EPA
600/4-85-013
(3rd edition)
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
EPA/600/4-85/013
March 1985
Research and Development
A66NCY
, FB4A9
Methods for
Measuring the Acute
Toxicity of Effluents to
Freshwater and
Marine Organisms
(Third Edition)
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EPA/600/4-85/013
March 1985
METHODS FOR MEASURING THE ACUTE TOXICITY OF EFFLUENTS
TO FRESHWATER AND MARINE ORGANISMS
(Third Edition)
Edited
by
William H. Peltier
Ecological Support Branch
Environmental Services Division
U.S. Environmental Protection Agency
Athens, Georgia 30605
and
Cornelius I. Weber, Ph.D.
Chief, Biological Methods Branch
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY - CINCINNATI
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
1985
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
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FOREWORD
Environmental measurements are required to determine the quality of
ambient water, the character of effluents, and the effects of pollutants
on aquatic life. EMSL-Cincinnati conducts research to develop, evaluate,
and promulgate methods to:
Measure the presence and concentration of physical, chemical and
radiological pollutants in water, wastewater, bottom sediments,
and solid waste.
Concentrate, recover, and identify enteric viruses, bacteria, and
other microorganisms in water.
Measure the effects of pollution on freshwater, estuarine, and
marine organisms, including the phytoplankton, zooplankton,
periphyton, macrophyton, macroinvertebrates, and fish.
Automate the measurement of the physical, chemical, and
biological quality of water.
Conduct an Agencywide quality assurance program to assure
standardization and quality control of systems for monitoring
water and wastewater.
The Federal water Pollution Control Act Amendments (Clean Water Act
or CWA) of 1977 (PL 95-217) explicitly state that it is the national
policy that the discharge of toxic substances in toxic amounts be
prohibited. Determination of the toxicity of effluents, therefore, plays
an important role in identifying and controlling toxic discharges to
surface waters. This report provides standardized methods for measuring
the acute toxicity of effluents to aquatic life, for use by the USEPA
regional and state programs, and National Pollutant Discharge Elimination
System (NPDES) permittees.
Robert L. Booth
Director
Environmental Monitoring and
Support Laboratory - Cincinnati
m
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PREFACE
This manual represents the third edition of the general purpose
effluent acute toxicity test manual initially published by
EMSL-Cincinnati in January, 1978. This edition of the manual was
reviewed by the Bioassay Subcommittee of the EMSL-Cincinnati Biological
Advisory Committee, USEPA headquarters and regional staff, state and
interstate water pollution control programs, environmental protection
groups, trade associations, major industries, consulting firms, academic
institutions engaged in aquatic toxicology research, and other interested
parties in the private sector.
EMSL-Cincinnati Bioassay Subcommittee Members
William Peltier, Subcommittee Chairman
Environmental Services Division, Region 4
Peter Nolan, Environmental Services Division, Region 1
Thomas Fikslin, Environmental Services Division, Region 2
Ronald Preston, Environmental Services Division, Region 3
Lee Tebo, Environmental Services Division, Region 4
Charles Steiner, Environmental Services Division, Region 5
David Parrish, Environmental Services Division, Region 6
James Lazorchak, Water Management Division, Region 6
Bruce Littell, Environmental Services Division, Region 7
Leo Mosby, Environmental Services Division, Region 7
Loys Parrish, Environmental Services Division, Region 8
Milton Tunzi, Environmental Services Division, Region 9
Joseph Cummins, Environmental Services Division, Region 10
Robert Schneider, National Enforcement Investigations Center, Denver
Cornelius Weber, EMSL-Cincinnati
Wesley Kinney, Environmental Monitoring Systems Laboratory - Las Vegas
Steven Schimmel, Environmental Research Laboratory - Narragansett
Douglas Middaugh, Environmental Research Laboratory - Gulf Breeze
Donald Mount, Environmental Research Laboratory - Duluth
Alan Nebeker, Western Fish Toxicology Laboratory - Corvallis
Rick Brandes, National Pollutant Discharge Elimination System
Technical Support Branch, Permits Division, Office of Water
Enforcement and Permits, (OWEP)
Edward Bender, Compliance Branch, Enforcement Division, Office of
Water Enforcement and Permits, (OWEP)
Thomas Murray, Monitoring Branch, Monitoring and Data Support
Division, Office of Water
Steven Ells, Environmental Effects Branch, Health and Environmental
Review Division, Office of Toxic Substances
Cornelius I. Weber, Ph.D.
Chairman, Biological Advisory Committee
iv
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ABSTRACT
This manual describes methods for measuring the acute toxicity of
effluents to freshwater and marine macroinvertebrates and fish. The
methods include a preliminary range-finding test, a screening test, and
multi-concentration (definitive) static and flow-through toxicity tests.
Also included are guidelines on laboratory safety, quality assurance,
facilities and equipment, effluent sampling and holding, dilution water,
test species selection and handling, data interpretation and utilization,
report preparation, organism culturing, and dilutor and mobile bioassay
laboratory design.
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CONTENTS
Foreword iii
Preface iv
Abstract v
Figures x
Tables xii
Acknowledgments :xiv
1. Introduction 1
Static Tests 2
Flow-through Tests 3
Test for Persistence of Effluent Toxicity 4
2. Health and Safety 5
General Precautions 5
Safety Equipment 5
General Laboratory and Field Operations 5
Disease Prevention 6
Safety Manuals 6
3. Quality Assurance 7
Effluent Sampling and Handling 7
Test Organisms 7
Facilities, Equipment, and Test Chambers 7
Dilution Water 7
Test Conditions 8
Reference Toxicants 8
Control Charts 8
Record Keeping 9
4. Facilities and Equipment 10
General Requirements 10
Effluent Dilution System 11
Test Chambers 11
Type 11
Cleaning 12
5. Test Organisms 13
Species 13
Source 13
Life Stage 16
Laboratory Culturing 16
Holding and Handling 16
Disease Treatment 17
Transportation to the Test Site and Acclimation to
Dilution Water 20
'6. Dilution Water 22
7. Effluent Sampling and Handling 25
Sample Type 25
Grab samples 25
Composite Samples 26
vii
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Test Type 26
Static Tests 26
Flow-through Tests 28
Effluent Handling 29
Samples Used in On-site Tests 29
Samples Shipped to Off-site Test Facility .... 29
8. Toxicity Test Procedures 30
Range-Finding Test 30
Screening Test 30
Definitive Test 31
Test Duration 32
Preparation of Test Solutions 32
Control 33
Number of Test Organisms 34
Replicate Test Chambers 35
Loading of Test Organisms 35
Test Temperature 35
Stress 35
Dissolved Oxygen Concentration ... 37
Illumination 37
Feeding 37
Persistence of Effluent Toxicity 38
Summary of Test Conditions for Commonly used Test
Organisms 38
9. Effluent Fractionation Procedure (by Gerald Walsh) ... 43
10. Test Data 45
Biological Data 45
Chemical and Physical Data 45
11. Toxicity Data Analysis 50
Introduction 50
All-or-Nothing Nature of Mortality Data from
Effluent Toxicity Tests 51
Variability in Toxicity Test Results 52
Manual Methods for Estimating the LC50 57
Graphical Method 57
Moving Average-angle Method 59
Litchfield-Wilcoxon Abbreviated Method 63
Probit Method 70
Computer Calculation of LC50s and Confidence
Intervals 77
12. Estimating the Potential for Acute and Chronic
Toxicity of Waste in Receiving Waters based
on the LC50 from Acute Toxicity (Lethality) Tests
(by Lee B. Tebo) 79
Estimating the Acute NOEC 80
Estimating the Chronic NOEC 81
13. Report Preparation and Data utilization 82
Selected References 84
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Appendices 91
A. Distribution, Life Cycle, Taxonomy, and Culture Methods . . 92
Daphnia (£. magna, D. pulex,) 92
Mysids (Mysidopsis Tjahia) 104
Fathead Minnows (Pimephales promelas)
(by Donald J. Klemm)TT 112
Silversides (Menidia) (by Douglas P. Middaugh) .... 126
Brine Shrimp 138
B. Dilutor Systems 141
