesis)
Rodent Acute
Toxicity
Rodent Acute
Toxicity
Microbial (Rodent Acute
Mutagenesis Toxicity)
Microbial
Mutagenesis
Cytoxicity
Cytotoxicity
(Cytotoxicity)
Cytotoxicity
Cytotoxicity
Ecology Effects Tests
Algal
Bioassay
Algal
Bioassay
Static
Bioassays
Static
Bioassays
Soil
Microcosm
Soil
Microcosm
Plant Stress
Ethylene
Soil
Microcosm
10
(a) (Recommended Test of Secondary Priority).
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TABLE 3. LEVEL 1 HEALTH AND ECOLOGICAL TESTS REQUIREMENTS
Test System/Level
Approximate
Time Required
(including analysis)
Sample
Microbial mutagenesis (Ames)
Cytotoxicity (mammalian cells)
Range finding toxicity (rats)
Freshwater static bioassay
Algal bottle assay (Freshwater)
Marine static bioassay
Algal Assay (Marine)
Plant stress ethylene
Soil-litter microcosm
2-5 days (3 wks)
2-4 days (3 wks)
14 days (4 wks)
4 days (1 wk)
10-14 days" (3 wks)
4 days (2 wks)
14 days (3 wks)
28 hrs (1 wk)
40 days (8 wks)
1 gram/50 ml
1/2 gram/50 ml
100 grams, 1 L
200 L
50 L
1360 L
1 gram/1 ml
23
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must be left to the discretion of the professional investigator. Some
guidelines are presented below and in Figure 3. Results of chemical
or physical tests done on-site and information from previously completed
biological tests may also be of assistance in making such decisions as
how much effluent to use and how to administer it.
3.2.2 Gaseous and Particulate Samples
Gas samples are to be tested by the plant stress ethylene test. Gas
containers obtained during sampling can be connected directly to an air-
handling system on the greenhouse exposure chambers to make the various
dilutions necessary, or the dilutions can be pre-mixed in a second con-
tainer and from this added to the exposure chambers.
Particulates can be incorporated into the bioassays as solid particles
or aqueous suspensions. In general, only respirable (< 5 (J*n) particles
should be tested as solid particles in the microbial mutagenesis or
cellular toxicity tests. Larger particulates hinder growth by inhibiting
adherence of cells to the flask or by other physical means and cannot be
ingested by cells. The BAH test is an exception and can incorporate the
large particulate fraction as solids. Farticulates of both fraction
sizes (respirable and non-respirable) can be administered to rodents in
a variety of ways including intubation, respiration, and skin painting
depending on the health problem expected to result from exposure to
particulates. For the soil microcosm test, solid particulates can be added
to the surface of the soil cores.
The XAD-2 sorbent is homogenized and extracted with pentane. The
pentane extract must then undergo solvent exchange with, preferably, dimethyl
sulfoxide (DMSO) or acetone before incorporation into the microbial
mutagenesis or cellular toxicity tests as a liquid. Depending on the
nature of the original sample, solvent exchange can be accomplished by
several ways. An equal volume of DMSO can be added to the pentane extract
and heated or evaporated at room temperature to remove the pentane, or
the pentane can be removed first and the residue resuspended in DMSO.
24
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SAMPLE FOR BIOLOGICAL ANALYSIS
CASKS AND SUSPIiNDKI)
PARTICULATE MATTER
LIQUIDS
Aqueous
(<0.2Z
organic)
Microbial
Mutagenesis
Microbial
Mutagenesis
Organic
With
Suspended
Solids
Microbial
Mucagenesis
Solvent:
Exchange
fo
ui
Filter
(2 mm sieve)
Plant Stress
Ethylene
(Microbial
Mutagenesis)
(Cytotoxicity)
(Rodent Acute
Toxicity)
Cytotoxicity
Soil
Microcosm
Microbial Mutagenesis
Cytotoxicity'i
Rodent Acute Toxicity
Algal Bioassay
Static Bioassay
Soil Microcosm
Cytotoxicity
Rodent Acute
Toxicity
FIGURE 3. BIOLOGICAL ANALYSIS OVERVIEW
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3.2.3 Liquid Samples
Generally, liquid containing < 0.2 percent organic solvents can be
added directly to the microbial mutagenesis test. If larger quantities
of various organic solvents are present, and if effects due to dissolved
substances are to be distinguished from those due to the solvent, solvent
exchange must be performed. Final concentrations in the test medium
of 0.5 percent DMSO or 0.01 percent acetone can be tolerated.
Liquids can be administered to rats by such methods as skin painting,
inhalation, ingestion, and injection. Greater concentrations of organic
materials can generally be tolerated with the skin painting technique.
For the ecological tests, samples should be added to the dilution
water without the use of solvents or additives, except water if necessary.
If additives are necessary, they should be kept to a minimum as they may
affect the pH of the test solutions. Organic solvents used to prepare the
sample should also be kept to a minimum. Concentration of solvent in any
test solution in the algal and static bioassays must not exceed 0.5 ml/1.
Temperature and salt content (if salt-water assay) of the effluent may have
to be adjusted before starting the test. Other adjustments may be necessary
depending on the results of on-site chemical tests. For instance, oxygen
may have to be bubbled through the test solution, but agitation of the
solution should be avoided. Samples that contain suspended solid material
can be filtered through a sieve as small as 2 mm.
Addition of organic material or highly acidic or basic additives to
the soil microcosm test should be minimized; however, no strict guidelines
are available at the present.
3.2.4 Solid Samples (Reference 37)
t* i
Solid samples should be finely ground (< 5 Mm particles) and incorporated
as are particulates in the microbial mutagenesis, cytotoxicity, and rodent
acute toxicity bioassays. Solid samples should be ground to < 1 mm particles
for the soil microcosm bioassay. Aqueous extracts of solid samples should
be added to the algal and static bioassays. Aqueous extracts are made
by vigorously shaking or stirring 1 part solid sample and 4 parts water for
30 minutes followed by 1 hour of settling and filtration or centrifugation
of the filtrate as appropriate.
26
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3.2.5 Ranee-finding and Definitive Tests
For all the following biotests, it is usually desirable to conduct a
range-finding or screening test to determine the concentrations that should
be used in the definitive test, unless the approximate toxicity of an
effluent is already known or unless the sample size is limiting. This
preliminary testing can save both time and money. Range-finding tests
should be conducted using three to five widely spaced effluent concentrations
(covering the entire range of 0 to 100 percent effluent, if possible).
Conditions for the range-finding and definitive tests should be kept as
similar as possible; the greater the similarity between the two tests, the
more useful the results of the range-finding tests will be.
3.3 HEALTH EFFECTS TESTS
3.3.1 Salmonella/Microsome Mutagenesis Assay (Ames1) (Reference 5)
The Ames assay is based on the property of selected Salmonella
typhimurium mutants to revert from a histidine requiring state to proto-
trophy due to exposure to various classes of mutagens. The test can detect
to nanogram quantities of mutagens and has been adapted to mimic some mammalian
metabolic processes by the addition of a mammalian liver 9,000 G microsomal
fraction (S-9). The test will be used as a primary screen to determine
the mutagenic activity of complex mixtures or component fractions. It
has recently been demonstrated that most carcinogens act as mutagens. In
extensive testing, the Ames' assay has demonstrated 90 percent accuracy in
detecting known carcinogens as mutagens. Certain known carcinogens are
negative in the test (e.g., asbestos, metals) or weakly positive. False
positives are also known which are mutagenic in the Ames1 system but which
have been found to be noncarcinogenic in mammals. Continued improvement
of the present bacterial strains, addition of new strains, and re-evaluation
of the conventional animal carcinogenesis data should reduce this level of
error, in the near future. The following is intended as a general descrip-
tion of the test; for detailed protocol see the published method. (Reference 5)
3.3.1.1 Method Description
Materials. The species of bacteria to be used are the Salmonella
typhimurium tester strains developed by Dr. Bruce Ames. Specifically,
these are TA-1535, TA-1537, TA-1538, TA-98, and TA-100. The tester strains
27
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are histidine deficient variants and are used to detect frameshift reverse
mutations (TA-1537, -98, and -1538) or base pair substitutions (TA-1535
and -100) as indicated by reversion to prototrophy. For general screening,
strain TA-1538 may be omitted.
Liver Microsome Preparations. The activation system for mutagenesis
screening consists of Aroclor 1254 induced S-9 fraction derived from rat
livers. Rats should be males (Sprague-Dawley) about 200 g each. Induction
is accomplished by a single intraperitoneal injection of Aroclor 1254
(diluted in corn oil to 200 mg/ml) into each rat five days before sacrifice
at a dosage of 0.5 mg/g of body weight. All rats are deprived of food
(not water) 12 hours before sacrifice. The rats are then stunned by a blow
on the head and decapitated. The following steps are carried out at 4 C
using cold sterile solutions and glassware.
The livers (10-15 g) are aseptically removed from the rats and placed
into a cold preweighed beaker containing 10-15 ml of 0.15M KC1. After the
livers are washed and weighed in this beaker they are removed with forceps to
a second beaker containing 3 ml of the KC1 solution per gram of wet liver
weight. The livers are then minced with sterile scissors, transferred to
a chilled glass homogenizing tube, and homogenized in an ice bath by
passing a low speed motor driven pestle through the livers a maximum of
three times. The chilled homogenates are then placed into centrifuge
tubes and centrifuged for 10 minutes at 9000 G at 4 C. The resulting
supernatant is decanted, transferred in 2-ml amounts to small storage tubes,
quickly frozen in dry ice, and stored at -80 C in a low temperature freezer.
This supernatant is known as the S-9 fraction. Sufficient S-9 for use each
day is thawed at-room temperature and kept on ice before and during use.
The extent of bacterial contamination of the S-9 fraction should be deter-
mined. The S-9 mix may be filter sterilized (0.45 Mm porosity filter) if
required. ' -
Metabolic Activation Mixture. The S-9 microsomal mix is prepared
according to the recommendations of Ames. The mix contains per ml: S-9
(0.04-0.1 ml), MgCl2 (8 kimoles.), glucose- 6-phosphate (5 Mmoles), nicotina-
mide adenine dinucleotide phosphate (NADP) (4 ^imoles), and sodium phosphate,
pH 7-4 (100 Mmoles). stock solutions of NADP (0.1 M) and glucose-6-phosphate
(l.OM) are prepared with sterile water,.in appropriate amounts, and maintained
28
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at -20 C. The stock salt solution (0.4M MgCl2 and 1.65 KC1) and phos-
phate buffer (0.2M pH, 7.4) are prepared, autoclaved, and refrigerated.
The S-9 mix is prepared fresh each day and is maintained on ice before
and during use. The activity of the S-9 preparation should be standard-
ized by measurement of its aryl hydrocarbon hydroxylase activity and by
determination of its total protein content.
Bacteriological Media. The minimal-glucose agar medium for histidine
requiring strains used in mutagenesis assays is a 1.5 percent Bacto-Difco
agar in Vogel-Bonner Medium E with 2 percent glucose. Top agar (0.6 per-
cent Difco agar, 0.5 percent NaCl) contains a trace of histidine and biotin
to permit the bacteria to undergo several divisions. The top agar medium
used in the (optional) toxicity assays is the same medium fortified with
f\
30 mg/ml of histidine. Fewer bacteria (about 10 cells/plate) are used in
the toxicity test.
Solvents and Positive Control Chemicals. The solvent for all stock
control chemicals is a spectrophotometric grade dimethyl sulfoxide. The
following compounds are examples of compounds used for positive control
assays. (Ref. 5) Others may be substituted. '
Indicator
Strain Nonactivation Assays Activation Assays
TA 1535 MNNG (N-Methyl-N'-nitro-N- 2-anthramine
nitrosoguanidine)
TA 1537 9- aminoacridine* 2-anthramine
TA 98 Daunomycin 2-anthramine
TA 100 MMS (Methyl methanesulfonate) 2-anthramine
*9-aminoacridine is dissolved in ethanol.
Methods. Each sample should undergo a mutagenesis test, an optional
toxicity test, a positive control test, and a sterility control test. The
plate incorporation test is recommended for routine use. The cells used
for the toxicity and mutagenesis determinations are derived simultaneously
from the same bacterial population. Viability in the toxicity assay will
be used to adjust counts of mutant colonies by the estimated proportion of
the surviving population.
In the plate incorporation assay the sample under investigation is
added directly to the molten top agar and is poured onto the plate along
with the indicator test organism and the liver S-9 activation system.
29
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It is recommended that the plate incorporation test be performed
(preferably in duplicate) initially at 0.01, 0.1, 1 and 10 mg/plate, and
that repeat studies be performed over a narrower concentration range show-
ing positive results in the initial test (up to a maximum concentration
of 20 mg/plate).
The test compound is evaluated with all tester strains in the presence
and absence of the liver S-9 activation system. Liquid samples are assayed
similarly with the maximum sample volume being about one ml. Controls are
included at all times and consist of a control for the spontaneous reversion
rate for each tester strain where the mutagen is omitted, a sterility
check of the mutagen solution, and a positive control consisting of com-
pounds which both do and do not require metabolic activation.
The number of spontaneous reversions/plate should be within the
following limits or those specified in Reference 5.
Strain Acceptable Spontaneous Revertants/Plate
TA-1535 20 ± 10
TA-1537 15 ± 10
TA-98 , 50 ± 25
TA-100 150 ± 75
A sample will be considered negative if the number of induced rever-
tants obtained as compared to the spontaneous revertants is less than
two-fold.
Compounds which indicate positive reversions (at least a two-fold
increase in reversion rate) will be examined by preparing dose response
curves with the tester strain(s) indicating high reversion rates. The
dose response curve will be performed in duplicate at five concentrations
of the sample found to be mutagenic. A two-fold increase in reversion
rate with evidence of increasing reversion with increasing sample concen-
tration shall constitute a positive test. In addition, the dose response
curve should be reproducible.
Sterility Controls. Each bioassay includes a sterility control check
for each test component. These include the S-9 mix, the compound under
test, and the solvent vehicle.
30
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Phenotype Monitoring. After the number of colonies on each plate is
counted, occasional samples (for example, 10) of the revertant colonies are
picked from each plate exhibiting a positive response. These colonies are
restreaked onto minimal agar to confirm if they are revertants or potential
phenocopies.
3.3.1.2 Results
The plate incorporation mutagenesis/toxicity test provides an indica-
tion of both mutagenicity and toxicity of the sample. Toxicity is evidenced
by a reduction in the number of colonies (viability) of bacteria. Counts1
of mutant colonies in the plate incorporation mutagenesis test are adjusted
by multiplying average counts by I/viability. Relative mutagenic activity
is derived from the adjusted average colony counts by representing the
control plate count (vehicle only) as 1.00 and then converting proportion-
ately the adjusted average counts from the plates which contain test sample.
3.3.2 Cytotoxicity Assays (References 6-11)
Cytotoxicity assays employ mammalian cells in culture to measure
quantitatively cellular metabolic impairment and death resulting from
exposure in vitro to soluble and particulate toxicants. Mammalian cells
derived from various tissues and organs can be maintained as short term
primary cultures or in some cases, as continuous cell strains or lines.