Solenoid and Vacuum Siphon Dilutor Systems 141
Solenoid System Equipment List 145
Vacuum System Equipment List 148
Dilutor Control Panel Equipment List 153
C. Bioassay Mobile Laboratory Plans 154
Tandem-axle Trailer 154
Fifth-wheel Trailer 157
D. Check Lists and Information Sheets 158
Bioassay Field Equipment List 158
Information Check List for On-site Industrial and
Municipal Waste Toxicity Tests 160
Daily Events Log 166
Dilutor Calibration Form 167
Daily Dilutor Calibration Check 168
E. Computer Programs for Calculating the LC50 and
95% Confidence Interval (by James Dryer) 169
Moving Average-angle Program 170
Probit Program 182
Trimmed Spearman-Karber Program 192
Program for Statistical Comparison of LC50s 202
TOXDAT Multi-method Program (Binomial,
Moving Average, and Probit Methods) 205
IX
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FIGURES
Number Page
1. Control chart 9
2. Approximate times required to replace water in test chambers
in flow-through tests 34
3. Rawson's nomogram for obtaining oxygen saturation values at
different temperatures at sea level 36
4. Diagram of effluent fractionation procedure 44
5. Data sheet for effluent toxicity tests 47
6. Check list on back of effluent toxicity data sheet 48
7. Linear plot of toxicity data 50
8. Comparison of angular, Logit and Probit transformations
of toxicity data 51
9. Plotted data and fitted line for log-concentration-versus-
percent-mortality method of determining LC50 58
10. Line fitted to data, and LC16, LC50, and LC84, as read from
the line 67
11. Nomograph for obtaining Chi2 from expected-percent-affected
and observed-minus-expected values 68
12. Nomograph for raising base S to a Fractional Exponent 69
13. Plot of toxicity test data for Probit analysis 72
14. Anatomy of female Daphnia pulex 94
15. Daphnia magna ephippium and eggs 94
16. Daphnia postabdomen 94
17. Lateral and dorsal view of the typical mysid 105
18. Morphological characteristics used in mysid
identification (Mysidopsis bahia) 105
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FIGURES (Cont.)
Number Page
19. Map showing distribution of the fathead minnow
in North America 113
20. Fathead minnow: adult female and breeding male 116
21. Silverside (Menidia) 128
22. Techniques for collection of silverside eggs in the field
and production of larvae in the laboratory 129
23. Holding and spawning system utilized in the culture of
silversides (Menidia) 132
24. Photographs of solenoid valve system and
vacuum siphon system 142
25. Solenoid valve dilutor system, general diagram 143
26. Solenoid valve dilutor system, detailed diagram 144
27. Vacuum siphon dilutor system, general diagram 146
28. Vacuum siphon dilutor system, detailed diagram 147
29. Effluent and dilution water chambers 149
30. Effluent chambers 150
31. Pre-mixing chamber 151
32. Dilutor control panel wiring diagram 152
33. Mobile bioassay laboratory, tandem-axle trailer,
external side view and internal view from above 154
34. Mobile bioassay laboratory, tandem-axle trailer,
external and internal end views 155
35. Mobile bioassay laboratory, tandem-axle trailer
internal views of side walls 156
36. Mobile bioassay laboratory, fifth-wheel trailer,
internal view from above 157
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TABLES
Number
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Recommended species, test temperatures, and life stages . . .
Recommended prophylactic and therapeutic treatments for
disease in freshwater fish
Preparation of synthetic fresh water
Preparation of synthetic sea water
Recommended test conditions for daphnids (Daphnia pulex
and D. magna.)
Recommended test conditions for mysids (Mysidopsis bahia) . .
Recommended test conditions for the fathead minnow
(Pimephases promelas)
Recommended test conditions for silversides (Menidia spp.)
Definition of fish behavior terms
Single laboratory precision of static acute toxicity tests
with aquatic organisms, using reference toxicants
Multi- laboratory precision of acute toxicity tests
with aquatic organisms, using reference toxicants
Multi-laboratory precision of acute toxicity tests
with Daphnia magna, using a standard effluent
Sample data for Litchf ield-Wilcoxon abbreviated method . . .
Corrected values for 0% or 100% effect
Values of Chi2 (p = 0.05)
Toxicity data for Probit Analysis
Empirical (observed) Probit percent mortality relationship .
Chi-square determination
Standard error of the login LC50 (M)
Page
14
18
23
24
39
40
41
42
46
53
54
56
59
65
66
66
71
71
74
75
XII
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TABLES (Cont.)
Number Page
21. Weighting factor (W) for the calculated Probit (Y) 75
22. Comparison of LC50s and 95% confidence limits obtained
with different computer programs and the graphic method . . 78
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ACKNOWLEDGEMENTS
The assistance of the following members of the staff of the Aquatic
Biology Section, EMSL-Cincinnati, is gratefully acknowledged: Dr. Donald
Klemrn prepared the section in the Appendix on culturing fathead minnows;
James Dryer provided the precision data on the fathead minnow nonrenewal
static toxicity tests, assisted in preparing other materials for the
section on test data analysis, provided listings of the computer programs
in the Appendix, and contributed to the figures for the dilutors and
bioassay trailers; the section in the Appendix on culturing Daphnia was
prepared in part from materials provided by Steven Pucke, Philip Lewis,
and Karen Brauer; Philip Lewis also provided precision data on the
Daphnia toxicity tests; and Cordelia Nowell and Diane Schirmann provided
valuable secretarial assistance.
Sections on facilities and equipment, test organisms, and test
procedures were adapted from, Methods for Acute Toxicity Tests with Fish,
Macroinvertebrates and Amphibians, National Water Quality Research
Laboratory, USEPA, Duluth, Minnesota, 1975. The sections on laboratory
safety and quality assurance were taken in part from, Interim NPDES
Compliance Biomonitoring Inspection Manual, Enforcement Division, Office
of Water Enforcement, USEPA, Washington, DC, 1979. The sections on
fractionating effluents and culturing silversides, were prepared by Drs.
Gerald Walsh and Douglas Middaugh, respectively, Environmental Research
Laboratory, USEPA, Gulf Breeze, Florida. Assistance in the preparation
of Section 10, on data analysis, was provided by Dr. Peter Gartside,
Biostatistician, Kettering Laboratory, University of Cincinnati,
Cincinnati, Ohio. Lee Tebo, Region 4, USEPA, Athens, Georgia, prepared
Section 12, on estimating the potential for acute and chronic toxicity of
waste in receiving waters. Dr. Thomas Waller and James Lazorchak,
University of Texas, Dallas, .provided information on the use of algae to
culture Daphnia.
In addition to the contributions of the Advisory Committee members
and others listed above, the editors wish to acknowledge the many helpful
comments, materials, and suggestions received from the following persons:
Joseph Arruda, Kansas Department of Health and Environment, Topeka,
Kansas; Bruce Barrett, U. S. Environmental Protection Agency, Washington,
D.C.; Alan Beck, U. S. Environmental Protection Agency, Narragansett,
Rhode Island; Nelson Cooley, U. S. Environmental Protection Agency, Gulf
Breeze, Florida; Jody Connor, New Hampshire Water Supply and Pollution
Control Commission, Concord, New Hampshire; Robert Cooner, Alabama
Department of Environmental Management, Montgomery, ALabama; Peter
Cumbie, Hunton and Williams, Washington, D.C.; Harold Cumiford,
xiv
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U. S. Environmental Protection Agency, Houston, Texas; G. Michael
DeGraeve, Battelle Laboratories, Columbus, Ohio; Kenneth Oickson, North
Texas State University, Denton, Texas; Philip Dorn, Shell Development
Company, Houston, Texas; Kenneth Dostal, U. S. Environmental Protection
Agency, Cincinnati, Ohio; James Fava, Ecological Analysts, Inc., Sparks,
Maryland; Henry Folmar, Mississippi Department of Natural Resources,
Jackson, Mississippi; John Gentile, U. S. Environmental Protection
Agency, Narragansett, Rhode Island; William Gulledge, Chemical
Manufacturers Association, Washington D.C.; David Gruber, Biological
Monitoring, Inc., Blacksburg, Virginia; Rebecca Hanmer, U. S.
Environmental Protection Agency, Washington, D.C.; John Howland, Missouri
Department of Natural Resources, Jefferson City, Missouri; Shing-Fu
Hsueh, New Jersey Department of Environmental Protection, Trenton, New
Jersey; Billy Isom, Tennessee Valley Authority, Muscle Shoals, Alabama;
Arthur Johnson, Massachusetts Department of Environmental Quality
Engineering, Westborough, Massachusetts; Suzanne Lassier, U. S.
Environmental Protection Agency, Narragansett, Rhode Island; Armond
Lemke, U. S. Environmental Protection Agency, Duluth, Minnesota; James
McCarty, U. S. Environmental Protection Agency, Corvallis, Oregon;
Charles McKenney, U. S. Environmental Protection Agency, Gulf Breeze,
Florida; Brian Melzian, U. S. Environmental Protection Agency, San
Francisco, California; Barry Mower, Maine Department of Environmental
Protection, Augusta, Maine; Harold Mullican, Tennessee Department Natural
Resources, Nashville, Tennessee; Delwayne Nimmo, U. S. Environmental
Protection Agency, Denver, Colorado; George Patton, American Petroleum
Institute, Washington, D.C.; James Polisini, California Department of
Fish and Game, Rancho Cordova, California; Landon Ross, Florida
Department of Environmental Regulation, Tallahassee, Florida; Carl Rybak,
U. S. Environmental Protection Agency, Cincinnati, Ohio; R. Schacht,
Illinois Environmental Protection Agency, Springfield, Illinois;
Carl-Axel Soderberg, Puerto Rico Environmental Quality Board, Santurce,
Puerto Rico; Bruce Sprague, U. S. Environmental Protection Agency,
Edison, New Jersey; Charles Stephan, U. S. Environmental Protection
Agency, Duluth, Minnesota; David Terpening, U. S. Environmental
Protection Agency, Seattle, Washington; Nelson Thomas, U. S.
Environmental Protection Agency, Duluth, Minnesota; Bruce Walker,
Michigan Department of Natural Resources, Lansing, Michigan; Stephen
Ward, U. S. Environmental Protection Agency, Edison, New Jersey; Gene
Welsh, Georgia Department of Natural Resources, Atlanta, Georgia;
Llewellyn Williams, U. S. Environmental Protection Agency, Las Vegas,
Neveda; John Youngerman, California State Water Resources Control Board,
Sacramento, California.
xv
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SECTION 1
INTRODUCTION
The Federal Water Pollution Control Act Amendments (Clean Water Act) of
1977 (PL 95-217), Section 10l(a)(3), in the Declaration of Goals and
Policy, states that "it is the national policy that the discharge of toxic
pollutants in toxic amounts be prohibited." Current Agency programs for
the control of toxic discharges are based in part on effluent limitations
for individual chemicals. However, data on the toxicity of substances to
aquatic organisms are available for only a limited number of elements and
compounds. Effluent limitations, therefore, may not provide adequate
protection for aquatic life where the toxicity of the components in the
effluent is not known, where there are additive, synergistic, or
antagonistic effects between toxic substances in complex effluents, and/or
where a complete chemical characterization of the effluent has not been
carried out. Since it is not economically feasible to determine the
toxicity of each of the thousands of potentially toxic substances in
complex effluents or to conduct exhaustive chemical analyses of effluents,
the most direct and cost-effective approach to the measurement of the
toxicity of effluents is to conduct an effluent toxicity test with aquatic
organisms. For this reason, there has been a steady increase in the use of
effluent toxicity tests within the Agency and state NPDES programs to
identify toxic discharges, and by permittees as a self-monitoring tool
(USEPA, 1979c). This manual was prepared to provide standardized methods
for effluent toxicity tests to minimize intralaboratory and interlaboratory
variability in toxicity tests conducted by USEPA regional and state
programs and NPDES permittees.
The first toxicity test methodology developed specifically for
effluents was provided in the previous editions of this manual (Peltier,
1978a,b), which were developed by the Bioassay Subcommittee of the
Biological Advisory Committee sponsored by EMSL-Cincinnati. The changes in
the membership of the Bioassay Subcommittee (see the Preface) and the
content of this manual reflect the considerable additional experience in
effluent toxicity testing gained by personnel within the USEPA since the
last (July 1978) edition of the manual was published.
The acute toxicity tests for effluents described in this report are
based on the cumulative experience of numerous USEPA regional and state
effluent toxicity monitoring programs, acquired during the past ten years.
These tests are used to determine the effluent concentration, expressed as
a percent volume, that is lethal to 50% of the organisms within the
prescribed period of time (LC50). Where death is not easily detected, such
as with the invertebrates, immobilization is considered equivalent to death.
In cases where an effect other than death (or immobilization) is used, the
results are expressed in terms of the median effective concentration, or
EC50.
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Acute toxicity tests are used in the NPDES Permit Program to identify
effluents containing toxic wastes discharged in toxic amounts. The data
are used to predict potential acute and chronic toxicity in the receiving
water, based on the LC50 and appropriate dilution, application, and
persistence factors (see Section 12). The tests are conducted as a part of
compliance sampling inspections, compliance biomonitoring inspections,
performance audit inspections, and during special investigations. The
samples are transported to a central laboratory for testing, or the tests
are conducted on-site by the regulatory agency or the permitee. Acute
toxicity tests can also be used in a toxicity reduction evaluation to
identify toxic waste streams within plants, which would aid in the
development and implementation of toxicity reduction plans, and can be used
to compare and control the effectiveness of various treatment technologies
for a given type of industry, irrespective of the receiving water
(USEPA 1984b).
Two types of acute toxicity tests are utilized — static and
flow-through. The selection of the test type will depend upon the
objectives of the test, the available resources, the requirements of the
test organisms, and effluent characteristics such as fluctuations in
effluent toxicity. Special environmental requirements of some organisms,
such as flowing water, or fluctuating water levels, may preclude the use of
static tests.
A negative result from an acute toxicity test with a given effluent
sample does not preclude the presence of chronic toxicity. Also, because
of the potential temporal variability in the toxicity of wastes, a negative
result with a particular sample does not preclude the possibility that
samples collected from that discharge at some other time might exhibit
acute (or chronic) toxicity.
The frequency with which these toxicity tests are conducted on an
effluent under a given NPDES permit is determined by the regulatory agency
on the basis of factors such as the variability and degree of toxicity of
the waste, production schedules, and process changes.
STATIC TESTS
Types of static tests include: (1) nonrenewal - the test organisms are
exposed to the same effluent solution for the duration of the test; and
(2) renewal - the test organisms are exposed to a fresh solution of the
same concentration of effluent every 24 h or other prescribed interval,
either by transferring the test organisms from one test chamber to another,
or by replacing all or a portion of the effluent solution in the test
chambers. Because of toxicant adsorption on the test chambers, uptake by
test organisms and the effect of metabolites on toxicity, the renewal
system is preferred.
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The advantages and disadvantages of static tests are as follows (also
see Buikema et al, 1982; Stephan, 1982):
Advantages:
1. Simple and inexpensive
2. Very cost effective.
3. Limited resources (space, manpower, equipment) required; would
permit staff to perform many sequential tests on samples collected
over time.