Primary cell cultures exhibit many of the metabolic and functional attri-
butes of the original tissues, some of which may be lost with prolonged
passage in culture. Both types of systems have been employed effectively
in eytotoxicity screening of, for example, pharmaceuticals (especially
antibiotics and anti-tumor agents), implantable medical polymers and min-
eral crystals and fibers. More recently, such systems have been applied
in evaluating the relative cellular toxicity of hazardous metallic salts
(Ref. 6) and industrial air particulates (Ref. 7). As compared to conven-
tional whole animal tests for acute toxicity, eytotoxicity assays are more
rapid, less costly and require significantly less sample. The tests can
provide useful information about the relative cellular toxicity of unknown
samples. However, it should be understood that because the assays employ
isolated cells and not intact animals, they can provide only preliminary
and imprecise information about the ultimate health hazards of toxic
chemicals.
31
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The cytotoxicity assays, to be evaluated as part of Level 1 analysis,
employ primary cultures of rabbit alveolar (lung) macrophages (RAM) and
maintenance cultures of strain WI-38 human lung fibroblasts. The alveolar
macrophage* constitutes as essential first line of pulmonary defense by
virtue of its ability to engulf and remove particulate materials which are
deposited in the deep lung. It is appropriate therefore that this cell
type be used to define the acute cellular toxicity of airborne particulates
and associated chemicals. It has been possible to develop a eytotoxicity
screening system -employing these cells to "rank" the toxicity of a series
of industrial particulates collected on a cyclone sampling train similar
to the SASS train (Ref. 7). The strain WI-38 human lung fibroblasts are
perhaps the best characterized diploid human cell available for cytotoxicity
screening. These cells exhibit the major pathways of DNA, RNA and protein
synthesis common to all dividing cells and can be shown to possess a number
of inducible enzyme systems.
For Level 1 assessment these two cytotoxicity assays will be performed
where possible on all solid and liquid effluents. In some cases, a clonal
toxicity assay will be employed for comparative purposes (utilizing as
appropriate cell type, e.g., CHO, L-929 cells).
3.3.2.1 Rabbit Alveolar Macrophage (RAM) Assay
Method Description. Male and female New Zealand white rabbits are
housed individually and fed antibiotic-free Purina Rabbit Chow and water
ad libitum. Clinically healthy rabbits weighing 1.5-2.0 kg are sacrificed
by injection of sodium pentobarbital (150 mg) into the marginal ear vein.
The animals are draped and the site of incision for tracheostomy liberally
irrigated with 70 percent ethanol. Lung lavage jLn situ is carried out
according to the procedure of Coffin et al., (Ref. 8) using prewarmed (37 C)
sterile 0.85 percent saline. The first 30 ml vol instilled into the lungs
is allowed to remain for 15 minutes; five subsequent instillations of 30 ml
each are withdrawn immediately. Lavage fluid found to contain blood or
mucous is discarded. The cellular composition of the pooled lavage fluid '
is determined from Giemsa stained smears and is routinely 95 percent
alveolar macrophages, 2-3 percent polymorphonuclear leukocytes, and 2 percent
lymphocytes.
32
-------
The cells are washed once by centrifugation at 365 G 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 percent), penicillin (100 units/
ml), streptomycin (100 Hg/ml), and kanamycin (100 l^g/ml). (Biologicals
available from Gibco, Grand Island, NY.) Cells are counted by means of
a hemocytometer or automatic cell counting device and diluted to approxi-
mately 1 x 10 cells per ml with supplemented medium. One ml of the cell
suspension is added to each well of 100 x 100 mm 4-place cluster dishes
(Falcon Plastics) containing the effluent sample, and sufficient medium
is added to bring the total volume per well to'2.0 ml. The cultures are
incubated, with rocking, for 20 hours at 37 C in a humidified atmosphere
containing 5 percent C02. At the end of this incubation period, the
cells are trypsinized and cell counts, cell viability, total protein, and
ATP determinations are performed on cells obtained from each well.
Viability Determinations. Viability determinations are performed
as follows:
The culture medium is poured off and retained separately in a culture
tube. Cells are dissociated by using 0.25 percent trypsin in Gibco solu-
tion A. The suspended cells are recombined with the original culture
medium and chilled. Appropriate dilutions, usually 4-fold, are made by
using cold 0.85 percent saline to yield a suspension of no more than
2 x 10 cells/ml. Trypan blue, freshly diluted with 0.85 percent saline
to a final concentration of 0.01 percent is added to an equal volume of
cell suspension for determination of cell viability. Simultaneous deter-
minations of cell viability and cell numbers per milliliter of cell
suspension are performed using a hemocytometer or Cytograf (Biophysics
Systems, Mohapac, NY). Viability is expressed as a percentage. The
viability of cells from control cultures is routinely 95 percent or greater.
Cell numbers are expressed as a percentage of the numbers of cells in
control cultures. Viability determinations are multiplied by total cell
numbers as a fraction of control cell numbers to yield the viability index,
or net number of viable cells as a percent of control.
.„. No. cells exptl
Viability index = Viability (7.) x No. cells control
33
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Protein Determinations. Total protein may be used in the place of
cell number. For determination of total culture protein, cells washed
twice withjjO.85 percent saline are lysed in 1.0 percent sodium deoxycholate
(Schwarz-Mann, Orangeburg, NY) and 0.1 ml aliquots are assayed according
to the method of Lowry et al. (Ref. 9) by using a bovine serum albumin
standard (Nutritional Biochemicals Corp., Columbus, Ohio).
Adenosine Triphosphate (ATP) Determinations. ATP is- determined
according to a procedure supplied with the DuPont model 760 Luminescence
Biometer. Dimethyl sulfoxide (0.4 ml) is used to extract ATP from a 0.1 ml
aliquot of trypsinized cell suspension containing 0.3-0.4 x 10 cells.
After 2 min at room temperature, 5.0 ml of cold 0.01 M morpholinopropane
sulfonic acid (MOPS) at pH 7.4 is added to buffer the extracted sample.
The tube containing the buffered sample is then placed in an ice bath.
Aliquots of 10 Hi are injected into the luminescence meter's reaction
cuvette containing 0.7 mM luciferin (crystalline), 100 units luciferase
(purified and stabilized)*, and 0.01M magnesium sulfate in a total volume
of 100 M-l of 0.01M MOPS buffer, pH 7.4 at 25 C. Light emitted from the
reaction cuvette is measured photometrically in the luminescence meter and
proportional to the ATP concentration of the sample. ATP values are
expressed per 10 cells and as a percent of the control cells.
Measurement of Phagocytic Activity. Phagocytic activity is measured
by addition of 1.1 M*n polystyrene latex particles (Dow Diagnostics,
Indianapolis, Indiana) to alveolar macrophages cultured in Lab-Tek (Miles
Laboratories, Inc. Naperville, 111.) four-chamber microslides (approxi-
mately 25 particles/cell in 1 ml of supplemented medium). Preparation and
maintenance conditions were as previously described. One hour after the
addition of latex particles, the slides are drained, air-dried, and exposed
for 3 min to concentrated Wright stain. The slides are then exposed for
an additional 5-6 min with a 1:1 aqueous dilution of Wright's stain. After
air drying, the slides are placed in xylene for 1 hr to dissolve extracellular
particles according to the procedure of Gardner et al. (Ref. 10). Following
*Unit (1) luciferaae = response to 1-* H«ol« ATP
response to 20 \i£i 14C calibrated light source
34
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an additional drying step, the slides were mounted in permount. Phagocytic
activity is determined under oil immersion by scoring a minimum of 200
cells. Each cell which contained at least one particle is considered
phagocytically active. Typically, 80-90 percent of the cells in control
cultures ingested one or more particles.
Sample Preparation and Handling. All samples are tested in a concen-
tration tested in duplicate. Solid samples are weighed directly into the
culture vessels so that the final particle concentrations are 10, 30, 100,
300 and 1000 M-g/ml of culture medium. Solid samples can be examined for
the presence of leachable materials, if necessary. Liquid samples are
added with and without sterile filtration to give a final conentration of
6, 20, 60, 200, and 600 Hi/ml (10X medium is used in order to add as much
sample as possible). The pH of the final incubation mixture is recorded
before and after incubation. No pH adjustments are made for the initial
testing. When pH adjustments are made, the sample is tested both with and
without adjustment.
Results. Samples found in the initial screening to significantly
affect the parameters being measured are retested for confirmation.
All of the above determinations are performed in duplicate. Since
cell viability could be considered a binomial response, the arc-sine trans-
formation is employed in the regression analysis. This technique helps to
linearize the data when viability, as a percentage, is plotted versus the
natural logarithm of the molar concentration. For data, the concentration
of the test compound that yielded a 50 percent response for any test
parameter (EC_n) should be obtained through inverse prediction of the
simple regression line.
3.3.2.2 Human Lung Fibroblast (WI-38) Assay
Materials and Methods. Human lung fibroblasts can be obtained from
the American Type Culture Collection, Rockville, Maryland, and should be
f\
maintained in 75 cm Falcon flasks. Cultures are subcultivated twice weekly
by use of 0.25 percent trysin in Gibco solution A with a 1:2 split ratio.
Cultures should not be employed beyond the 35th subcultivation. Cultures
for experimentation are seeded at 1.75 x 10 cells/ml (4.0 ml total volume)
in 25 cm2 Falcon flasks and maintained in Basal Medium Eagle (BME) with
35
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Earles salts plus 10 percent fetal bovine serum (virus screened) 2 pmole/
ml L-glutamine, 100 units/ml penicillin, 100 p-g/ml streptomycin, and 2.5
p,g/ml amphotericin-B. Cells maintained under these conditions show a period
of rapid growth from 24 of 72 hours after subcultivation during which time
the experiments are performed. Routinely antibiotics should be removed
from the maintenance to determine the presence of contaminating micro-
organisms and mycoplasma.
The growth inhibition assay is performed by planting 1.5 to 2.0 x 10
o
cells per flask in 25 cm Falcon flasks and adding dilutions of the
effluent test material 24 hours after the cells have adhered to the flask
surface. The cultures are incubated with closed caps for 20 hours at
37 C. At the end of this incubation period, the cells are trysinized and
and cell counts, cell viability, protein and ATP determinations are per-
formed as described previously.
Sample Handling and Preparation. All samples are tested in a concen-
tration response fashion (as described previously for the rabbit alveolar
macrophages) with each concentration tested in duplicate.
Results. See description for the rabbit alveolar macrophage test.
3.3.2.3 Clonal Toxicity Assay
The clonal assay (e.g. using L-929, CHO or other suitable cell) in-
volves the plating of specified numbers of cells (generally 100 to 1000 in
increments of 100) per 100 mm or 60 mm tissue culture dish and the attach-
ment of these cells for a 24-hour period. Replicate plates then are exposed
to particulate or soluble (aqueous or limited organic) toxicants for 24-48
hours. The cultures then are washed free of toxicant, refed with fresh
medium, and allowed to develop discrete "clonal" colonies of cells. After
10-16 days (time depends upon the cell line) the cultures are fixed and
stained.
3.3.3 Acute In Vivo Test in Rodents (References 12-14)
Since the major objective of the Level 1 biological testing procedure
is to identify toxicology problems at minimal cost, it is recommended that
a two-step approach be taken to the initial acute jji vivo toxicology
evaluation of unknown compounds. The first is based on the quantal (all-
36
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or-none) response and the second on the quantitative (graded) response.
Normally, the quantal test is used to determine the necessity to carry
out the quantitative assay.
3.3.3.1 Method Description
Quantal. Five male and five female young adult rats (approximately
250 g) will be purchased from the supplier and conditioned at the labora-
tory for a minumum of five days. A single dose of the test material
undiluted if a liquid, diluted with a biologically inactive solvent if a
solid will be administered by gavage to this population of animals in a
single dose of 10 ml/kg (if a solvent is used for a solid, the diluent
utilized should be the minimum quantity to effectively administer the
test substance). Immediately following administration of the test substance
and at frequent intervals during the first day, observations will be re-
corded on all toxic signs or pharmacological effects (Table 4). The
frequency and severity of the signs will be scored. Particular attention
will be paid to time of onset and disappearance of signs. Daily observa-
tions will be made on all animals through a 14 day observation period.
Effluent samples which produce harmful effects in vivo and do not result
in deaths, may be further investigated. At termination of the observation
period, all surviving animals will be killed and necropsies will be performed.
Similarly, necropsies will be performed on all animals that die during the
course of this study.
Should no mortality occur in the quantal study, no further work need
be done on the test substance and the LD50 should be reported as greater
than 10 g/kg.
Quantitative. If a single animal in the quantal study dies in the
14 day observation period, then a quantitative study will be performed.
Eighty animals (See 3.3.3.1) equally divided by sex will be used for this
study add maintained for 7 days in quarantine. Having determined good
health in the study population, the animals will be randomly divided into
four groups of five male and five female animals per group. The test sub-
stance treated as before will be administered in graded dosages according
to the following schedule: 3.0, 1.0, 0.3, and 0.1 g/kg. A different
dosage schedule may be selected depending on the results of the quantal
study in relationship to the numbers of animals that died and severity and
37
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TABLE 4. 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
Genitour inary
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
Perineal 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, cons tipation
Flatulence, contraction
Unformed, black or clay colored
Swelling
Prolapse
Soiled
Reddening, flaccid skinfold,
eruptions, piloerection
Discharge, congestion,
hemorrhage cyanosis, jaundice
Exophthalmus, hys tagmus
Opacities
Subnormal, increased
Swelling
Abnormal posture, emaciation
38
-------
type of signs. Observations and duration of study as well as necropsy
procedure will be carried out as indicated above. The LD50 will be calcu-
lated by the method of Horn (Reference 14). If the data are not suitable
for calculation of a precise LD50, i.e., total mortality occurs in the low
dosage level, an estimate of the LD50 should be made or the LD50 could be
expressed as greater than 3 g/kg or less than 0.1 g/kg. Occasionally it
may be necessary to do higher dosage, lower dosages, or another series at
intermediate dosages depending on the results of the above data at the
discretion of the Project Officer.
*
3.3.3.2 Reports
Reports will be provided giving a statement of the methods used, the
results obtained, and a statement of conclusions reached with regard to
the toxicity of the test substances.
3.3.3.3 Discussion
Due to the complex mixture of chemical compounds in the sample and
its subsequent potential additive or synergistic action, the rodent in
vivo screen is one of the necessary test procedures.
Because of the availability of uniform strains of mice and rats, ease
of housing, size, relatively low cost, and a large amount of published
toxicologic data, these two species are usually the animals of choice for
the measurement of acute toxicity.
The advantages of the in vivo toxicity assays are embodied mainly
in the fact that the toxicological assessment is performed in whole animals.
There is a significant background of test data on a wide range of toxicants
for the rodent systems, thus supplying needed information for reliable
interpretation of results with complex effluents.
The disadvantages of the acute rodent toxicity studies are that they
by definition may not satisfactorily predict long-term/low-level exposures
to toxic materials. An additional consideration is the need for multi-gram
quantities of test material which may prohibit testing of small amounts
of relatively purified toxic components of complex mixtures such as found
in the particulate and gaseous samples.
39
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3.4 ECOLOGICAL EFFECTS TESTS
3.4.1 Freshwater Algal Assay Procedure; Bottle Test (References 14, 15)
3.4.1.1 Introduction
tit
An algal assay is based on the principle that growth is limited
by the nutrient that is present in shortest supply with respect to the
needs of the organism. The test is designed to be used to quantify the
biological response (algal growth) to changes in concentrations of
nutrients and to determine whether or not various effluents are toxic
or inhibitory to algae. These measurements are made by adding a selected
test alga to the test water and determining algal growth at appro-
priate intervals. Several methods that may be used for determining
growth are listed in the results section.