4. Small volume (1 to 20 L) of effluent required.
5. Provides some measure of persistence of toxicity, i.e. aging of the
effluent (nonrenewal static test).
Disadvantages:
1. Results do not reflect temporal changes in effluent toxicity.
2. Dissolved oxygen (DO) depletion may result from high chemical oxygen
demand (COD), biological oxygen demand (BOD), or metabolic wastes.
3. Possible loss of toxicants through volatilization and/or adsorption
to the exposure vessels.
4. Generally less sensitive than flow-through test, because the toxic
substances may degrade or be adsorbed, thereby reducing the apparent
toxicity. Also, there is less chance of detecting slugs of toxic
wastes, or other temporal variations in waste properties.
FLOW-THROUGH TESTS
Two types of flow-through tests are in common use: (1) effluent is
pumped continuously from the sampling point directly to the diTutor system;
and (2) effluent grab or composite samples are collected periodically,
placed in a tank adjacent to the test laboratory, and pumped continuously
from the tank to the dilutor system. The flow-through method employing
continuous effluent sampling is the preferred method for on-site tests.
Because of the large volume (often 400 L/day) of effluent normally required
for flow-through tests, it is generally considered too costly and
impractical to conduct these tests off-site at a central laboratory.
However, a mini-dilutor requires substantially smaller volumes and may be
practical to use off-site (Benoit et al., 1982).
The advantages and disadvantages of flow-through tests are as follows:
Advantages:
1. Provide a more representative evaluation of effluent acute toxicity,
especially if toxicity varies with time.
2. DO concentrations are more easily maintained in the test chambers.
3. A higher loading factor (biomass) may be used.
4. The possibility of loss of toxicant due to volatization, adsorption,
degradation and uptake is reduced.
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Disadvantages:
1. Large volumes of effluent and dilution water are required.
2. Test equipment is more complex and expensive, and requires more
maintenance and attention.
3. More space is required to conduct tests.
4. Does not measure the effect of aging of the waste on toxicity.
5. Because of resources required, it would be very difficult to
perform multiple or overlapping sequential tests.
TEST FOR PERSISTENCE OF EFFLUENT TOXICITY
The persistence of the toxicity of an effluent is of interest in
assessing the potential effects of the effluent on the receiving water. If
the toxicity of the waste has a half-life of less then 96 h, the toxicity
can be considered "non-persistent" (p. 122, USEPA, 1973). Methods for
determining the persistence of toxicity are discussed on p. 38.
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SECTION 2
HEALTH AND SAFETY1
GENERAL PRECAUTIONS
Collection and use of effluents in toxicity tests may involve
significant risks to personal safety and health. Personnel collecting
effluent samples and conducting toxicity tests should protect themselves
from injury by taking all safety precautions necessary for the prevention
of bodily injury, or inhalation or absorption of corrosive or toxic
substances through skin contact, and the prevention of asphyxiation due to
lack of oxygen or presence of noxious gases.
Prior to sample collection and laboratory work, personnel should make
sure that all necessary safety equipment and materials have been obtained
and are in good condition.
SAFETY EQUIPMENT
1. Personal Safety Gear
Personnel should use safety equipment, as required, such as rubber
aprons, lab coats, respirators, gloves, safety glasses, hard hat,
and safety shoes.
2. Laboratory Safety Equipment
Each laboratory (including mobile laboratories) should have safety
equipment such as first aid kits, fire extinguishers, fire blankets,
etc.
Mobile laboratories should be equipped with a telephone to enable
personnel to summon help in case of emergency.
GENERAL LABORATORY AND FIELD OPERATIONS
1. Work with effluents should be performed in compliance with accepted
rules pertaining to the handling of hazardous materials. It is
recommended that personnel collecting samples and carrying out
toxicity tests should not work alone.
2. Because the chemical composition of effluents is usually only poorly
known, they should be considered as potential health hazards, and
exposure to them should be minimized.
"•Adapted from: USEPA, 1979c.
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3. It is advisable to cleanse exposed parts of the body with soap and
water immediately after collecting effluent samples.
4. All containers should be adequately labeled to indicate their
contents.
5. Good housekeeping contributes to safety and reliable results.
6. Electrical equipment or cords not having Underwriters Laboratories
approval should not be used. Ground-fault interrupters should be
installed in all "wet" labs where electrical equipment is used.
7. Mobile laboratories should be properly grounded to protect against
electrical shock.
8. Staff training in basic first aid and cardio-pulmonary resusitation
is strongly recommended.
DISEASE PREVENTION
Personnel handling samples which are known or suspected to contain
human wastes should be immunized against tetanus, typhoid fever, and polio.
SAFETY MANUALS
For further guidance on safe practices when collecting effluent samples
and conducting toxicity tests, see permittee and general industrial safety
manuals, including USEPA, 1977, and Walters and Jameson, 1984.
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SECTION 3
QUALITY ASSURANCE 1
Quality assurance (QA) practices for effluent toxicity tests include
all aspects of the test that affect the accuracy and precision of the data,
such as: (1) effluent sampling and handling; (2) the source and condition
of the test organisms; (3) condition of equipment; (4) test conditions; (5)
instrument calibration; (6) use of reference toxicants; (7) record keeping;
and (8) data evaluation. For general guidance on good laboratory practices
related to toxicity testing, see: FDA, 1978; USEPA, 1979d, 1980a, and
1980b; and DeWoskin, 1984.
EFFLUENT SAMPLING AND HANDLING
If effluent samples are collected for off-site testing, the samples
should be preserved as described in Section 7.
TEST ORGANISMS
Test organisms listed in Table 1 (Section 5) may be used. The
organisms should be free of disease, as indicated by minimal rates of
mortality in holding tanks and in test controls, should not have been
exposed to pollutants or other stress prior to testing, and are to be in an
early, life stage, as specified in Table 1.
FACILITIES, EQUIPMENT, AND TEST CHAMBERS
Laboratory and bioassay temperature control equipment must be adequate
to maintain recommended test water temperatures. Thermometers, pH meters,
diTutors, and other measuring devices must be calibrated by following the
manufacturer's recommended methods before, and at appropriate intervals
during, use. Recommended materials must be used in the fabrication of the
test equipment which comes in contact with the effluent (see Section 4).
DILUTION WATER
The dilution water used in the tests will depend on the objectives of
the study and logistical constraints. Receiving water collected upstream
from the discharge point should be used as dilution water wherever
possible. Depending upon the objectives, it may be desirable to use
receiving water as the dilution water even when it is contaminated by
"upstream" sources. These objectives may include determination of the
toxicity of the receiving water upstream from the discharge and the
additive toxicity of the waste and the contaminated intake water used for
plant processes. If the presence of contaminants in the receiving water
makes it unsuitable as dilution water, other uncontaminated surface water
such as a nearby tributary, or ground water, or synthetic water may be
employed.
Adapted from: USEPA, 1979c.
-------
TEST CONDITIONS
Water temperature ranges for the test organisms (Table 1) must be
maintained within the recommended limits. Dissolved oxygen concentrations
should be checked at the beginning of the test and regularly throughout the
test period. Aeration is employed if necessary to ensure that the DO does
not fall below 40% saturation for warm-water species and 60% saturation for
cold-water species.
REFERENCE TOXICANTS
A reference toxicant is to be used to establish the validity of
effluent toxicity data generated by toxicity test laboratories. Three
reference toxicants, sodium dodecylsulfate, sodium pentachlorophenate, and
cadmium chloride, are available from EMSL-Cincinnati. Instructions for
their use and the expected LC50 values are provided with the samples.
Factors affecting the accuracy of the data include test organism age,
condition, and sensitivity, water temperature, etc. If the laboratory does
not have an ongoing culturing program and obtains the test organisms
periodically in large numbers (batches) from an outside source, the
sensitivity of each batch of test organisms must be evaluated with a
reference toxicant within the seven days immediately preceding an effluent
toxicity test or concurrently with the test. Laboratories that obtain test
organisms from outside sources and conduct less than one toxicity test per
month may find it convenient to run all sensitivity tests concurrently with
effluent toxicity tests. If the laboratory maintains breeding stock, the
sensitivity of the offspring should be checked with a reference toxicant at
least once each month. If preferred, this reference toxicant test may be
run concurrently with an effluent toxicity test.
CONTROL CHARTS
A control chart (USEPA, 1979a) should be prepared for each reference-
toxicant/organism combination, and successive LC50s plotted and examined to
determine if the results are within prescribed limits (Fig. 1). In this
technique, a running plot is maintained for the LC50s from successive tests
with a given reference toxicant. This type of control chart, called a
cumulative-summation (cusum) chart, evaluates the_ cumulative trend of the
statistics from a series of samples. The mean (X) and upper and lower
control limits (+_ 2S) are recalculated with each successive point, until
the statistics stabilize. Outliers, which are values which fall outside
the upper and lower control limits, and trends of increasing or decreasing
sensitivity are readily identified.
If the LC50 of reference toxicant does not fall in the expected range
for the test organisms, the sensitivity of the organisms and the overall
credibility of the test system are suspect. In this case, the test
procedure should be examined for defects, and a different batch of test
organisms should be employed in repeating the reference toxicant and
effluent toxicity tests.
-------
<=>
in
CJ
1=1
n
UPPER CONTROL LIMIT(X+ 2S)
CENTRALTENDENCY
LOWER CONTROL LI MIT (X - 2S)
i i i i i i i i i i i i i i i i i i i
I
I
05 10 15 20
TOXICITY TEST WITH REFERENCE TOXICANTS
Figure 1. Control chart.
n- 1
Where:
X-j = Successive LCSO's from toxicity tests,
n = Number of tests.
X = Mean LC50.
S = Standard deviation.
RECORD KEEPING
Proper record keeping is very important. Bound notebooks should be
used to maintain detailed records of test organisms such as species, age,
size, source, date of receipt, culture maintenance, and disease treatment,
and information on the calibration of equipment, test conditions employed,
and test results. Annotations should be made on a real-time basis to
prevent the loss of information.
-------
SECTION 4
FACILITIES AND EQUIPMENT
GENERAL REQUIREMENTS
Effluent toxicity tests may be performed in a fixed or mobile
laboratory. Depending upon the scope of the toxicity screening program,
facilities may include equipment for rearing, holding, and acclimating
organisms. Temperature control can be achieved using circulating water
baths, heat exchangers, or environmental chambers. Water used for rearing,
holding, acclimating, and testing organisms may be ground water, surface
water, dechlorinated tap water, or synthetic water. It is recommended that
receiving water be used for acclimation and testing wherever possible. Tap
water can be dechlorinated by active aeration (using air stones) for 24 h,
filtration through actived carbon, or the use of sodium thiosulfate.
Aeration and treatment with sodium thiosulfate may not remove toxic
halogenated organics produced by chlorination, whereas filtration with
actived carbon will remove both the chlorine and the halogenated organics,
and is the preferred treatment.
Equipment should be available for water temperature control and
aeration. Air used for aeration must be free of oil and fumes. Test
facilities must be well ventilated and free of fumes. During holding,
acclimating, and testing, test organisms should be shielded from external
disturbances.
Some organisms may have special environmental requirements such as
flowing water, fluctuating water levels, or substrate, which must be
provided. During holding, acclimating, and testing, immature stream
insects should always be in flowing water (Nebeker and Lemke, 1968). Since
cannibalism can occur among many species of shrimp, crabs and decapods,
they should be isolated by some means (e.g., with screened compartments),
or the claws of crabs and crayfish should be bound.
Materials used for diTutors, exposure chambers, tubing, etc., which
come in contact with the effluent should be carefully chosen. Tempered
glass, No. 304 (for freshwater) or No. 316 (or higher grade) stainless
steel (for saline water), and perfluorocarbon plastics (e.g., TEFLOlP)
should be used whenever possible to minimize sorption and leaching of toxic
substances. These materials may be reused following decontamination.
Plastics such as polyethylene, polypropylene, polyvinyl chloride, TYGOlP,
etc., may be used as test chambers and to store effluents and dilution
water or to convey them to a test system. However, caution should be
exercised in the use of plastics because they could introduce toxicants
when new or carry over toxicants from one test to another. New plastics
products of a type not previously used should be tested for toxicity before
initial use. Small, inexpensive plasticware (except that of
perfluorocarbon) which comes in contact with effluents should be discarded
after each test. Equipment (large tanks, pumps, valves, etc.) which cannot
10
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be discarded after each use because of cost, must be decontaminated
according to the cleaning procedures listed below. Fiberglass, in addition
to the previously mentioned materials, can be used for holding, acclimating,
and dilution water storage tanks, and in the water delivery system. All
materials should be flushed or rinsed with the test media for a period of
time before using in the test.
Copper, galvanized material, rubber, brass, and lead, must not come in
contact with holding, acclimation, or dilution water, or with effluent
samples and test solutions. Some materials, such as several types of
neoprene rubber (commonly used for stoppers) may be toxic and should be
tested before use.
EFFLUENT DILUTION SYSTEM (Flow-through test only)
Although many types of diTutors have been designed (Brungs and Lemke,
1978), the flow-through, proportional dilutor is the preferred system for
routine effluent toxicity tests conducted in both fixed and mobile
laboratories.
The following factors should be considered in designing the dilutor:
(1) whether the apparatus will be installed and used in a fixed or mobile
laboratory; (2) the existence of adequate space and/or structural
requirements for the dilutor, test chambers, effluent and dilution-water
storage; (3) the applicability of the dilutors to specific effluent
characteristics (high suspended solids, volatiles, etc.); (4) the system's
dependability, durability, flexibility, and ease of maintenance, cleaning,
and replacement; (5) the ability to perform within the flow rate and
concentration accuracy limitations; and (6) the cost of the system.
Until recently, it was necessary to custom-make all dilutors. However,
prefabricated dilutor systems are now commercially available (e.g. Ace
Glass1; Specialized Environmental Equipment, Inc.2). Two examples of
custom-made proportional dilutors -- the solenoid valve system and the
vacuum siphon system -- are described in the Appendices.
TEST CHAMBERS
Type
The test chambers most commonly used in static tests for fish are
wide-mouth, 0.95 L (1 qt), 3.8 L (1 gal) or 19.0 L (5 gal) soft-glass
bottles or aquaria. Containers such as 10 to 20 cm (4 or 8 in.) diameter
culture dishes or beakers may be more suitable as test chambers for fish
eggs and/or larvae and small Crustacea. Special glass or stainless steel
test chambers can be constructed to accommodate test organisms requiring
particular physical conditions (large surface-area-to-volume ratio).
^Ace Glass Company, Vineland, New Jersey.
^Specialized Environmental Equipment, Inc., Easley, S.Carolina.
11
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Test chambers used in flow-through tests are constructed of 3-mm
(0.125-in.), double strength, or 6-mm (0.25-in.) tempered plate glass held
together with clear silicone adhesive. Silicone adhesive absorbs some
organochlorine and organophosphorus pesticides, which are difficult to
remove. Therefore, as little of the adhesive as possible should be in
contact with water; extra beads of adhesive inside the containers should be
removed. Stainless steel (No. 304 or No. 316) can be used in the
construction of test chambers, but must be of welded, not soldered,
construction.
In the event several species are to be tested simultaneously, the lest
chamber may be partitioned using dividers made from NITEX® 1, TEFLON®,
stainless steel, or glass. Also small cups made of NITEX®, TEFLONR or
glass may be used to segregate different species within the same test
chamber. Commercially available test chambers may be used if they meet the
above requirements.