3.4.1.2 Method Description
Apparatus
Constant temperature room or equivalent incubator arrangement
capable of providing temperature control at 24 ± 2 C.
Illumination - "cool white" fluorescent lighting to provide
4,304 lux (400 ft-c) ± 10 percent or 2,152 lux (200 ft-e) ±
10 percent measured adjacent to the flask at the liquid level.
Shaking apparatus for test culture flasks capable of 110 oscilla-
tions per minute.
Analytical balance capable of weighing 100 g. with a precision
of ± 0.1 rag.
Oven - dry heat capable of temperature of 120 ± 1 C.
Culture flasks - Erlenmeyer, Pyrex or Kimax type glass. While flask
size is not critical due to C02 limitation, the surface to volume
ratios are. Recommended ratios are 40 ml of sample in 125 ml flask.
Culture flask closures - foam plugs, loose-fitting aluminum foil,
or inverted beakers.
Millipore filter apparatus - for use with 47-mm pre-filter pads
and 0.45)J. porosity membrane filters.
Autoclave or pressure cooker - capable of producing 1.1 kg/cm2
40
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(15 psi) at 121C.
Sampler - non metallic.
Sample bottles - borosilicate glass, linear polyethylene, polycar-
bonate or polypropylene, capable of being autoclaved.
Light meter capable of being calibrated against a standard
source or light meter.
Microscope and illuminator - good quality, general purpose.
Hemocytometer or plankton counting slide.
Centrifuge capable of a relative centrifugal force of at least
1,000 G.
pH meter having a scale of 0 - 14 pH units with an accuracy of
+0.1 pH unit.
Spectrophotometer or colorimeter for use at 600 to 750 mg..
Pipettes - pre-sterilized, disposable.
Reagents
Algal Nutrient Medium - Prepare a stock solution of each of the
macronutrient salts listed in Table 5 in 1000 times the specified final
concentrations in glass distilled and/or deionized water.
TABLE 5. MACRONUTRIENTS NEEDED FOR ALGAL NUTRIENT MEDIUM
Macronu tr ient
Compound
NaN03
K2HP04
MgCL2
MgS04 '. 7H20
CaCl2 . 2H20
NaHCCL
Stock Solution
Concentration
mg/1
25.500
1.044
5.700
14.700
4.410
15.000
Elements
N
Na
P
Mg
S
Ca
K
Resulting
Concentrations
mg/1
4.200
11.001
0.186
2.904
1.911
1.203
0.468
•^— ^— j
Prepare a single stock mix of the micronutrients listed in Table 6
at 1000 times the final specified concentrations in glass distilled
and/or deionized water. The trace metal FeCl3 and the EDTA must be
combined in a single stock mix at 1000 times the final concentrations.
41
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TABLE 6. MICRONUTRIENT STOCK SOLUTION
Compound
H^
MnCl2
ZnCl2
CoCl2
CuCl2
Na2Mo04 • 2H2C
FeCl3
Na2EDTA • 2H2(
Preparation
Concentration
M.g/1
185.64
264.27
32.70
0.78
0.009
) 7.26
96.0
) 1000.0
of Glassware. Carefully
Element
B
Mn
Zn
Co
Cu
Mo
Fe
Fe
check all
Resulting
Concentration
Jig/1
33.0
114.0
15.0
.35
.003
2.88
33.0
33.0
glassware for era
or leakage around screw-caps and thoroughly wash it in hot lab soap or
phosphorus-free detergent. The laboratory sink should be cleaned
previously with hot soap and water and rinsed with 20 percent hydro-
chloric acid to remove residual chemicals or other contaminants. Following
washing, rinse glassware well in hot tap water and give at least one
full rinse in 20 percent concentrated HCl solution. Rinse all glassware
thoroughly at least five times in distilled and deionized water.
Sterility Techniques. When bacterial growth may interfere with
the test, use the following, sterile techniques for preparing the culture
medium.
1. Sterilize a suitable quantity of double-distilled water.
2. Prepare suitable autoclave-sterilized containers.
3. Pre-rinse sterile 0.22 M> porosity membrane filters by passing
sterile double-distilled water through them.
Preparation of Algal Culture Medium. Prepare medium by adding 1 ml
of each of the macronutrient stock solutions in the order listed to 900
ml of glass distilled and/or deionized water, mixing between each
42
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addition. Filter-sterilize by passing through the sterile, pre-rinsed,
0.22^ porosity membrane filter into the sterile container. Add 1 ml
of the micronutrient stock solution and 1 ml of the FeCl3-EDTA mixture
which have been sterilized in the same manner and then make up to 1 liter
with sterile distilled and/or deionized water.
When additional sterile procedures are required, consult Reference 15.
Recommended Test Algae
Selenastrum capricornutum Printz., 10 cells/ml
Microcystis aeruginosa Kutz. emend Elenkin (Anacystis cyanea
Drouet and Daily, 50 x 10^ cells/ml
o
Anabaena flos-aquae (Lyngb.) Debrebisson, 50 x 10J cells/ml
3
Diatom - Cyclotella sp. 10 cells/ml
3
Nitzschia sp. 10 cells/mL
These algae are recommended because they provide a representative cross-
section of the types of algae likely to be found in waters of differing
nutritional status. Other algae that may be used for special test
purposes include diatoms where they may be a consideration in relation
to water supply problems and naturally occurring mixtures of species.
Algal cultures may be secured from National Eutrophication Research
Program, Pacific Northwest Environmental Research Laboratory, U.S.
Environmental Protection Agency, 200 S.W. 35th Street, Corvallis,
Oregon 97330, and from Department of Botany, University of Indiana,
Bloomington, Indiana.
Maintenance of Stock Cultures> Culture S. capricornutum and diatoms
under continuous, "cool white" fluorescent lighting at 4304 lux (400 ft-c)
± 10 percent at 24 ± 2 C. and M. aeruginosa and A. flos-aquae at 2152 lux
(200 ft-c) ± 10 percent; shake at 110 oscillations per minute.
After receiving, transfer the inocula species aseptically to
filter-sterilized algal nutrient media (e.g., 1 ml of inoculum to 30 ml
of algal nutrient medium in 125 ml Erlenmeyer flask or similar ratios
of inoculum to medium in larger flasks). Repeat daily thereafter for
7 days. On the eighth day, the initial transfer culture will be 7-days
old and this culture will then be used as the inoculum for the next transfer.
Thereafter, only 7-day old cultures will be used for inocula. These stock
cultures should be incubated under the same conditions as prescribed for the
test flasks.
43
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Procedures
Sampling and Storage. Samples for the test may be (1) surface
samples from lakes, rivers, or other bodies of water; (2) wastewaters;
(3) substances of concern that may ultimately reach surface waters, and
(4) any sample to which nutrients or other substances are added or from
which they are removed.
In the case of surface waters, collect them directly in polyethylene
containers leaving a minimum of air space. Samples from desired depth should be
collected with a non-metallic water sampler bottle and then transferred
to polyethylene bottles. Samples of other substances to be tested should
be collected and stored in containers fabricated of materials that do not
alter the character of the sample.
Transport samples to the laboratory in the dark and at ice tempera-
ture. Temporary storage in the laboratory should occur under similar
conditions.
Preparation of Filtered Sample. As soon as possible, but not to
exceed 2 days after collection, begin the test procedure. Test each
sample in triplicate, but for statistical purposes divide each into three
aliquots before filtration and thereafter treat as separate samples.
Pretreat the 0.45u porosity membrane filter by passing 50 ml of distilled
water through it and discard the filtrate. Then, filter a quantity of
the sample as needed under reduced pressure of 0.5 atmosphere. Use the
resulting filtered sample water in testing. If suspended matter in the
sample requires it, the 0.45u> porosity membrane filter should be pre-
ceded by an appropriate pre-filter pad (for example, fiber glass) which
is also pretreated as described above.
Preparation of Inoculum. After no less than three and no more than
seven weekly transfers, use the cultures to inoculate the test waters.
Centrifuge cells from a 7-day old stock culture and discard the super-
natant. Resuspend the sedimented cells in an appropriate volume of a
15,000 Hg/1 NaHC03 solution and recentrifuge. Then resuspend sedimented
cells in the NaHCOo solution and count. Pipet a portion into each test
water as the inoculum.
Amount of Inoculum. Count the cells suspended in the bicarbonate
solution and pipet into the test water to give a starting cell concen-
tration in the test waters as follows:
44
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3
S. capricornutum 10 cells/ml
3
M. aeruginosa 50 X 10 eelIs/ml
3
A. flos-aquae 50 X 10 cells/ml
3
Diatoms 10 cells/ml.
The volume of the transfer is calculated to result in a concentration
3
of 10 cells/ml in the test flasks (e.g., 5 X 105 cells/ml in stock
culture requires a 0.2 ml transfer per 100 ml test water). Transfer
should not exceed 1 ml per 100 ml (a minimum 1 X 10 cells/ml in stock
culture is required).
Preparation of Test Flasks. Add filtered sample water in equal
amounts to the sets of test flasks. The amount of sample water added
is dependent upon the size of the test flask used, but should be such
that good agitation is realized when shaking (e.g., a 30 ml water sample
in a 125 ml Erlenmeyer flask).
Simultaneously, test a triplicate set of flasks containing the
algal nutrient medium for algal growth comparison. These flasks should
contain the same volume of medium and inoculum as the flasks containing
the sample water.
Depending upon the type and amount of information being sought,
incorporate other sets of test flasks for comparison. These may include
such as the following:
• Sample water supplemented with phosphorus
(Example: 0.1 mg P/l),
• Sample water supplemented with nitrogen
(Example: 1.0 mg N/l),
• Sample water supplemented with phosphorus and nitrogen
(Example: 0.1 mg P/l and 1.0 mg N/l),
• Sample water diluted with distilled water to give various
concentrations
(Example: 10 and 50 percent dilutions).
Incubation. Incubate test flask cultures at the following controlled
environmental conditions:
•' Temperature - 24 C ± 2 C
• Illumination - continuous "cool white" fluorescent
lighting to provide 100 ft-c (1076 lux) at midpoint
(from top to bottom) of culture flasks of Anabaena and
and Microcystis and 400 ft-c (4304 lux) of culture
flasks of Selanastrum.
45
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• Shaking - continuous reciprocating or gyratory, 80 strokes
per minute.
3.4.1.3 Results
Observations. Depending upon the method for evaluating growth,
choose the method used to determine growth response during incubation
from among the following. Count cells on a microscope, using a hemacyto-
meter or a Palmer-Maloney or Sedgwick-Rafter plankton counting chamber.
Determine the amount of algal biomass by measuring the absorbance of the
culture at 600 to 750 my, with a colorimeter or spectrophotometer. Measure
the amount of chlorophyll contained in the algae either directly (in
vivo) by fluorometry or after extraction. An electronic particle counter
provides an accurate and rapid count of cells. All methods used for
determining the algal biomass should be related to a dry weight measure-
ment (mg/1) determined gravimetrically.
Growth Measurement. Two parameters are used to describe the growth
of each test alga: maximum specific growth rate and maximum standing
crop. The maximum specific growth rate (n max) for an individual flask
is the largest specific growth rate (M>) occurring at any time during
incubation. The [i max for a set of replicates is determined by averaging
the V- max of the individual flasks. The specific growth rate, p., is
defined by
In (X2/XL)
where,
X = biomass cone, at the end of the selected time interval
X. = biomass cone, at the beginning of the selected time
t_ - t. = elapsed time (days) between selected determination of biomass.
Because the maximum specific growth rate (M> max) occurs during the
logarithmic phase of growth (usually between day 0 and day 5), the biomass
must be measured at least daily during the first 5 days of incubation.
The maximum standing crop in any flask is defined as the maximum algal
biomass (dry weight) achieved during incubation. For practical purposes,
the maximum standing crop can be assumed to have been achieved when the
rate of increase in biomass has declined to less than 5 percent per day.
46
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After the maximum standing crop has been achieved, determine the dry
weight of the algal biomass gravimetrically by either the aluminum dish or
filtration technique. If biomass is determined indirectly, convert the
results to an equivalent dry weight using appropriate conversion factors.
Following determination of the maximum standing crop and maximum
specific growth rate, compare these values for the effluent tests
and standard culture medium tests (controls) to determine the EC50. The
EC50 represents that concentration at which either 50 percent of the maximum
standing crop or maximum specific growth rate is obtained, as compared
with controls.
Calculation of the EC50. The EC50 is the concentration at which
growth is 50 percent of the control. It may be estimated by interpolation by
plotting the data on semilogarithmic coordinate paper with concentrations
on the logarithmic axis and percentage growth on the arithmetic axis. A
straight line is drawn between the 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.
Stimulation. If growth was stimulated by the effluent, report the
percentage stimulation at each concentration.
Cell Count Measurement. Any method of count (i.e., cell number/ml)
determination is applicable, including direct microscopic counting
(hemocytometer), Palmer-Maloney slide, or the use of electronic particle
counters. Make counts at least every other day during log-phase growth.
These may be less frequent after the log phase. Preferred counting
days are: 3, 5, 7, 9, 13, 17, and 21. For detailed procedures of
plankton counting and chlorophyll analysis, see Reference 15.
Gravimetric Procedure. This method is particularly useful for
assessing the growth of Anabaena flos-aquae. The cells of the alga grow
in filaments and it is difficult to obtain accurate cell counts. The
method may also be used with j>. capricornutum. M. aeruginosa, and other
species of algae. In any case, use it only with either relatively dense
cultures or large volumes of thinner cultures. Otherwise, the error
may be large. Two methods may be employed.
Method I. Centrifuge a suitable portion of algal suspension. Use
a continuous-flow centrifuge, if available. Discard the supernatant,
resuspend the sedimented cells .in distilled water, transfer to tared
47
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crucibles or aluminum cups, dry overnight in a hot air oven at 105 C, cool
in a desiccator over Drierite for 1 hour, and weigh.
Aluminum cups should be pretreated before taring by adding approxi-
mately 15 ml of distilled water and drying at 105 C in the oven. Repeat
this pretreatment three times; then the cups can be tared accurately.
Method II. This method involves filtering a measured portion of
algal suspension through a tared Millipore® filter, preferably a type AA
filter with an 0.80 micron pore size.
The method is as follows:
a. Dry filters for several hours at 90 C in an oven/- ' The
filters may be placed in folded sheets of paper upon which
the weights or codes may be written.
b. Cool filters in a desiccator containing desiccant.
c. Filter a suitable measured aliquot of the culture under
a vacuum of 0.5 atmosphere. Normally 10 ml is sufficient,
but in thin cultures more may be required.
d. Rinse the filter funnel with 50 ml distilled water using
a wash bottle and allow the rinsing to pass through the
filter. This serves to transfer all of the algae to the
filter and to wash the nutrient salts from the filter.
e. Dry the filter in its paper folder at 90 C, cool in the
desiccator, and weigh.(2)
f. To collect for loss of weight of filters during washing,
wash two blank filters with 50 ml of distilled water,
pouring it through slowly under reduced vacuum. Dry
and weigh filters and record weight loss. This correction
is not large, but is essential for meaningful results on
thin cultures. For example, if 10 ml have been filtered
and yield a difference between tare and final of 1.10 mg
and the blank has lost 0.02 mg, then the culture contains
(1.10 + 0.02) x 100 = 112 mg/1 dry weight.