The size of the chambers is varied according to the size of the test
organism. All test chambers should be covered to prevent accidental
contamination. The incidence of "floaters" (organisms trapped on the
surface film) in Daphnia test chambers can be reduced by using opaque covers.
Cleaning
New plasticware used for sample collection or organism exposure vessels
does not require rigorous cleaning. It is sufficient to rinse the new
containers once with sample before use. New glassware, however, should be
soaked overnight in acid (see Paragraph 3 below). It is also recommended
that all sample containers, test vessels, and other equipment that has come
in contact with effluent be washed after use in the manner described below
to remove surface contaminants:
1. Soak 15 min, and scrub with detergent in tap water, or clean in an
automatic dishwasher.
2. Rinse twice with tap water.
3. Rinse once with fresh, dilute (20%, V:V) nitric acid or hydrochloric
acid (add 20 mL of concentrated acid to 80 ml of distilled water) to
remove scale, metals, and bases.
4. Rinse twice with tap water.
5. Rinse once with full-strength, acetone to remove organic compounds.
6. Rinse well with tap water.
7. Rinse well with dilution water.
When feasible, the above cleaning procedure must also be used for other
equipment that comes in contact with the effluent, such as the dilutor
system, pumps, tanks, etc. All test chambers and equipment must be
thoroughly rinsed with the dilution water immediately prior to use in each
test.
1NITEX® is available from Tobler, Ernst, and Trabor, 2434 Dempster St.,
Des Plaines, Illinois 60016 (312-299-1051), or Tetco, Inc., 420 Sawmill
River Rd., Elmsford, New York 10523, (914-592-5010).
12
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SECTION 5
TEST ORGANISMS
SPECIES
The species used in characterizing the toxicity of an effluent will
depend on the objectives of the test and the requirements of the regulatory
agency. The organisms used in effluent toxicity tests must be identified
to species. If there is any doubt as to the identity of the test
organisms, they should be sent to a taxonomic expert for examination.
Species that have been widely used in toxicity tests and are acceptable
test organisms are listed in Table 1. For practical reasons, the number of
test species commonly used in effluent toxicity tests has been limited
primarily to those organisms that are easily cultured in the laboratory.
Although it might seem appropriate to use marine organisms in toxicity
tests of effluents discharged to saline waters, effluents generally consist
of altered freshwater, and it would be appropriate to use freshwater
organisms in the tests. The selection of freshwater and/or marine
organisms for these tests may be dictated by site-specific conditions
and/or may be specified in state water quality standards.
SOURCE
Although it might be desirable to use test organisms residing in the
receiving water, it is usually not practical for the following reasons:
(1) sensitive organisms may not be present in the 'receiving water because
of previous exposure to the effluent or other pollutants; (2) it is often
difficult to collect organisms of the desired age and condition from the
receiving water; (3) most states require collection permits, which may be
difficult to obtain. Therefore, it is usually more cost effective to
obtain tests organisms from private, state, and Federal sources. Care
should be taken to ensure that test organisms are free of signs of stress
and disease. Some species of test organisms, such as trout, can be
obtained from "disease-free" stocks.
Some fish, such as the fathead minnow and silverside, and
invertebrates, such as daphnids, midges, and mysid shrimp, are easily
reared in the laboratory. Invertebrates and fish not amenable to
laboratory rearing and not available from commercial or government sources,
may be obtained in some cases by collecting them directly from wild
populations. For methods of collecting fish and invertebrates see Weber
(1973). However, test organisms obtained from the wild must be observed in
13
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TABLE 1. RECOMMENDED SPECIES, TEST TEMPERATURES, AND LIFE STAGES.
Species
Test
Temperature
(°C)a
Life
Stageb
Freshwater
Vertebrates
Cold Water
Brook trout:
Coho salmon:
Rainbow trout:
Warm Water
Salvelinus fontinalis
Oncorhynchus kisutch
Salmo gairdneri
Bluegill: Lepomis macrochirus
Channel catfish: Ictalurus punctatus
Fathead minnow: Pimephales prome1asc
12
12
12
20
20
20
Invertebrates
Cold Water
Stoneflies:
Crayfish:
Mayflies:
Warm Water
Pteronarcys spp.
Pacifastacus jeniusculus
Baetis spp. or Ephemerella spp.
12
12
12
30 - 90 days
30 - 90 "
30 - 90 "
1 - 90 days
Larvae
Juveniles
Nymphs
Amphipods:
Cladocera:
Crayfish:
Mayflies:
Midges:
Hyalella, spp.,
Gammarus lacustris, G. fasciatus,
or G. pseudolimnaeus
Daphnia magna or D. pulex,^
Ceriodaphnia spp.
Orconectes spp., Cambarus spp.,
Procambarus spp.,
Hexagenia limbata or H. bilineata
Chironomus spp.
20
20
20
20
20
20
20
20
20
Juveniles
n
1 - 24 h
1 - 24 h
Juveniles
Nymphs
Larvae
a. To avoid unnecessary logistical problems in trying to maintain
different test temperatures for each test organism, it would be
sufficient to use one temperature (12°C) for cold water organisms and
one temperature (20°C) for warm water organisms.
b. The optimum life stage is not known for all test organisms.
c. Mayes et al., 1983, found no significant difference in the sensitivity
of fish ranging in age from 10 to 100 days, in tests with nine
toxicants.
d. Daphnia pulex is recommended over j). magna because it is more widely
distributed in the United States, test results are less sensitive to
feeding during tests, and it is not as easily trapped on the surface
film.
14
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TABLE 1. (CONTINUED)
Species
Marine and estuarine
Vertebrates
Cold Water
English sole:
Sanddab:
Winter flounder:
Warm Water
Flounder:
Longnose killifi
Mummichog:
Pinfish:
Test
Temperature Life
(°c) Stage
Parophrys vetulus
Citnarichthys stigmaeus
Pseudopleuronectes
americanus
Paralichthys dentatus
P. lethostigma
sh: Fundulus similis
Fundulus heteroclitus
Lagodon rhomboides
Sheepshead minnow: Cyprinodon variegatus
Silverside:
Spot:
Threespine
stickleback:
Invertebrates
Cold Water
Dungeness crab:
Oceanic shrimp:
Green sea urchin
Purple "
Sand dollar:
Warm Water
Blue crab:
Mysid:
Grass shrimp:
Penaid shrimp:
Sand shrimp:
Pacific oyster:
American oyster:
Menidia spp.
Leiostomus xanthurus
Gasterosteus aculeatus
Cancer magjster
Pandalus jordani
: Strongylocentrotus
droebachiensis
: S. purpuratus
Dendraster excentricus
Callinectes sapidus
Mysidopsis spp.
Neomysis spp.
Palaemonetes spp.
Penaeus setiferus
P. duorarum
P. aztecus
Crangon spp.
Crassostrea ^icjas
Crassostrea virginica
12 1-90 days
12
12 Post-metamorphosis
20 1-90 days
20
20
20
20
20
20
20
12 Juvenile
12
12 Gametes/embryo
12
12
20 Juvenile
20 1-5 days
20
20 1-10 days
20 Post larval
20
20
20
20
20 Embryo/larval
15
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the laboratory for one week, prior to use, to assure that they are free of
disease and not suffering from stress due to capture and handling. Fish
captured by electroshocking must not be used in toxicity testing.
LIFE STAGE
Young organisms are often more sensitive to toxicants than are adults.
For this reason, the use of early life stages, such as first instars of
daphnids and mysids, and juvenile fish, is recommended for all tests.
There may be special cases, however, where the limited availability of
organisms will require some deviation from the recommended life stage. In
a given test, all organisms should be approximately the same age and should
be taken from the same source. For the commonly used test organisms, the
maximum difference in age within a batch is as follows: fathead minnow and
silverside, +_ 3 days; daphnids, ±_ 12 h; mysid, +_ 24 h. Since age may
affect the results of the tests, it would enhance the value and
comparability of the data if the same species in the same life stages were
used throughout a monitoring program at a given facility.