1. The drying temperature has been selected to avoid damage to
filters.
2. In weighing, do not hang the filters over the edge of the pan or
electrostatic attraction of the filter to the balance will result
in an error.
48
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Absorbance Measurements. Measure absorbancc initially and after each
24 hours with a spectrophotometer or loloriffiete? at a wavelength of
600 mu. In reporting results, specify the Instrument make or model,
the geometry and path length of the cuvette, and the equivalence
between absorbance and some other measure of cell quantity.
Absorbance, as defined by the Beer-Lambert Law, A « logl£ =
abc(l) (I0 = intensity of incident light, Ix = intensity of transmitted
light, a = absorbancy index characteristic for the solution, b =
length of cell path, c = concentration of solute in solution) is usually
derived for absorption of light by molecules of solute in homogeneous
solution. It can be derived also for a suspension of uniform particles,
but with some necessary added restrictions. For particles of
bacterial or algal cell size, Ix is less than I by virtue of absorp-
tion and also by virtue of scattering caused by cell reflections and
refractions. The fraction of the latter which reaches the light
measuring receiver depends upon the instrument design. A large
receiver close to the cuvette catches much of the scattered light
(i.e., is insensitive to scattering). A small receiver far from the
cuvette in a long-focused or diaphragmed optical path catches very little
scattered light (i.e., is very sensitive to scattering) (Ref. 14).
Most current instruments are likely to be more sensitive to scatter-
ing than to absorption as evidenced by effect of wavelength. A simple
test is: for a green alga, light absorption by its pigments in vivo
will show relative absorbance ratios such as 70: 500: 1, for 600:
680: 750 m|J. light (ratios probably correct in order of magnitude).
In practical measurements, without elaborate precautions to avoid
effects of scattering, the ratios will always be very much less.
In any photometric measure of absorbance, considerations of
precision lead to a simple rule of thumb that measurements be limited
to a range of 0.05
-------
A common and necessary check upon instrumentation is to measure A on
various dilutions of an algal suspension. By this means absorbance can be
calibrated against any other measure of cell quantity (X) such as cell
number of dry weight. There is no assurance that the relation between
A and X will be constant and independent of culture conditions. As noted
above, the absorbance measured is a complex function of volume, size,
and pigmentation of the cells. Hence, the relation between A and X should
be examined on different batches of algae which best simulate actual con-
ditions of the test.
Report. In the final report, include the following specific infor-
mation as well as the general information provided in all analyses
(see Section 3.5):
1. The EC50 at 12 days and other days of importance to be decided
upon by the shape of the growth curve.
2. The specific growth rate between days 3 and 12 and any other
period that should be reported depending upon the shape of
the growth curve.
3.4.1.4 Discussion
The bottle test is the only algal bioassay that has undergone
sufficient evaluation and refinement to be considered reliable. Follow-
ing years of intensive evaluation, participating laboratories, have
obtained excellent agreement in results. For more detailed information
concerning bioassays, consult Reference 15.
3.4.2 Acute Static Bioassays
With Freshwater Fish and Daphnia (References 16-34)
3.4.2.1 Introduction and Rationale
l~™"—™"™""^^^™^^~ * ^^^^^^^
Biological testing must be considered as well as chemical and physical
parameters when assessing the potential impact of complex industrial or a
combination of municipal and industrial wastes on the aquatic environment.
Biological testing usually involves performing toxicity tests on samples of
treated wastes.
In a toxicity test, two or more treatments are used to study the effect
of a toxic agent on test organisms which are usually all of the same species.
Aquatic organisms will integrate synergistic and antagonistic effects of all
the components over the duration of the exposure.
50
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Although toxicity tests with aquatic organisms can be conducted by
applying the toxic agent directly to the test organisms, such as by in-
jection or in food, most tests are conducted by exposing the test organisms
to test solutions containing various levels of a toxic agent. One or more
control treatments are used to provide a measure of the acceptability of
the test by giving some indication of the healthiness of the test organisms
and the suitability of the dilution water, test conditions, handling proce-
dures, etc. A control treatment is an exposure of the test organisms to
dilution water with no toxic agent added. The other treatments are exposures
of the test organisms to dilution water with toxic agent added. Generally
the most important data obtained from a toxicity test are the percentages of
test organisms that are affected in a specified way by each of the treatments.
The result derived from these data is a measure of the toxicity of the toxic
agent to the test organisms under the conditions of the test or, in other
words, a measure of the susceptibility of the test organisms to the toxic
agent.
Acute toxicity tests are generally used to determine the level of toxic
agent that produces an adverse effect on a specified percentage of the test
organisms in a short period of time. Because death is normally an easily
detected and obviously important adverse effect, the most common acute toxi-
city test is the acute mortality test. Experimentally, 50 percent effect is the
most reproducible measure of the toxicity of a toxic agent to a group of
test organisms, and 96 hours is often a convenient, reasonably useful exposure
duration. Therefore, the measure of acute toxicity most often used with fish
and macroinvertebrates is the 96-hour median lethal concentration (96-hr LC50).
Thus the result of an acute mortality test is the statistically derived best
estimate of the LC50, which is the concentration of toxicant in dilution water
that is lethal to exactly 50 percent of the test organisms during continuous exposure
for a specified period of time, based on data from one experiment. However,
the measure of acute toxicity most often used with daphnids and midge larvae
is the 48-hour median effective concentration (48-hr EC50) based on immobili-
zation. The terms median lethal concentration (LC50) and median effective
concentration (EC50) are consistent with the widely used terms median lethal
dose (LD50) and median effective dose (ED50), respectively. However, whereas
"concentration" refers to the concentration of toxicant in the test solution,
"dose" refers to the amount of toxicant that enters the test organism.
51
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The static technique provides the easiest measure of toxicity and is
recommended for the waste assessment studies. Fathead minnow (Pimephales
promelus) and Daphnia pulex are the test animals.
The following procedure is based primarily on the methods given in
Reference 19. In many instances, sentences and/or complete paragraphs
are used verbatim.
The primary objections to the following procedure, are that the recommended
dilution water may not closely simulate receiving water characteristics and
the fathead minnow may not be representative of the most sensitive species in
a given geographical area. However, the procedure will adequately serve to
develop relative toxicity data for the purpose of ranking industries based
on the toxicity of their effluents.
3.4.2.2 Method Description
Equipment
Facilities. The facilities should include tanks for holding and ac-
climating test organisms, and a constant-temperature area or recirculating
water bath for the test chambers. There should be a dilution water tank
that may be used to prepare reconstituted water. This is sometimes elevated so
dilution wafer can flow by gravity into holding and acclimation tanks and
test chambers. Holding, acclimation, and dilution water tanks should be
equipped for temperature control and aeration. A-f" used for aeration must
be free of oil and fumes; filters to remove oil and water are desirable.
During holding, acclimation, and testing, test organisms should be shielded
from disturbances. The test facility must be well ventilated and free of
fumes. A 16-hour light and 8-hour dark photoperiod should be provided.
Construction Materials. 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 sortition
of toxicants from water. Glass, #316 stainless steel, and perfluoro-
carbon plastics must be used whenever possible to minimize leaching,
dissolution, and sorption. Unplasticized plastics can be used for holding
and acclimation tanks and in the water supply system. Rubber, copper,
brass, and lead must not come in contact with dilution water, stock
solutions, effluent samples, or test solutions.
52
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Test Chambers. Test chambers for the fathead minnow should be 19.6-
liter (five gallon), widemouth, soft-glass pickle jars containing
15 liters of solution, or 30 cm x 60 cm x 30 cm deep, all-glass chambers.
The all-glass chambers can be made by welding, not soldering, stainless
steel or by gluing double-strength or stronger window glass with clear
silicon adhesive. Silicon adhesive sorbs some organochlorine and organo-
phosphorus pesticides which are then difficult to remove. Therefore,
as little of the adhesive as possible should be in contact with water;
extra beads of adhesive should be on the outside of chambers rather than
on the inside. The test solution should be between 15 and 20 cm deep.
Daphnids may be exposed in 3.9-litter (1-gallon), widemouth, soft-glass
bottles, or battery jars containing 2 to 3 liters of solution. Loosely
covered 250-ml beakers containing 200 ml of solution may also be used.
Cleaning. Test chambers must be cleaned before use. New ones must be
washed with detergent and rinsed with 100 percent acetone, water, acid (such
as 5 percent concentrated nitric acid), and twice with tap or other clean
water. At the end of every test, if the test chambers are to be used again,
they should be (a) emptied, (b) rinsed with water, (c) 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), and (d) rinsed twice with water. Acid is useful for removing
mineral deposits, and 200 mg of hypochlorite/liter is useful for removing
organic matter and for disinfection. However, acid and hypochlorite must
not be used together. Test chambers must be rinsed with dilution water just
before use.
Dilution Water
General Requirements. An adequate supply of a dilution water that is
acceptable to the test organisms and the purpose of the test must be avail-
able. For acute toxicity tests, a minimal criterion for an acceptable di-
lution water is that healthy test organisms will survive in it for the dura-
tion of acclimation and testing without showing signs of stress, such as dis-
coloration or unusual behavior. Because daphnids are more sensitive to many
toxicants than most other freshwater aquatic animals, a more realistic cri-
terion for an acceptable freshwater dilution water is that first instar
daphnids will survive in it for 48 hours without food. A more stringent
criterion for an acceptable dilution water is that test organisms will sur-
vive, grow, and reproduce satisfactorily in it. Water in which daphnids
53
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will survive and reproduce satisfactorily should be an acceptable dilution
water for most tests with freshwater animals. The dilution water should be
intensively aerated prior to the introduction of the toxicant. Adequate
aeration will bring the pH and the concentration of dissolved oxygen and
other gases into equilibrium with air, and will minimize oxygen demand and the
concentration of volatiles. The concentration of dissolved oxygen in the
dilution water should be between 90 percent and 100 percent saturation, and
water that may be contaminated with undesirable microorganisms should be
passed through a properly maintained ultraviolet sterilizer equipped with an
intensity meter.
Recommended Dilution Water. The dilution water should be of constant
quality and should contain less than the following maximum accepted levels
of substances:
Suspended solids 20 mg/1
Total organic carbon 10 mg/1
Un-ionized ammonia 20 ug/1
Residual chlorine 3 ug/1
Total organophosphorus pesticides 50 ng/1
Total organochlorine pesticides
plus PCB's 50 ng/1
The dilution water is considered to be constant quality if the monthly
ranges of the hardness, alkalinity, and specific conductance are less than 10
percent of their respective averages and if the monthly range of pH is less
than 0.4 unit. The dilation water 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 either must be shown that first
instar daphnids can survive in it for 48 hours without food or residual
chlorine must be measured. When possible, dilution water with a hardness of
100 mg/1 - 10 percent as CaC6_ should be used to minimize problems that occur
in data interpretation and comparison.
Test Organisms
Species. The fathead minnow Pimephales promelus should be the primary
animal used in all tests carried out under this protocol for the environmental
assessment studies. A secondary choice is Daphnia pulex , which should be used
if additional toxicity data are desired or if it is impossible to use the
fathead minnow. These species were chosen for their ready availability,
heartiness, and the ease, convenience, and economy with which they can be
maintained in the laboratory.
54
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Source. Usual sources of fish are private, state, and Federal fish
hatcheries. Fathead minnows may be captured from wild populations in rela-
tively unpolluted areas. However, collecting permits may be required by
local and state agencies. Fish collected by electroshocking should not be
used. Daphnia should be reared in the testing facility from laboratory
cultures. Daphnia from cultures in which ephippia are being produced should
not be used. All animals in a test should be from the same source and as
healthy and uniform in size and age as possible.
Size. Very young (not yet actively feeding), spawning, or spent fish
should not be used in the testing being done using this protocol. The fish
should weigh between 0.5 and 1.0 g. In any single test, all fish should be
from the same year class, and the standard length (tip of snout to end of
caudal peduncle) of the longest fish should be no more than twice that of the
shortest fish.
The Daphnia pulex should be in the first instar stage of their life
cycle.
Care and Handling. To avoid unnecessary stress, organisms should not
be subjected to rapid changes in temperature or water quality. In general,
aquatic organisms should not be subjected to more than a 3 C change in water
temperature in any 12-hour period. Holding and acclimation tanks should be
sterilized with an iodophor or with 200 mg of hypochlorite/liter for 1 hour,
scrubbed well once during the hour, and then rinsed well between groups of
test organisms. When organisms are first brought into the facility, they
should be quarantined at least until they appear to be disease free. To main-
tain organisms in good condition during holding and acclimation, crowding
should be avoided and the dissolved oxygen concentration must be maintained
between 60 and 100 percent saturationj gentle aeration may be used
if necessary. Organisms should be fed at least once a day and the tanks
scrubbed at least twice a week. Careful observations should be made during
holding and acclimation for signs of disease, stress, physical damage, and
mortality. Dead and abnormal individuals must be discarded.
Organisms should be handled as little as possible. When handling is
necessary, it should be done as gently, carefully, and quickly as possible,
so that the organisms are not unnecessarily stressed. Organisms that touch
dry surfaces or are dropped or injured during handling must be discarded.
Small dip nets are best for handling the fish. These are commercially avail-
55
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able, or they can be made from small-mesh nylon netting, nylon or silk bolting
cloth, plankton netting, or similar material. Smooth glass tubes with rubber
bulbs should be used for transferring the Daphnia. Equipment used to handle
aquatic organisms should be sterilized between uses with an iodophor, 200 mg
of hypochlorite/liter, or 30 percent formalin plus 1 percent benzalkonium
chloride. Hands should be washed or sterilized before handling or feeding
test organisms.
Disease Treatment. Freshwater fish may be chemically treated to cure
or prevent diseases by using the treatments recommended in Table 7; but if
they are severely diseased, it is often better to destroy the entire lot.
Until acceptable treatments have been proven effective, all other diseased
animals should be discarded. Generally the fish should not be treated
during the first 16 hours after arrival at the facility because they are
probably stressed due to collection or transportation and some are treated
during transit. Tests must not be begun with treated fish for at least 4
days after treatment. Tanks and test chambers which may be contaminated with
undesirable microorganisms should be sterilized for 1 hour with an iodophor
or with 200 mg of hypchlorite/liter.
Holding and Acclimation. After collection or transportation, the fish
or Baphnia should be held in and acclimated to the dilution water for at least
2 days before beginning a test. They should be held if possible under stable
conditions of temperature and water quality in a flow-through system with a
flow rate of at least two water volumes per day or in a recirculating system
in which the water flows through a carbon filter and an ultraviolet sterilizer.
When possible, the fish should be held in the dilution water at the temperature
at which they are to be tested. During long holding periods, however, it is
generally easier and safer to hold them at a lower temperature (10-15 C) rather
than higher temperatures (20-25 C) because the metabolic rate and the number
and severity of disease outbreaks are reduced.
A group of animals must not be used for a test if individuals appear to
be diseased or otherwise stressed or if more than 5 percent die during the
48 hours immediately prior to the beginning of the test. If a group fails to
meet these criteria, all individuals must be discarded or treated and held an
additional 4 days. Usually it is more practical to discard the entire group
and begin over.
The fish should not be fed for 48 hours before the beginning of a test.
However, the Daphnia may be fed up to the beginning of the test.
56
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TABLE 7. RECOMMENDED PROPHYLACTIC AND THERAPEUTIC
TREATMENTS FOR FRESHWATER FISH3
Disease
Chemical
Concentration
(mg/1) Application
External
bacteria
Monogenetic
trematodes,
fungi, and
external
protozoad
Parasitic
copepods
Benzalkonium chloride
(Hyamine 1622R)
Nitrofurazone (water mix)
Neomycin sulfate
Oxytetracycline hydrochloride
(water soluble)
Formalin plus zinc- free
malachite green oxalate
Formalin
Potassium permanganate
Sodium chloride
AI)
Trichlorfpn
1-2 AIb 30-60 minC
3-5 AI
25
25 AI
25
0.1
150-250
2-6
15,000-30,000
2000-4000
20
0.25 AI
30-60 min
30-60 min£
30-60 minC
1-2 hours
30-60 min
30-60 min°
5-10 min dip
e,c
30-60 min°
f
SThese recommendations do not imply that these treatments have been cleared
or registered for these uses. Appropriate state and federal regulatory
agencies should be consulted to determine if the treatment in question
can be used and under what conditions the uses are permitted. These
treatments should be used only on fish intended for research. They
have been found dependable, but efficacy against diseases and toxicity to fish
may be altered by temperature or water quality. Researchers are
cautioned to test treatments on small lots of fish before making large-
scale applications. Prevention of disease is preferred, and newly
acquired fish should be treated with the formalin-malachite green
combination on three alternate days if possible. However, generally
fish should not be treated on the first day they are in the facility.
This table is merely an attempt to indicate the order of preference of
treatments that have been reported to be effective. Before a treatment
is used, additional information should be obtained from sources such as
Davis (1953), Hoffman and Meyer (1974), Reichenbach-Klinke and Elkan (1965),
Snieszko (1970), and van Duijn (1973). (Ref. 20, 23, 25, 26, 32.)
57
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TABLE 7. (Continued)
AI - active ingredient.
^
Treatment may be accomplished by (1) transferring the fish to a static
treatment tank and back to a holding tank; (2) temporarily stopping
the flow in a flow-through system, treating the fish in a static manner,
and then resuming the flow to flush out the chemical; or (3) continuously
adding a stock solution of the chemical to a flow-through system by
means of a metered flow or the technique of Brungs and Mount (Ref. 16).
One treatment is usually sufficient except for "Ich", which must be
treated daily or every other day until no sign of the protozoan remains.
This may take 4 to 5 weeks at 5 to 10 C and 11 to 13 days at
15 to 21 C. A temperature of 32 C is lethal to Ich in one
week.
fiinimum of 24 hours but may be continued indefinitely.
Continuous treatment should be employed in static or flow-through systems
until no copepods remain, except that treatment should not be continued
for over 4 weeks and should not be used above 27 C.
58
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Test Procedure
Experimental Design. At least 10, but preferably 20, organisms must be
exposed to each treatment. They may be divided between two or more test
chambers. The use of more organisms and replicate test chambers for each
treatment is desirable. Randomization of the treatments is desirable; if
replicates are used, random assignment of one test chamber for each treatment
in a row, followed by random assignment of a second test chamber for each
treatment in another or an extension of the same row, is recommended rather
than total randomization. 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 which consists of the same dilution water,
conditions, procedures, and organisms 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. 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 percent 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 results of the test.
Dissolved Oxygen Concentration. Aeration of the test solutions during
the test should be avoided if possible. If necessary, aeration should be
done with minimal agitation of the test solution.
Test Temperature. The test with fathead minnows should be run at 22 C
and with Daphnia at 17 C with no more deviation than 1 C during the test.
Loading. The grams of organism per liter of solution in the test
chambers must not be so high that it affects the results of the test. There-
fore the loading must be limited to insure that the concentration of dissolved
59
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oxygen and toxicant is not decreased below acceptable levels, that the con-
centration of metabolic products does not increase above acceptable levels,
and that the organisms are not stressed due to crowding. The loading in the
test chambers must not exceed 0.8 g/liter. A lower loading rate should be
used if low dissolved oxygen problems may occur. If the dissolved oxygen
concentration is less than 60 percent saturation in any test chamber, it may
be desirable to conduct the toxicity test with a modified procedure by slowly
bubbling air or oxygen through the solutions in the test chambers during the
test. If the modified procedure is used, the exact methodology must be des-
cribed in detail in all reports of the test. In some instances, it may be
necessary to run an aerated and a non-aerated test side by side.
Toxicant. The toxicant is a sample of an effluent. 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 2 mm or larger holes. Samples must
be covered at all times and violent agitation must be avoided. Undissolved
materials must be uniformly dispersed by gentle agitation immediately before
any aliquot of the sample is taken for use. The timing of the test and the
collection of samples should be based on an understanding of the short and
long-term operations and schedules of the discharger if possible.
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 on composite samples may be
desirable in some cases. Tests should be begun as soon as possible, but tests must
be begun within 8 hours after the sample is obtained. The temperature of
the sample should be adjusted 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.
Beginning the Test. The animals should be put in the test chambers with-
in 30 minutes after the effluent sample is added to the dilution water.
Feeding. The animals must not be fed while in the test chambers.
Duration. A test begins when the organisms are first exposed to the
effluent. Daphnla must be exposed for 48 hours and fathead .minnows for
96 hours. If time and resources permit, observations may be extended for ,
longer time periods.
60
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3.4.2.3 Results
Biological Data. The number of dead or affected organisms in each
chamber must be counted every 24 hours after the beginning of the test. More
frequent observations are desirable, especially near the beginning of the test.
Dead animals must be removed as soon as they are observed.
Death is the adverse effect most often used to study acute toxicity with
aquatic organisms. The criteria for death are usually the lack of movement,
especially the absence of gill movement in fish, and the lack of reaction to
gentle prodding. However, because death is not easily determined for some
invertebrates, an EC50 is often determined rather than an LC50. The effect
generally used to determine the EC50 for Daphnia is immobilization which is
defined as the lack of movement except for minor activity of appendages.
General observation of such things as erratic swimming, loss of reflex, dis-
coloration, behavioral changes, excessive mucous production, hyperventilation,
opaque eyes, curved spine, and hemorrhaging should be reported.
The weights and standard lengths of the fathead minnows should be deter-
mined by measuring representative animals before the test or the control
animals after the test. Fish that are to be used in a test must not be weighed
or measured after acclimation has begun.
Chemical and Physical Data. The dissolved oxygen concentration must be
measured at the beginning of the test and every 24 hours thereafter to the
end of the test in the control and the high, medium, and low effluent concen-
trations. The pH and specific conductance must be measured at the beginning
of the test in the control and the high, medium, and low effluent concentrations
The temperature should be monitored continuously during the test
with a recording thermometer submerged in one test chamber. Chemical
analysis should be performed on the dilution water to indicate that it meets
the recommended specifications.
Methods used for analysis of water quality must be those specified in
Reference 21, 24, and 31. Residual chlorine can be measured to 3 p.g/1
using a modified amperometric method (Ref. 22). The concentration of
un-ionized ammonia can be calculated from the concentration of total ammonia,
pH, and temperature according to Table 8.
Range-finding Test. Unless the approximate toxicity of the effluent
is already known, it is usually desirable to conduct a range-finding test
to determine the concentrations that should be used in the definitive test.
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TABLE 8 . PERCENTAGE OF AMMONIA THAT IS DN-IONIZED IN
DISTILLED WATER AT DIFFERENT TEMPERATURES
AND pH'S (Ref. 30)
Temperature gH
C 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
7 0.01 0.05 0.15 0.46 1.45 4.44 12.8 31.7 59.5
12 0.02 0.07 0.22 0.68 2.13 6.44 17.9 40.8 68.5
17 0.03 0.10 0.32 1.00 3.08 9.14 24.1 50.2 76.1
22 0.04 0.14 0.45 1.43 4.39 12.7 31.5 59.2 82.1
27 0.06 0.21 0.65 2.03 6.15 17.2 39.6 67.4 86.8
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Generally groups of five organisms are exposed to three to five widely spaced
effluent concentrations and a control for 8 to 24 hours. The greater the
similarity between the range-finding test and the definitive test, the more
useful the results of the range-finding test will be.
Meaningful range-finding tests may often be difficult to conduct,
because the characteristics of the effluent may vary significantly within
short periods of time. However, many nonchlorinated effluents have an LC50
between 2 percent and 100 percent. If a range-finding test is to be conducted
with the same grab sample of the effluent with which a definitive test is to
be conducted, the range-finding test can last 8 hours at the most.
Definitive Test. For the determination of an LC50 or an EC50, a control
and at least five concentrations of effluent in a geometric series should be
used. More treatments are desirable to insure the acceptability of the test
and to provide additional data for various lengths of exposure. A definitive
test must meet both of the following criteria so that the LC50 can be calcu-
lated with reasonable accuracy:
(1) Except for the controls, the concentration of toxicant in each
treatment must be at least 50 percent of the next higher one.
(2) One treatment other than the control must have killed or
affected less than 35 percent of the organisms exposed to it,
and one treatment must have killed or affected more than 65
percent of the organisms. This requirement does not apply if
100 percent effluent does not kill or affect more than 65 per-
cent of the organisms exposed to it.
If an LC or EC near the extremes of toxicity is to be calculated, such
as an LC10 or an EC90, at least one treatment must have killed or affected
a percentage of test organisms, other than 0 percent and 100 percent, near
the percentage for which the LC or EC is to be calculated. This requirement
might be met in a test to determine an LC50 or an EC50, but special tests with
appropriate toxicant concentrations will often be necessary.
Other ways of providing information concerning the extremes of toxicity
are to'report the highest concentration of toxicant that actually killed or
affected no greater a percentage of the test organisms than did the control
treatment in the toxicity test or to report the lowest concentration of toxi-
cant that actually killed or affected all of the test organisms exposed to it.
These alternatives are normally more informative than reporting a result such
as an LC2 or an EC98 unless several partial kills or effects are obtained
close to 2 percent or 98 percent.
63
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Calculations. Graphical interpolation provides the simplest means of
obtaining an estimated LC50 value. The data are plotted on semilogarithmic
coordinate paper with concentrations on the logarithmic axis and the per-
centages of affected fish on the arithmetic axis. A straight line is drawn
between two points representing death in concentrations that were lethal to more
than half and less than half of the animals. The concentration at which the
line crosses the 50 percent death line is the LC50 value. If 50 percent of
the animals are not killed by the highest concentration, the percent killed
should be reported. Graphical interpolation does not, however, provide any
confidence limits for the LC50 value.
For the purpose of determining the relative toxicity of industrial ef-
fluents, the graphical interpolation procedure is sufficient. However, if
desirable, there are a variety of methods that can be used to calculate an
LC50 and EC50 or any other LC or EC values (Ref. 24-25) and confidence limits.
The most widely used are the probit, logit, moving average, and Litchfield-
Wilcoxon (Ref. 27) methods. For each set of data, the LC50 or EC50 value and
its 95 percent confidence limits can be calculated using the initial volume
percent of the effluent in the test solutions. The percentage of test animals
that are in the control treatment must not be used in the calculation of the
results. The raw data should always be available so that the statistical
methods can be applied if desired.
Reporting. Any deviation from these methods should be reported as well
as the following specific information:
1. The chemical characteristics of the dilution water*
2. Detailed-Information about the test organisms, including
scientific name, standard length, weight, age, life stage,
source, history, observed diseases, treatments, and accli-
mation procedure used.
3. A description of the experimental design and the test
chambers, the way the test was begun, the number of
organisms per treatment, the loading, the lighting.
4. Definition of the criterion used to determine the effect
and a summary of general observations on other effects
or symptoms.
5. Percentage of organisms that died or showed the effect
in the control treatment.
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6. For Daphnia the 24- and 48-hour and for the fathead
minnow the 24-, 48-, and 96-hour LC50 or EC50 values and
the method(s) used to determine them. If 100 percent
effluent does not kill or affect more than 65 percent of
the test organisms, report the percentage of organisms
killed or affected by various concentrations of the
effluent.
7. Methods used for, and the results of, all chemical
analyses of water quality and toxicant concentration,
including validation studies and reagent blanks.
8. The average and range of the acclimation temperature
and the test temperature.
3.4.3 Bioassav With Unicellular Marine Aleae
3.4.3.1 Introduction
The community of unicellular algae is a very important constituent
of marine ecosystems. It is comprised of a variety of species that have
different growth rates, photosynthetic rates, nutrient requirements, and other
functions that regulate species composition and diversity in the community in
relation to environmental parameters. The algal community, through photo-
synthesis, produces most of the food and oxygen used in the marine ecosystem.
It is well known that algal species and communities are sensitive to
environmental changes. Species may be inhibited or stimulated by pollutants.
In a community, a pollutant may affect some species but not others, thereby
causing changes in species diversity and composition. This can be followed
by changes in composition of the animal community and altered routes of flow
of energy and materials. Often, the altered ecosystem is undesirable from
the human standpoint.
It is necessary, therefore, that a bioassay program designed to study
effects of suspected pollutants include adequate research on unicellular algae.
3.4.3.2 Method Description
Facilities for Growth of Algae. Experimental and stock algal cultures
must be maintained under carefully controlled environmental conditions. The
easiest way to do this is to keep them in a walk-in controlled-environment
chamber with shelves that allow for illumination of cultures from above by
"cool white" fluorescent tubes. Light intensity should be approximately 4000
lux and temperature should be 20 * 2 C. The lighting cycle should be 14 hours
of light followed by 10 hours of darkness.
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Algal Species. Algal species vary in their responses to toxicants. For
example, a material may be highly toxic to one species but not another, while
a second material may have the opposite effect. It might be best if species
indigenous to an industry's area were used, but, because of variation in
species response, it is recommended that Skeletonema costatum be used for
ranking purposes. This alga is available from the Culture Collection of Algae,
Department of Botany, University of Texas, Austin, Texas 78712.
Culture Medium. Since the main purpose of industrial waste bioassays is
the ranking of industries according to toxicity of wastes, it is recommended
that a single, artificial seawater salt mix be used. Although fortified
natural seawater is a good medium for algal testing, geographic variation in
composition may affect growth and thus affect ranking. Also, undiluted
waste will be tested and it will be necessary to add solid salt mix to it.
The amount of salt added to test media approximates that found in low-
salinity estuarine areas for two reasons:
1. Presumably, toxic effluents will usually be at their highest
concentrations there •
2. The effect of a toxicant may be greatest when algae are under
low-salinity stress.
Toxicity Tests. The recommended species grow well at low salinity.
Saline culture media are to be prepared by adding a commercial salt mix (Rila
Products, Teaneck, NJ 07666) to effluent to a concentration of 10 parts per
thousand (ppt). Add approximately 10 gm of Rila Salts, with gentle swirling,
to each liter of undiluted effluent. When the salt has dissolved, check the
salinity by means of an instrument such as an American Optical Co. refracto-
me ter fitted with a salinity scale. Because artificial sea salts are hygro-
scopic, it will probably be necessary to add more salt. Do so carefully until
a salinity of 10 ppt is reached after all salt is dissolved.
i
Diluting fluid is artificial seawater of 10 ppt salinity prepared
with glass-distilled or deionized water. This will also be used as
growth medium for control cultures.