LABORATORY CULTURING
Instructions for culturing fathead minnows, silversides, Daphnia, and
mysids, are included in Appendix A.
HOLDING AND HANDLING
Except for new tanks, or where there is no previous history of disease
problem, holding tanks or test chambers are disinfected before use with
0.5% commercial bleach for one hour (5 mL of bleach added to 1 L of
water). Brush thoroughly with the disinfectant and rinse well between
groups of organisms. Other equipment used to handle organisms must be
disinfected with 0.556 commercial bleach. Note: Bleach is highly toxic.
All vessels cleaned with bleach must be thoroughly rinsed with tap water,
thiosulfate solution (50 mg/L), and then with uncontaminated holding water
before the final filling and introduction of the test organisms.
To avoid unnecessary stress after collection, and during transportation
and acclimation, organisms should not be subjected to changes of more than
3°C in water temperature or three parts per thousand (ppt) in salinity in
any 12 h period.
Organisms should be handled as little as possible. When handling is
necessary, it should be done as gently, carefully, and quickly as possible
to minimize stress. Organisms that are dropped or touch dry surfaces or
are injured during handling must be discarded. Dipnets are best for
handling larger organisms. These nets are commercially available or can be
made from small-mesh nylon netting, silk bolting cloth, plankton netting,
or similar material. Nets coated with urethane resin are best for handling
catfish. Wide-bore, smooth glass tubes (4 to 8 mm inside diameter) with
rubber bulbs or pipettors (such as a Propipette^ or an electric pipettor)
should be used for transferring smaller organisms such as daphnids, mysids,
and larval fish.
16
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Holding tanks are supplied with a good quality water (see Section 6)
with a flow-through rate of at least two tank-volumes per day. Otherwise,
use a recirculation system where the water flows through an activated
carbon or undergravel filter to remove dissolved metabolites. Culture
water can also be piped through high intensity ultraviolet light sources
for disinfection, and to photodegrade dissolved organics.
When test organisms are obtained from a source known to have a healthy
stock, a minimum observation period of 48 h is required. If the organisms
are obtained from a source where the health of the stock is unknown, or
obtained from the wild, a minimum observation time of seven days is
required before use. The need for a lengthy observation period for
organisms obtained from the wild may preclude the use of resident species
in on-site toxicity tests.
Crowding should be avoided (see guidelines for loading, Section 8).
The DO must be maintained at a minimum of 40% of saturation for warm water
species and 60% of saturation for cold water species. Aeration is used if
necessary.
Fish should be fed as much as they will eat at least once a day with
live or frozen brine shrimp or dry food (frozen food should be completely
thawed before use). Brine shrimp can be supplemented with commercially
prepared food such as Tetramin^ flakes, BioRil^, or equivalent. Excess
food and fecal material should be removed from the bottom of the tanks at
least twice a week by siphoning. Organisms should be observed carefully
each day for signs of disease, stress, physical damage, and mortality.
Dead and abnormal specimens should be removed as soon as observed. It is
not uncommon to have some fish mortality in a holding tank because of
individuals that refuse to feed on artificial food and, as a result, die of
starvation.
A daily log of feeding, behavioral observations, and mortality should
be maintained.
DISEASE TREATMENT
If fish are obtained from the wild, or from some source that is not
certified as disease-free, it is recommended that prophylactic treatments
be routinely utilized for external bacteria, trematodes, and protozoa. If
fish are from inhouse cultures, or from sources certified as disease-free,
therapeutic treatments should be used only when disease is observed. If
the disease is not serious and responds to treatment, the fish may be used
after a disease-free period of seven days.
Recommended prophylactic and therapeutic treatments for freshwater fish
are listed in Table 2. Some of these chemicals can also be used to treat
saltwater fish. When fish are severely diseased, the entire lot should be
discarded. Invertebrates which become diseased should be discarded. Tanks
which are contaminated with disease-causing microorganisms must be
disinfected with 0.5% commercial bleach and thoroughly rinsed (see p. 16).
17
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TABLE 2. RECOMMENDED PROPHYLACTIC AND THERAPEUTIC TREATMENTS FOR
DISEASE IN FRESHWATER
Disease
Chemical
Concentration0
(mg/L)
Duration
of Treatment
External Terramycin (oxytetracycline
bacteria hydrochloride, water soluble)
Penicillin G Procained
(In dihydrostreptomycine
Sulfate Solution)
Benzalkonium chloride
(Hyamine 1622R or 3500R)
25C
(3mL/100gal)
1-2C
30-60 min
48-72 h
30-60 min
Furanace
0.05 - 0.1
1.0
Indefinite
5-10 min
Monogenetic
trematodes
fungi, and
external
protozoa6
Formalin
Potassium permanganate
Sodium chloride
250
2-6
up to 60 min
30-60 min
15,000-30,000 5-10 min dip
2,000-4,000 24 h minimum
Parasitic
copepods
Trichlorfon
(MasotenR)
0.25C
Continuous"*"
This table indicates the order of preference of treatments that have been
reported to be effective, but their efficacy against disease and toxicity to
fish may be altered by species, temperature or water quality. CAUTION:
TREATMENTS SHOULD BE TESTED ON SMALL LOTS OF FISH BEFORE MAKING LARGE-SCALE
APPLICATIONS. Additional information may be obtained from sources such as
Davis (1953), Hoffman and Meyer (1974), Reichenbach-Klinke and Elkan (1965),
Sniewzko (1970), van Duijn (1973), and Herwig (1979).
18
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TABLE 2. Footnotes (Continued)
b. 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 (1967).
c. Active ingredient.
d. Treatment of fish with Penicillin G Procaine immediately after they are
brought into the lab may be necessary to prevent outbreaks of bacterial
infections caused by stress from handling. Penicillin can be obtained from
veterinarians, and from animal feed and supply stores.
e. Frequently, treatments for trematodes and protozoans need to be continued
for three successive days. Ichthyophthirius ("Ich") must be treated daily
or every other day until no sign of the protozoan remains.
f. Continuous treatment should be employed in static or flow-through systems
until no copepods remain, except that treatment should not be continued for
more than four weeks and should not be used above 27°C.
19
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TRANSPORTATION TO THE TEST SITE AND ACCLIMATION TO DILUTION WATER
When organisms are to be transported from the base laboratory to a
remote test site, it is convenient to move them in plastic bags placed in
styrofoam coolers. Adequate DO is maintained by replacing the air above
the water in the bags with oxygen from a compressed gas cylinder, and
sealing the bags. Another method commonly used to maintain sufficient DO
during shipment is to aerate with an airstone which is supplied from a
portable pump. During transport and acclimation, the organisms should not
be subjected to a change of more than 3°C in water temperature or 3 ppt
salinity in 12 h. The DO concentration must not fall below 40% of
saturation for warm water species and 60% of saturation for cold water
species.
Upon arriving at the test site, the organisms are acclimated to the
test dilution water and temperature by changing from holding water to
dilution water. All but a small volume (approximately 5%) of the holding
water is removed by siphoning, and replaced slowly over a 10 to 15 min.
period with dilution water. If receiving water is used as dilution water,
caution must be exercised in exposing the test organisms to it, because of
the possibility that it might be toxic. For this reason, it is
recommended that only 50% of the test organisms be exposed initially to the
dilution water. If the mortality does not exceed 5% in 24 h, the remainder
of the test organisms are acclimated to the dilution water. All organisms
must be exposed to 100% dilution water, within the temperature range of the
test, for a minimum of 24 h before they are used in the effluent toxicity
tests.
A group of organisms must not be used for a test if they appear to be
diseased, discolored, or otherwise stressed (Sprague, 1969), or if more
than 5% die during the 24- to 48-h period immediately preceding the test.