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Nutrients must be added to all control, diluted, and undiluted media.
To each liter of medium, add 15 ml of metal mix, 1.0 ml of minor salt
mix and 0.5 ml of vitamin mix as described in Table 9. The final concen-
trations of these nutrients are approximately one-half those needed
for maximum growth and are used here so that enhancement of growth by
effluent may be detected. Chelating agents should not be added to
the media.
Stock Culture Medium. Algal stock cultures are to be maintained in
autoclaved artificial seawater medium of 10 ppt salinity prepared from
glass-distilled or deionized water. Nutrient concentrations should be
the same as above. Stock cultures are grown in 150 ml of medium in
500 ml Erlenmeyer flasks and transferred every 10-14 days. Stock cultures
must be manipulated according to standard microbiological techniques
to insure a minimum of contamination by bacteria.
Glassware. All glassware must be of high-grade borosilicate glass
(Pyrex or Kimex). Great care must be taken during cleaning to insure
that contaminants from previous tests are not present. Each piece of
glassware is first soaked in detergent for several hours and then hand-
brushed, rinsed thoroughly with glass-distilled or deionized water, and
rinsed three times with pesticide grade acetone. It is then soaked
overnight in 10 percent nitric acid, again rinsed throughly with glass-distilled
or deionized water, and oven-dried. When not in use, glassware must be
stored in relatively dust-free containers or else covered by aluminum
foil.
3.4.3.3 Procedure
Preparation of Test Algae. The algal bioassay method described here
is the simplest one available and is designed to yield a large amount of
data in as short a time as possible. It is not as precise as other methods
in which cell counts are made each day or where actual living biomass
is estimated, but it should be very adequate for ranking purposes.
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TABLE 9 . COMPOSITION OF MIXES TO BE ADDED TO
ALGAL GROWTH MEDIA
Nutrient Mixes
Amount/Liter
Metal Mix
(a)
ZnSO.-TH-O
CuSO.-SH-O
Glass-distilled or deionized water
y itfliniri fitt-^
Thiamin hydrochloride
Biotin
B12
Glass-distilled or deionized water
Minor Salt Mix*C*
Glass -distilled or deionized water
0.480g
0.144g
0.045g
0.157mg
0.404mg
0.140g
1 liter
50 mg
O.Olmg
O.lOmg
100ml
3.0g
50. Og
20.Og
1 liter
(a) Add 15 ml/1 of test solution.
(b) Add 0.5 ml/1 of test solution.
(c) Add 1.0 ml/1 of test solution.
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One week before a test is to begin, start an algal culture by adding,
sterilely, 1.0 ml of a well-grown culture to 40 ml of 10 ppt artificial
seawater with nutrients. Allow the algae to grow while being shaken
under 4000 lux illumination (approximately) from "cool white" flour-
escent tubes on a 14-hours light, 10-hours darkness cycle. On the day
the test is set up, dilute this culture in sterile tubes with sterile
artificial seawater to an optical density of 0.100 at 525 M-m. A Fisher
Electrophotometer II® (Fisher Scientific Co.) is convenient to use
here because it accepts calibrated test tubes of 25 x 200 mm size (Bellco
Glass Co., Vineland, NJ 08360). The calibration procedure for optically
matching the tubes must be done in the testing laboratory.
Range-finding Tests. Range-finding tests are needed in bioassays to
determine the concentrations of effluents to be used in definitive tests.
Dilute the effluent that was taken to 10 ppt salinity with 10 ppt
artificial seawater that contains nutrients to 0.01, 0.1, 1.0, and 10.0 percent
Filter-sterilize the dilutions by passing them through a sterile
membrane filter such as the 0.22^, porosity filter manufactured by the
Millipore Filter Corp., Bedford, MA 01730. At the same time, filter artificial
seawater growth medium to be used for control cultures and undiluted
effluent in a similar fashion.
Then add filter-sterilized medium aseptically to a sterile
automatic pipette (such as Ace Glass, Inc. No. 8004-A fitted with No.
8004-36 volumetric bulb) and 25 ml added to each of three autoclaved
25 x 200 mm calibrated test tubes fitted with microbiological closures.
This will yield 18 test culture tubes with three tubes each for control
and 0.01, 0.1, 1.0, 10.0, and 100 percent waste. Add, aseptically,
0.5 ml of the cell suspension of 0.100 absorbance. Place the tubes on a
shaker at approximately 60 excursions per minute. Model G2 Gyratory
shaker fitted with No. AG2-TA25 platform (New Brunswick Scientific Co.,
New Brunswick, CN 08903) works well.
"Read the absorbance of each tube against filtered medium on
the fifth day after inoculation. Calculate the average absorbance
for each group and the percentage growth in waste media as compared to
the control. If growth in the lowest concentration is strongly inhibited,
repeat the test with more dilute media. If inhibition of
growth is found, estimate the concentrations, as percentage of controls,
that will inhibit growth by approximately 65 and 35 percent.
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Definitive Tests. For determination of the EC50, at least one control
group and five concentration groups must be used. The five concentrations
must be in a geometric series and include concentrations that inhibit
growth by approximately 65 and 35 percent.
If growth stimulation occurred in the range- finding tests, use five
concentrations in a geometric series between a concentration without
effect and 100 percent waste.
The definitive tests are set up in the same way as range- finding tests
except for different concentrations of effluent. Determine absorbance of
the cultures every day between days 3 and 12. Plot the average absorbance
for each day on graph paper and examine the shape of the curve. Some
toxicants inhibit growth in the early stages of a test and it is necessary
to be careful in interpretation of data.
Use the expression
l
M- = ~
where \i> = specific growth rate
X^ = absorbance on day ti
X2 = absorbance on day t£
to calculate the specific growth rate between any days.
Estimate final biomass on the 12th day by weighing an aliquot of
each culture. To do this, pass 200 ml of glass-distilled or deionized
water through a 0.45t* porosity membrane filter (Millipore). Dry the filter
in an oven for 4-6 hours at 90 C. The filter may be dried on a sheet of
paper that contains an identifying number. Cool the filter to room
temperature in a desiccator and weigh it to the nearest one-tenth of a
milligram.
Pass a suitable measured aliquot, usually 10 ml, of each culture
through the filter under a vacuum of 0.5 atm. Do not use a greater vacuum
because cells may break. Quickly, wash the cells with 5 ml of glass-
distilled or deionized water to remove salts. Dry the filter with its
attached algae for 4-6 hours at 90 C, cool to room temperature in a
desiccator, and weigh to the nearest one- tenth of a milligram. Subtract
the weight of the filter from the weight of the combined filter and algae
and express algal weight to the nearest milligram.
Do the above for all tubes, calculate the average for each group, and
express it as percentage of the control group.
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3.4.3.4. Results
Calculation of the EC50. The EC50 is the concentration at which
growth was 50 percent of the control. It may be estimated by interpolation
by plotting the data on semilogarithmic coordinate paper with concentra-
tions on the logarithmic axis and percentage growth on the arithmetic axis.
To do so, draw a straight line between the 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.
Stimulation. If growth was stimulated by the effluent, report the
percentage stimulation at each concentration.
Report. In the final report, the following specific information
should be included along with the general information provided for all
bioassays:
1. The EC50 at 12 days and other days of importance to be decided
upon by the shape of the growth curve.
2. The specific growth rate between days 3 and 12 and any other
period that should be reported depending upon the shape of the
growth curve.
3.4.4 Static Bioassays With Marine Animals (References 36-38)
3.4.4.1 Introduction
The method recommended for static bioassays on marine animals is the
simplest, least expensive one available, and is designed to give a large
amount of data in the shortest possible time. It incorporates methods
given in Reference 36 and Reference 16. Juvenile sheepshead minnows
(Cyprinodon variegatus) and adult grass shrimp (Palaemonetes pugio or
^. vulgaris) are the test animals. These species adapt easily to a wide
range of salinity and temperature in static bioassays and several phases
of the life cycles can be studied.
The main objections to the method are that receiving water
characteristics are not closely simulated and the test organisms may not be
representative of the most sensitive species in a given geographical area.
However, the method should be satisfactory for the purpose of ranking
industries according to toxicity of their effluents.
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3.4.4.2 Method Description
Facilities. Before tests are done, it is necessary to acclimate and
observe the animals for several days. This requires tanks or live cars
for holding in natural flowing seawater and tanks for acclimation. It is
absolutely necessary that temperature be controlled in the holding and
acclimation tanks and during the bioassay. The tanks must be constructed
of materials that do not leach into, or sorb toxicants from, the water.
Glass, #316 stainless steel, or perfluorocarbon plastics should be used
whenever possible. Also, holding tanks should be constructed of plywood
coated with fiberglass resin. Rubber, copper, brass, galvanized metal,
or lead may not be used in any part of the test. Holding and acclimation
tanks must be in a well-ventilated area that is free of fumes. They must
be equipped for aeration, and the air, introduced via an air stone or
glass tube, must be taken from a well-ventilated fume-free area. It may
be pumped by an oilless rotary or piston type air compressor.
During holding, acclimation, and testing, the animals must not be
disturbed unnecessarily, either by excessive handling or excessive move-
ment around the tanks. When they must be handled, it should be as
gently, carefully, and quickly as possible.
Species. Species to be used are juvenile sheepshead minnows
(Cyprinodon variegatus) and adult grass shrimp (Palacmonetes pugio or P.
vulgaris). Test shrimp may be collected from wild populations in
relatively unpolluted areas, purchased from commercial suppliers, or
cultured in the laboratory. Whatever the source, all animals used in
testing should be healthy and as uniform in size as possible.
Juvenile sheepshead minnows are to be obtained from laboratory
populations according to the method of Schimmel et al. (Ref. 37) •
Test Containers. The static bioassays are to be done in 19.6-liter
(5-galIon), widemouth, soft-glass bottles that contain 15 liters of test
medium or in 30 x 60 x 30 cm deep, all-glass test chambers. If the
rectangular test chambers are used, their sides should be bonded with clear
silicon adhesive. As little adhesive as possible should be in contact
with the water, and extra beads of adhesive should be on the outside of
the containers rather than on the inside.
The test containers must be cleaned scrupulously before use. New
containers must be washed with detergent, rinsed with tap water, then
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pesticide-free acetone, and again with tap water. They must then be
soaked for at least six hours in 10 percent hydrochloric acid, and rinsed with
glass-distilled or deionized water. Between tests, the vessels must be
treated in the same way, except they must be soaked for 2-4 hours in a
solution of 200 tog hypochlorite/liter of glass-distilled or deionized
water. This solution can be made by mixing six volumes of household
chlorine bleach with 94 volumes of water. Never use hypochlorite when
acid is present because hazardous fumes may be generated.
Containers must be rinsed thoroughly after cleaning. Just before use
in a test, they must be rinsed with artificial seawater (see Diluted
Effluent below).
Test Medium. The test medium is prepared from liquid effluent by
addition of a commercial artificial sea-salt mix (Rila Products, Teaneck,
NJ 07666). Salinity is 10 ppt because it is probable that industrial
effluent will be in highest concentration in low-salinity water in
estuaries.
Undiluted Effluent. Check the salinity of the effluent and add an
appropriate amount of Rila Salts to each liter to yield a salinity of
10 ppt as determined by an American Optical Co. refractometer, or its
equivalent, fitted with a salinity scale. Gently swirl or mix the effluent
while adding salt.
Diluted Effluent. Prepare a Rila S?lt solution of 10 ppt in glass-
distilled or deionized water. Use this artificial seawater to dilute the
salt-containing medium prepared with undiluted effluent. Dilutions will
be prepared as percentages of undiluted medium.
Acclimation. Test the animals at 20 + 2 C. If they are collected
at another temperature (the fishes will be raised at 22 C), they should
not be subjected to more than a 3 C change in temperature in any 1-hour
period or to more than a 5 ppt change in salinity (the fishes will be
raised at 10 ppt) in any 24-hour period. Avoid crowding during acclimation.
Feed the animals a commercial flake food (Longlife Aquarium Products,
Harrison, NJ 07029) once a day. Clean the acclimation tanks after each
feeding.
The acclimation water is 100 percent dilution water. The animals
should show no signs of stress, such as discoloration, altered behavior,
or disease and must be kept for at least 2 days in acclimation tanks.
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Remove dead and abnormal animals as soon as they are noticed. If more
than 5 percent of a group dies in the 48 hours previous to testing, discard
the whole group.
Treatment of Effluent Samples. Before testing, effluent samples must
not be aerated or altered in any way. They should be covered at all times
and never agitated. However, they may be swirled gently before a test to
suspend particles or to aid in solution of Rila Salts. Initiate tests as
soon as possible after the sample is taken and after range-finding tests
are completed. Store the samples at 20 + 2 C.
3.4.4.3 Procedure
Range-finding Tests. Because toxicity of an effluent probably will
not be estimated, it will be necessary to run range-finding tests before
definitive EC50 values can be calculated. Range-finding tests must be
done in two ways, one with and one without aeration. In the test with
aeration, introduce clean air at the rate of 100 + 15 bubbles/minute from
a glass tube of one mm diameter.
Use effluent concentrations of 0.01, 0.1, 1.0, 10.0 and 100 percent.
If more than 50 percent of the animals die at 0.01 percent effluent,
conduct a new range-finding test at lower concentrations such as 0.0001
and 0.001 percent. Conduct a control test of 100 percent dilution water
at the same time. The pH of the test media and controls must be taken
before and after the test.
Carefully place five animals in test containers at a weight not
exceeding 0.8 gm/liter of medium. Determine weights by weighing represen-
tative members of the populations to be used before acclimation. Indivi-
duals that are to be used in a test must not be weighed or measured after
acclimation has begun. Watch the animals for death or stress for 24 hours.
i
In some cases, an 8-hour observation period may be sufficient but it
should be recorded. Remove dead animals as soon as possible after death.
Definitive Tests. Concentrations of test effluent for definitive
tests that yield LC50 or EC50 values will be determined from results of
the range-finding tests. In the definitive tests, use a control and six
concentrations of effluent in a geometric series with the concentration
in each treatment at least 50 percent that of the next higher one. One
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treatment must kill more than 65 percent of the animals and one treatment
must kill less than 35 percent.
Do not feed animals during the definitive tests.
The bioassay exposures consist of two separate 96-hour bioassays,
one with aeration and one without. This is because some effluents can be
expected to have high BOD. Aeration is to be identical to that described
in the range-finding test. Determine oxygen content of the water at
24-hour intervals by polarigraphic methods, and record pH and temperature
for the same periods.
Observe the animals frequently throughout the 96 hours and record
the number of dead animals for each 24-hour period. An animal should be
considered dead if it does not respond to gentle prodding.
For shrimp, it may be necessary to calculate an EC50 value (effective
concentration) rather than a LC50 (lethal concentration). This is be-
cause shrimp may not die from toxicants but may be immobilized and unable
to move anything but the appendages. Loss of equilibrium may be another
criterion for effect on shrimp.
For both shrimp and fish, report such things as erratic swimming,
loss of reflex, discoloration, behavioral changes, excessive mucous pro-
duction, hyperventilation, opaque eyes, curved spine, hemorrhaging, molting,
and cannibalism.
3.4.4.4 Results
Calculation of LC50 or EC50. An LC50 is a concentration at which
50 percent of the experimental animals died and an EC50 is a concentration
at which 50 percent of the experimental animals were affected. Either
may be an interpolated value based on percentage of animals dying or
affected at two concentrations. To estimate the EC50 or LC50, plot the
data on semi-logarithmic coordinate paper with concentrations on the
logarithmic axis and percentages of affected animals on the arithmetic
axis. Draw a straight line between two points representing death or
effect in concentrations that were lethal to or effective against more
than half and less than half of the organisms. The concentration at
which the line crosses the 50 percent mortality or effect line is the
LC50 or EC50 value. If 50 percent of the animals were not affected by
the highest concentration, the percent affected should be reported.
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In some cases, only the 96-hr EC50 can be calculated because of color
or suspended material that hide the animals in the waste.
Report. Any deviation from the above method should be reported. All
reports should also include:
1. Weight of animals in each container.
2. Definitions of the criteria used to determine the effect, and a
summary of general observations on other effects or symptoms.
3. Percentage of control organisms that died or appeared abnormal.
4. The 24-, 48-, and 96-hour LC50 or EC50.
5. Results of all pH, temperature, and oxygen measurements.
3.4.5 Stress Ethylene Plant Response (References 39,40) •
This test is based on the well-known plant response to environmental
stress: release of elevated levels of ethylene. Under normal conditions
plants produce low levels of ethylene. The test is designed to expose
plants to various levels of gaseous effluents under controlled conditions.
The ethylene released during a set time period is then measured by gas
chromatography to determine toxicity of the effluent.
3.4.5.1 Method Description
Materials. The recommended plant for this test is 6-week-old soybean,
Glycine max (Dare variety). Other plants could be substituted, but all
tests should be performed using one species, as plants vary in sensitivity
to stress. Soybean is known to be very sensitive and is readily available
in most areas. Optimum age of plants used for the test is also somewhat
variable, but all plants used should be of the same age. Very young plants
and, especially, senescent plants should not be used. Plants should be
selected for uniformity of sice and number of leaves. Great care should
be taken during growth and exposure to insure that plants are not injured
or stressed except by the feedstock or waste stream.
Greenhouse or growth chamber facilities with controlled temperature
and lighting are necessary for growing the plants. It is suggested that
the plants be grown under supplemental lighting to give a 16-hour day and
under mmrlimmi day/night temperatures of 26 ± 3 C and 18 ± 2 C, respectively.
Greenhouse exposure chambers equipped with an overhead light bank and
an air-handling system, similar to those described in Reference 40 and
a dark room at 26 C are also needed.
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Procedure. The procedure used in this bioassay is a modification of
that used in Reference 39. Because of limitations in gas sample size,
Level 1 testing will be done using a static rather than a flow-through
exposure. In addition, at this time the stress ethylene bioassay is
under revision; therefore, what follows may be altered in the future.
Plants are exposed to various concentrations of gaseous effluent for
2 hours in the greenhouse exposure chambers. Control plants must also be
treated to determine normal ethylene levels. Immediately following expo-
sure, each plant plus its pot are enclosed in a bag of known volume and
incubated for 22 hours in a dark room at 26 C. The duration of ethylene
release is usually short and increases with higher pollutant concentration.
Ethylene samples are taken from the bag by inserting a syringe needle,
and the concentration is measured using a gas chromatograph equipped with
a flame ionization detector. The presence of ethylene is confirmed by
co-chromatography with standard ethylene. A variety of chromatographic
conditions can be used for measuring ethylene. Analysis should be done
as soon as possible after incubation as some ethylene will diffuse through
the bag with time. The range for normal ethylene production in Glycine
max is 25-40 nl ethylene/1 air as reported in Reference 39. As most work
has been done on the effects of known pollutants such as ozone, it is
difficult to predict the levels of ethylene released in response to
exposure to a grab sample. The exposure to 0.4 M-l/1 and 0.8 |il/l ozone
causes release of 250 and 600 nl/1 ethylene, respectively (Ref. 39).
It should be noted that very high levels of pollutant may cause death
of plant tissue. In this case, little ethylene will be released as only
wounded, living plant tissue produces ethylene. Plants should be inspected
for such damage, and it should be reported if observed.
For this test, at least three levels of the sample and a control should
be run. The sample gas should be pure (undiluted) and in dilutions of
0.5 and 0.25. If adequate gas sample is not available, the test must be
run on more diluted samples. Four to ten plants should be exposed to each
experimental condition and the control.
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3.4.5.2 Results
Results from varying concentrations of gas exposure are plotted on
semi-log paper and fitted by the least squares method to the following
formula:
InC = In (A) + BX.
C = total ethylene concentration (nanoliters/liter).
A = amount of ethylene produced by a non-stressed plant.
B = measure of increase in stress-induced ethylene produced as a
function of pollutant concentration.
X = pollutant concentration.
This formula takes into account the low levels of ethylene produced by
plants under normal circumstances, and the use of natural logarithms
stabilizes the variance of ethylene measurements at higher concentrations.
In addition to reporting the slope parameter, (B), and its 95 percent
confidence limit, specific information about the plants used and their
growth conditions should be supplied for each sample.
3.4.5.3 Discussion
Release of ethylene is a well documented plant response to various
kinds of stress. Advantages of this test include reliability, sensi-
tivity, speed and economy. Disadvantages center on the extreme care
needed in handling the plants to prevent any physical damage or stress
which would confuse the test results.
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3.4.6 Soil Microcosm Test (References 41-49)
3.4.6.1 Introduction
The Level 1 testing yields a rapid, yes-no solution to assessment
queries. It quantifies the relationship between contaminant dose and
transport, accumulation, and short-term effects. Since terrestrial
accumulation sites and remineralization processes are predominantly
within soil, intact soil microcosms excised from representative target
systems are used as test units.
Techniques and procedures following have resulted from several
individual experiments exploring the utility of microcosms as test
systems for assessing potentially hazardous materials. These procedures
have been tested using a variety of toxicants and have been compared to
field derived data. (Ref. 41-44)
In order to implement an adequate, efficient assessment, some back-
ground data are needed. First, what is the mode of entry into the target
ecosystems? If discernable, what is the frequency of discharge? Second,
what are the chemical characteristics of the substance? Third, what
information is available on direct toxicity to laboratory personnel?
Fourth, what are the target ecosystems?
The immediate questions which must be addressed in assessing potentially
hazardous substances are: (1) Where does it go? and (2) What and how much
alternation of ecosystem processes and population occurs?
Initially, we must discern if there is significant transport and whether
significant, short-term effects can be shown. These questions call for a
rapid, highly accurate, binary (yes/no) experimental design. The key to
answering this question is establishing a dose-transport and dose-effect
relationship for the specific substance of interest. Substances should be
tested on those ecosystem types (northern hardwood forest, pasture or
desert, e.g.) which will be the likely recipient.
The soil microcosm test results in specific answers to five questions.
These are: (1) Is the substance (or transformation or degradation product)
mobile? (2) Is the primary mode of soil export gaseous, dissolved, or
particulate? (3) Is nutrient cycling disruption indicated? (4) Are soil
biota affected? (5) Is mobility, export mode, nutrient cycling disruption,
or population effecting a function of dose?
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The soil microcosm test has been shown rapid and accurate in answering
these questions compared to total system microcosm and field studies
(Ref. 42-45) This allows the user to: (1) Determine dose ranges
for further testing; (2) discern whether there is reason for further
terrestrial testing (substance not highly mobile) or whether aquatic
studies are needed (substance highly mobile); and (3) rank substances
being tested for priorities of Phase 2 and 3 testing.
3.4.6.2 Method Jtescrlptlon
Obtain soil cores 5 cm diameter x 5 cm depth from representative
terrestrial ecosystems. Remove above ground higher plant tissue.
Encase the cores in 1 mil thick teflon within 2 mil thick shrinkable
polyvinyl chloride and gently heat shrink until a tight bond with the
core (minimum boundary flow) is achieved. Leave enough lining above the
soil surface to use gaseous export traps if necessary. Mount on glass
funnels in test tubes. Cover sides with opaque wrapping to negate abnormal
algae growth. Place in environmental chamber under as near the field con-
ditions as possible (Figure 4).
Equilibrate 3 weeks, if possible. Leach with rainwater or reconsti-
tuted water (known water chemistry) 2-3 times (enough to obtain 20-30
ml/date during equilibration). Determine Ca and dissolved organic carbon
(DOC) concentrations in these samples. If possible, use alkali traps to
determine daily C02 efflux 3-10 days during equilibration. Use these data
to discard dissimilar replicate soil cores and establish behavior of
individual replicates.
Experimental design is preferably a randomized complete block and,
if possible, factorial treatment arrangement of dosages with a minimum of
three cores per dose per terrestrial ecosystem tested. Randomized incom-
plete block designs can be used to simultaneously test a large number of
contaminants. Dosages of a wide range should be used for this phase to
maximize the clarity of dose dependent observations.
Add the test contaminant to the surface of the cores in a carrier,
such as soil (taken from replicate cores to those used as experimental
units). Dosages might be 0, 10, 100, 1000 ppm, for example, based on
core weights. Total amendment to all cores should be equivalent, so that,
for example, a control replicate would receive carrier (such as soil)
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Teflon Lined
PVC Tube
Soil Depth
0-5 cm.
Teflon Ring
Inverted Beaker
Alkali Trap
Glass Funnel
Leachate Collection
Tube
FIGURE 4. DIAGRAM OF A SOIL MICROCOSM UNIT
81
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equivalent to the carrier plus contaminant received by any treatment
dose. The following are weights of carrier and contaminant added to soil
cores approximatly 180 g each unit weight, 150 g air dry weight.
Dosage (ppm) Carrier (me) Contaminant (me)
0 180 0
10 178.2 1.8
100 162 18
1000 0 180
Contaminant and carrier should be mixed, and the particulate ground
to 1 ran particle size. Deposition of treatments onto cores should be as
even across the surface as possible.
Set gas traps to monitor C0£ efflux. Standard alkali traps, such
as 0.2 N KDH can be used for C0£ recovery. Collect traps and titrate
with 0.1 M HC1 at 24, 48, or 72 hour intervals. The more frequent the
measurement, the more complete the data analysis can be; however,
diurnal rhythms make daily observation the minimum useful time period.
Traps to absorb volatile contaminants may be used to discern the
existence and relative importance of gaseous export of the contaminant.
TENEX®absorbs most organics quantitatively and can be eluted to recover
them for detection.
After a week, add sufficient rainwater or reconstituted water (known
water chemistry) to collect approximately 20 ml leachate per core.
Analyze Ca and DOC concentration. Determine contaminant concentration
using standard chemical techniques. On days 14 and 21 repeat leaching.
Perform intact extraction technique for pools of nutrients left
within the core to estimate mass balances for microcosm units. (Ref. 46)
This technique is the addition of 200 ml of 1.0 M KC1 or NaHC03 to cores
and measurement of Ca, DOC and the contaminant in the leachate respectively.
Biotic analyses should also be conducted. Core samples 1 cm in
diameter should be removed from the soil microcosms using a cork borer.
The hole should be filled by a glass rod of the appropriate size if extrac-
tion for contaminant and nutrients is to be performed.
Biotic analysis should use the ATP assay, which would allow relative
microbial pools to be compared across treatment levels. The procedure for
ATP analysis is to add 1 g soil (wet weight) to 6 ml 7.4 pH TRIS buffer
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with 0.06 g ethylene diamine tetraacetic acid (EDTA). Vortex briefly.
Add 3 ml chloroform. Vortex again. Sonify in ice water 2-5 minutes.
Centrifuge (preferably at low temperature) at 1000 G for 2-10 minutes.
Transfer buffer to new tube. Add 3 ml CCl^. Recentrifuge briefly.
Sample buffer phase. Assay at 340 nm using standard hexokinase reaction
(commercial kit available from Calbochem Corporation (Ref. 47, 48).
Divide cores into 1 cm depths. Within each depth measure the amount
of contaminant by radioisotope techniques or stable chemistry. This is
an optional step, useful if the distribution of the contaminant is to
be estimated.
3.4.6.3 Results
Two types of data will be produced: monitoring data and harvest data.
Monitoring and harvest data will be available on nutrient processes as
well as on contaminant transport and fate.
Monitoring Results. Calculate the total export of Ca, DOC and con-
taminant for each microcosm by date using concentrations detected
and volumes of leachate collected. Calculate mean export (with standard
error) by treatment dose for each contaminant. Plot these data as cumula-
tive export as a function of time. (See Figure 5). C0£ efflux or other
gaseous export data can be similarly summarized and presented. Statistical
comparison can be made by covariance to determine the effect of treatment
on export of nutrients and contaminant. Previous studies show that often
CC>2 efflux and nutrient export increases as a function of dose. Transport
of the contaminant is usually greater with increasing dose.
Harvest Results. Calculate the extractable Ca, DOC, or contaminant
based on concentrations of ions measured multiplied by extractant volumes.
Calculate means (with standard errors) across replicates for each-treatment
dose. Use standard analysis of variance and Duncan's Range Tests to deter-
mine differences due to treatment. The following is an example of data
which would result from extraction procedures at harvest.
Contaminant Extractable Ca
Dosage (ppm) (me/microcosm)
0 250 ± 23
10 260 ± 10
100 106 ± 20
1000 63 ± 40
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n
o
I
u
t-t
3
o
FIGURE 5. SAMPLE PLOT OF CUMULATIVE CALCIUM LOSS AS A FUNCTION OF
TIME FOR 4 DOSAGE LEVELS OF A CONTAMINANT.
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Biotic data can be summarized as ATP per gram soil by 1 cm depth
intervals within each dosage. ATP concentration may be increased or de-
creased by contaminant, depending upon specific microbial group impacted.
Reporting Data. Data summaries should include information on source
of soil cores, dates taken from the field, whether equilibration was made
before treatment, and conditions under which cores were maintained. An
example data summary sheet is given in the Appendix.
3.4.6.4 Discussion (Ref. 45)
Replicabilitv. In order to establish quality control of screening
procedures, three criteria must be met. First', the parameters must be
predictable through the experimental period. Usually, this requirement
necessitates equilibration of the experimental units prior to treatment
with test substance. Second, confidence limits must be established for
the measured parameters. Unique microcosms for each substance, or each
dose rate are inapplicable. Third, reproducibility of parameters between
control microcosms between comparable experiments must be reasonable.
Replicability among experimental units within a treatment class has
been calculated during experiments using intact, homogenized, and sand-
soil substrates. We have found that replicability is dependent upon
substrate type and upon the parameter estimated. For example, C02 efflux
was replicable in all substrates, but DOC efflux varied greatly among
homogenized and sand-soil replicates at each sampling date and through
time. Similar results were obtained for nine nutrients. Results from
grass microcosms established using these substrates were similar. We
conclude that replicability of intact soil microcosms is greater than
other substrates.
Reproducibilitv. Several comparable experiments have been conducted
using the intact soil profile, total system microcosms, and field studies.
Overlapping error terms for common parameters measured in control units of
these experiments allow us to conclude reproducibility of the technique.
Accuracy. The criteria for determining microcosm result accuracy are
dual. First, parameters indicative of ecosystem processes should be
similar to those measured within comparable strata of the intact ecosystem.
Second, the distribution and the transport rates of the test substance
should mimic those which would occur in the source ecosystem. These
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criteria are the most difficult of those needed to establish the utility
of microcosms in screening potentially hazardous substances. Only limited
data have been collected to address this issue. The comparisons below
represent our findings to date.
Evidence was obtained from forest microcosm experiments. (Ref. 42)
Using comparable data for cadmium, zinc, copper, and lead, microcosm
data were compared to the forest ecosystem under stress from the heavy
metal deposition. (Ref. 49) Enrichment ratios, ER, were computed by
dividing the heavy metal concentration of the treated microcosm component
by the same metal concentration of the control microcosm component.
Greatest enrichment of metals occurred in litter strata. Cadmium had
greater ER for soil than did other metals, although Pb was greatly enriched.
Values compared to those near the heavy metal source, were similar to the
sampled ecosystem component. (Ref. 42)
More conclusive proof of accuracy was the nonsignificant difference
in field and microcosm data taken on similar dates, for parameters such as
C(>2 efflux, calcium, and DOC.
Problems« Several problems remain in the evaluation of the utility
of microcosms as screening tools to assess transport, fate and effects of
potentially hazardous substances. Four problems seem important. These
are: (1) the rate of ecosystem degradation cannot be calculated from loss
rates of nutrient elements as yet, although in theory it is possible to do
so; (2) the microcosms have not been tested using organic effluents,
singly or in combination; (3) absolute accuracy of microcosm results has
not been proven; and (4) reproducibility among microcosm studies conducted
using intact profiles excised during different seasons has not been
established.
Some information is needed to conduct adequate screening of potentially
hazardous substances. The mode of entry of the substance into the system is
needed. The direct toxicity of the substance to research workers is needed
in order to practice safe laboratory experiments. The potentially targeted
ecosystems should be identified if possible and used as sources of microcosm
units. Lacking this information, forest or pasture systems should be used
since more than 80 percent of the non-urban U.S. is one of these ecosystems.
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3.5 REPORTING FORMAT
The report of the results of any bioassay should include the
following:
(1) Name of the laboratory and investigator conducting the
test.
(2) Dates of the test.
(3) Detailed description of the material tested including
its source, date and time of collection, and any known
information on its physical and chemical properties
obtained from observation and on-site testing (e.g.,
color, turbidity, pH, and solubility).
(4) Detailed description of the testing procedure including
information on the test organisms, materials, and
apparatus used for the test, and on sample handling
(e.g., dilution and solvent factors, mode of
administering).
(5) Results of the tests including all preliminary tests
on range finding or mode of administering plus all
control tests performed. Qualitative as well as
quantitative observations should be reported. Raw
data in the form of a table or graph should be
included along with final calculations such as per-
cent inhibition at various effluent concentrations,
EC50 or LC50, and maximum tolerated dose estimate
plus statistical analysis where appropriate. For
some tests the final form of the data will be a
table or graph.
(6) Any other relevant information.
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CHAPTER IV
APPROACH TO LEVELS 2 AND 3 BIOLOGICAL TESTING
4.1 INTRODUCTION (Reference 3)
Level 1 analysis provides a survey of effluent streams which permits rank-
ing on a priority basis or distinguishing extremely hazardous streams from
those which are less hazardous or innocuous. Level 1 sampling and analysis
procedures are suitable for analyzing a variety of materials and should
ensure a high probability of detecting potential environmental problems; how-
ever, the procedures will not necessarily provide information on specific
substances.
With the information provided by Level 1 analysis, a more detailed, more
quantitative analysis of the streams in order of their relative importance can
be performed (Level 2). Level 2 sampling and analysis programs are directed
towards a confirmation of Level 1 results as well as a more detailed character-
•
ization of t*»* biological effects of the more toxic streams. They are not
as broad as Level 1 in that resources are expended to improve information on
streams of a critical nature. Additional sampling of other streams is de-
ferred because Level 1 information has indicated a less significant level of
environmental impact. In some cases, Level 2 may make use of the same pro-
cedures or modifications of the procedures used in Level 1. In many cases,
more sophisticated techniques may be required. A major difference In the
two levels is that at Level 2, greater attention is given to acquiring repre-
sentative samples and to more replication of samples. Furthermore, Level 2
analysis will not be conducted via a prescribed series of tests. Each sample
will require selection of appropriate techniques based on the information
developed in Level 1 and the information requirements of the assessments.
Level 2 analyses are the most critical of all three levels because they
must provide a valid estimate of toxicity. It Is equally important,
however, that the analyses are conducted in an Information effective man-
ner because increasing specificity and/or accuracy result in cost escala-
tions. Due to the multiplicity of analytical techniques required and
the potential for unnecessarily high resource expenditures, the
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Level 2 analyses should be managed by experienced analytical personnel working
in well-equipped laboratories. Furthermore, the analyses must be conducted
with full awareness of the information requirements of an environmental assess-
ment program. Biological analysis as well as chemical and physical analysis
should be performed at Level 2. Suggestions for possible Level 2 bioassays
are included in this chapter.
Level 3 sampling and analysis is designed to monitor a limited number
of selected compounds and to define accurately chronic sublethal effects of
the selected compounds. In vivo monitoring should be incorporated, if
possible. Since the analytical procedures are highly process and site
specific, it is not possible to define their exact form. In general, analysis
will be optimized to a specific set of stream conditions, and therefore,
Level 3 tests will most probably not be as complex or expensive as Level 2
tests. It should be stressed that the Level 2 and 3 biological tests
listed here are suggestions only. They are not recommended or required tests
and should be considered only as examples of the kind of test appropriate
to a particular level.
4.2 APPROACH TO HEALTH EFFECTS TESTING
Possible Level 2 bioassays include in vitro and in vivo tests to confirm
possible toxicity or mutagenicity detected at Level 1. Any biotesting to be
done at Levels 2 and 3 will be done through the Office of Health and Ecologi-
cal Effects, Research Triangle Park. Suggested Level 3 tests include in vivo
analyses for chronic toxicity, mutagenesis, carcinogenesis, teratogenesis,
and metabolism. Such procedures provide more quantitative information on
possible health risks.
4.3 APPROACH TO ECOLOGICAL EFFECTS TESTING
Examples of Level 2 bioassays include bioaccumulation and persistence
studies to provide a more detailed analysis of the effluent streams. Possible
Level 3 bioassays include tests of toxic substance removal by waste treatment
facilities, mutagenicity, and ecosystems analysis.
One possible terrestrial ecological effect bioassay at Levels 2 and 3
is phytotoxicity of airborne substances. Toxicity is indicated by any
foliar lesion produced by exposure to the test substance. Level 2 testing
would be designed to establish relative toxicity using selected plant
species. Level 3 would be designed to establish the threshold for plant
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injury. At both levels only direct effects on plants are considered
by this test. Not considered are such indirect effects as: (1) altera-
tion by a toxicant of disease susceptibility, (2) accumulation of a
toxicant on plant growth and yield. Possibly bioassays taking such
factors into account could be developed in the future.
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Soil Cores to Assess Impact of Chemical Toxicants on Soil. Pedobiol.
(in press).
47. Bastick, W. D. and Ausmus, B. S. A Technique for Determination of
Adenosinco. Submitted to Anal. Letters.
48. Ausmus, B. S. and O'Neill, E. G. The Relationship Between Forest and
Microcosm Soil Carbon Dynamics. Soil Biol. Biochem. (submitted)
49. Jackson, D. R. and Watson, A. P. Disruption of Macronutrient Pools
in Forest-Floor Litter Near a Lead Smelter. J. Environ. Qual. (in press)
50. Gray, T. R. C., and Parkinson, D. (editors) International Symposium
on the Ecology of Soil Bacteria. Univ. of Toronto Press, 1968.
51. Loomis, T. A. Essentials of Toxicology. 2nd edition, Lea and Febiger,
Philadelphia, 1964.
52. Rubenstein, R., et al. "Test Methods for Assessing the Effects of
Chemicals on Plants", NTIS PB-248-198, 1975, 246 p.
94
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APPENDIX
BIOASSAY RECORD SHEETS
95
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GENERAL FORMAT FOR RECORDING TOXICITY OR MUTAGENICITY DATA
Test Condition
Assay
Amount
Sample Type (lift/plate )
Revertanta on Individual Plates
Number Number Repeat Repeat
1234
Average
1&2 36,4
••••M ^^^HM
Adjusted
Average
Revertanta
1&2 3&4
^•^•^ ••^^•B
Index of
Relative
Mutagenlcttv
1&2 3&4
Nonaetlvatlon
Positive control
Solvent control
Sample
Induced activation
*
Positive Control
Solvent control
Sample
-------
WI-38 CELLULAR TOXICITY TESTING
Sample No. EC^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. ES via"
Tube Hg/ml After Cell No. as Viable bility
No. or tel/ml) Initial Incub. 7, of Control Cells Index ATP Protein
97
-------
ALVEOLAR MACROPHAGE TOXICITY TESTING
Sample No. DIFFERENTIAL
Date Rec'd Macrophages
Description of Sample Neutrophils
Other
Date Tested Incubation Time
Date Report Out EC5Q Values_
No. Rabbits Used Cell Count
Remarks About Rabbits Viability_
Viability Index_
Total No. Cells Recovered Protein_
Seeding Population of Cells Other
TEST RESULTS
Eg Via-
Tube M>g/ml After Cell No. as Viable bility
No. or (tH/ml) Initial Incub. I of Control Cells Index ATP* Protein
*ATP/106 cells as % of Control
98
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STRESS ETHYLENE PLANT RESPONSE RECORD SHEET
Sample No._
Date Rec'd
Description of Sample_
Date Tested
Date Report Out_
Plant Material Used:,
Age of Plants ^_
Growth Conditions Light Cycle:
Light Intensity:_
Day/Night Temp.:_
Cones, of Gas Sample Used:
No. of Plants Tested/Cone.:
TEST RESULTS
Plant No.
Cone.
Percent
99
Ethylene Release
Nanoliters/liter
-------
ALGAL BIQASSAY DATA SHEET
LABORATORY
DATE
INVESTIGATOR^
TEST NO.
TEST SAMPLE SOURCE
CULTURE MEDIUM_
pH OF MEDIUM
TEMPERATURE
COLLECTION DATA (TIME-DATE)
ILLUMINATION
OTHER PERTINENT SAMPLE INFO.
RESULTS
Sample
Flask
No.
Cone.
INCUBATION TIME-DAYS
Cells/ml
3
Dry wt.
mg/L
Other
Cells/ml
5
Dry st.
mg/L
Other
7
Cells/ml
•
Dry wt.
mg/L
Other
(etc for
9,13,17,
21 Days)
REMARKS:
Maximum Specific Growth Rate:
Maximum Standing Crop:
EC 50(12 Day or other dyas of importance)
100
-------
B10ASSAY RECORD SHEET
Dilution Water Analysis
t
Hardness
»g/l
as CaCo3
Alkalinity
ng/1
as CaCo \
Specific
Conductance
>>H
Suspended
solids
P.K/1
TOC
Bg/1
un- Ionized
ammonia
PR/1
Residual
chlorine
M/gl
Total organo
phos. pesti-
cides
ng/1
Total organo
t:lil»r . post 1-
cides * I'CB's
nu/1
Date
-------
STATIC BIOASSAY RECORD SHEET
Series:
Municipality
or
Company:
Date:
Technician:
Starting hour:
Material being tested:
Source:
Source of dilution water:_
Test species:
Temp. range:
No. individuals per percent waste:
Start
Percent waste
DO
Temperature
PH
Specific
conductance
Number
surviving
% survival
DO
Temperature
pH
Number
surviving
% survival
DO
Temperature
pH
Number
surviving
% Survival
DO
Temperature
pH
24 hours
48 hours
t
96 hours
Control
*
*
LC50/EC50
* Method used for calculating.
102
-------
SAMPLE DATA SUMMARY FOR SOIL MICROCOSM TEST
Date of Report: Investigator:
Contaminant: Source: Date:
Microcosm design
Treatment levels (dosages)
Replications
Carrier
Microcosm Source Date Taken
Equilibration Period_
C02 Efflux (X ± S.E.) mg/day
Ca Export (x ± S.E.) ng/week
D.O.C. Export (* ± S.E.) ng/week
Treatment Date
Monitoring Data:
flux:
dose x ± S.E.(mg/day) cumulative loss(mg)
C02 Efflux:
Ca Export:
dose x ± S.E.(mg/week) cumulative loss(mg)
DOC Export:
dose x ± S.E.(mg/week) cumulative loss(mg)
103
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SAMPLE DATA SUMMARY FOR SOIL MICROCOSM TEST
(Continued)
Monitoring Data:
Contaminant Export:
Detection method
dose i ± S.E.(ng/week) cumulative loss(rag)
Harvest Data:
Extractable Ca: Extractant
dose mg/g soil
Eztractable DjO.C. Extractant
dose mg/g soil
Ertractable Contaminant. Extractant
dose mg/g soil
Distribution of Contaminant:
depth (cm) dose
mg/g soil
1
1-2
2-3
3-4
4-5 104
-------
SAMPLE DATA SUMMARY FOR SOIL MICROCOSM TEST
(Continued)
ATP Concentration:
dose
depth(cm) mg/g soil
0-1
1-2
2-3
3-4
4-5
Summary;
Contaminant ( ) increased/decreased/did not change monitored para-
meters, and increased/decreased/did not change harvest parameters.
105
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/7-77-043
2.
3. RECIPIENTS ACCESSION'NO.
. T,TLE AND SUBTITLE
Procedures Manual: Level 1
Environmental Assessment Biological Tests For Pilot
Studies
5. REPORT DATE
April 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
K.M.Duke, M.E.Davis, and A. J. Dennis
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
BatteUe-Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
10. PROGRAM ELEMENT NO.
EHE623
11. CONTRACT/GRANT NO.
68-02-2138
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Research and Development'
Industrial Environmental Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
Task Final: 6/76-3/77
14. SPONSORING AGENCY CODE
EPA/600/13
15 SUPPLEMENTARY NOTES T£RL-RTP project officer for this report is Larry D. Johnson,
Mail Drop 62, 919/549-8411 Ext 2557.
is. ABSTRAcrTne manuai gives Level 1 biological testing procedures (recommended by
Industrial Environmental Research Laboratory—Research Triangle Park) for per-
sonnel experienced in conducting bioassays on samples from industrial and energy
producing processes. The phased environmental assessment strategy provides a
framework for industry, process, and stream priorities on the basis of a staged
sampling and analysis technique. Level 1 is a screening phase that characterizes
the pollutant potential of process influent and effluent streams. The manual presents
the strategy of the phased approach. It also presents the basic sampling procedures
and the Level 1 protocol for the biological tests used to analyze the samples. It
briefly discusses possible bioassay procedures for Levels 2 and 3. The manual is a
companion to TERL-RTP Procedures Manual: Level 1 Environmental Assessment,'
EPA-600/2-76-160a, June 1976.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
Air Pollution
Bioassay
Sampling
Analyzing
Air Pollution Control
Stationary Sources
Biological Tests
Environmental Assess-
ments
13B
06A
14B
18. DISTRIBUTION STATEMENT
Unlimited
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
114
20. SECURITY CLASS (Thispage)
Unclassified
22. PfllCE
EPA Form 2220-1 (9-73)
106
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