If the organisms fail to meet these criteria, the entire group must be
discarded and a new group obtained. As stated above, the mortality may be
due to the presence of toxicity in the dilution (receiving) water, rather
than a diseased condition of the test organisms. If the acclimation period
is repeated with a new group of test organisms and mortality exceeds 10%,
it is recommended that an alternative source of dilution water be used.
The acclimation of marine organisms for effluent toxicity tests poses
special problems because most effluents discharged into the marine
environment consist of altered freshwater. Therefore, when the effluent is
diluted with the receiving water (salt water), the higher percent effluent
volumes will have a low salinity (the salinity will be inversely
proportional to the percent volume of effluent). If the effluent is
essentially freshwater, it is obvious that 100% effluent cannot be used in
a flow-through system with marine test organisms. In this case, the test
is conducted without the inclusion of 100% effluent as one of the
treatments. However, depending upon the objectives of the test and the
policy of the regulatory agency, the problem of salinity control may be
avoided altogether by using a freshwater test species and freshwater as the
diluent.
20
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In static tests, marine organisms can be used at all concentrations of
effluent by adjusting the salinity of the effluent to a standard salinity
(such as 20 ppt) or to the salinity of the receiving water, by adding
sufficient dry ocean salts, such as Instant Ocean (or equivalent)^ . Saline
dilution water can also be prepared by adding dry salts to the receiving
water or to distilled water or a freshwater such as well water or a suitable
surface water. Care must be taken to ensure that the added salts are
completely dissolved before the test organisms are placed in the solutions
(see discussion in Preparation of Test Solutions, Section 8). For tests in
which dry ocean salts are used to obtain the desired salinity, the test
organisms should be acclimated in synthetic saline water prepared with the
dry salts. However, addition of dry ocean salts to dilution water may result
in an increase in pH. It may be desirable to lower the pH of the
salinity-adjusted effluent, before using it in the test, by the addition of
(IN) hydrochloric acid.
If it is not possible to maintain the same salinity in all effluent
concentrations because of the large volumes of water required, such as in
flow-through tests, the highest effluent concentration (lowest salinity) that
could be tested would depend upon the salinity of the receiving water and the
tolerance of the test organisms. The tidewater silverside and mysid, for
example, are known to tolerate a salinity range of 5 to 35 ppt, and the
inland silverside has a salinity tolerance range of 0 to 30 ppt. However,
the tolerances of other marine species would have to be determined by the
investigator in advance of the test.
Because of the circumstances described above, when performing
flow-through tests of effluents discharged to saline waters, it is advisable
to acclimate groups of test organisms to each of three different salinities,
such as 10, 20, and 30 ppt, prior to transporting them to the test site (see
p. 20). It would also be advisable to maintain cultures of these test
organisms at a series of salinity levels, including at least 10, 20, and 30
ppt, so that the change in salinity upon acclimation at the desired test
dilutions does not exceed 6 ppt.
It may be desireable to conduct static tests at several salinities as
described above.
i Table dry ocean salts, and sources of supply include:
(a) Instant Ocean^. Aquarium Systems, 8141 Tyler Blvd., Mentor, Ohio
44060 (216-255- 1 997 ) .
(b) Marine Environment^. Import Associates, Inc., P.O. Box 16350, San
Francisco, California, 94116 (415-591-2200).
(c) h.w. Marine Mix^. Hawaiian Marine Import, Inc., P.O. Box 218687,
Houston, Texas 77218 (713-492-7864).
(d) Forty Fathoms^. Marine Enterprises, Inc., 8755 Mylander Lane,
Towson, Maryland, 21204 (301-321-1189).
(e) Jungle Ocean 50^. Jungle Laboratories Corp., Box 630, Cibolo,
Texas, 78108 (800-327-2200, or 512-658-3503).
(f) Rila Marine MixR. Rila Products, P.O. Box 114, Teaneck, New Jersey,
07666 (201-836-0855 ) .
(g) Kahl Sea Salt^. Kahl Scientific Instrument Corporation, P.O. Box
1166, El Cajon, California, 92202-1166 (619-444-2158 or 5944).
21
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SECTION 6
DILUTION WATER
Dilution water used in the tests will depend on the objectives of the
study and other factors. Receiving water collected upstream from the
discharge point should be used as dilution water wherever possible.
Depending upon the objectives, it may be desirable to use receiving water
as the dilution water even when it is contaminated by "upstream" sources;
e.g. if the objective of the test is to determine the effect of multiple
point sources. However, if because of the study objectives the presence of
contaminants in the receiving water makes it unsuitable as dilution water,
or it is not economically feasible to collect and ship receiving water to a
remote laboratory for off-site testing, other surface waters such as nearby
tributaries, or ground water, or synthetic water may be employed.
When the dilution water is taken from the receiving water, it should be
collected at a point as close as possible to the outfall, but upstream from
or outside of the zone influenced by the effluent. The water used to make
up the effluent dilutions should be collected immediately prior to the
test, but never more than 96 h before the test begins.
If contaminant-free water is required in the test, dilution water is
acceptable if healthy test organisms survive in it without signs of stress
and mortality does not exceed 5% during the acclimation period.
Recommended procedures for the preparation of synthetic freshwater and
sea water are given in Tables 3 and 4, respectively. A more convenient
method of preparing synthetic salt water is to add commercially available
dried ocean salts (see Section 5) to distilled or de-ionized water.
However, addition of dry ocean salts to dilution water may result in an
increase in pH. If this occurs, the pH of the salinity-adjusted water
should be lowered to the desired level for the test by the addition of (IN)
hydrochloric acid.
In an estuarine environment, the investigator should collect uncon-
taminated water having a salinity as near as possible to the salinity of
the water at the receiving site. Water should be collected at slack high
tide, or within one hour after high tide. If there is reason to suspect
contamination of the water in the estuary, it is advisable to collect
uncontaminated water from an adjacent estuary. At times it may be
necessary to collect water at a location closer to the open sea, where the
salinity is relatively high. In such cases, uncontaminated freshwater is
added to the saline water to dilute it to the required test salinity.
22
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When necessary, the dilution water should be pretreated by filtering through
a nylon sieve having 2- to 4-mm holes to remove debris and/or break up large
floating or suspended solids. If a mini-dilutor employing capillary delivery
tubes is used, it may be necessary to filter the effluent through glass wool
prior to entering the dilutor system.
It is preferable to pump the dilution water continuously to the acclimation
tank and dilutor. However, it may be more practical to transport batches of water
to the testing site in tanks, and continuously pump water from the tanks to the
acclimation chamber and dilutor.
TABLE 3. PREPARATION OF SYNTHETIC FRESH WATER*.
Reagent Added (mg/L)b
Final Water Quality
Water
Type NaHC03 CaS04'2H20 MgS04
Very soft
Soft
Moderately Hard
Hard
Very hard
12.0
48.0
96.0
192.0
384.0
7.5
30.0
60.0
120.0
240.0
7.5
30.0
60.0
120.0
240.0
KC1
0.5
2.0
4.0
8.0
16.0
pHc
6.4-6.8
7.2-7.6
7.4-7.8
7.6-8.0
8.0-8.4
Alka-
Hardness^ linity
10-13
40-48
80-100
160-180
280-320
10-13
30-35
60-70
110-120
225-245
a. Taken in part from Marking and Dawson (1973).
b. Add reagent grade chemicals to distilled or deionized water
c. Approximate equilibrium pH after aerating 24 h.
d. Expressed in mg/L as CaC03.
23
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TABLE 4. PREPARATION OF SYNTHETIC SEA WATER.
Chemical Amount
NaF 3 mg
SrCl2-6H20 20 mg
HsBOs 30 mg
KBr 100 mg
KC1 700 mg
CaCl2-2H20 1.47 g
Na2S04 4.00 g
MgCl2-6H20 10.78 g
NaCl 23.50 g
Na2Si03-9H20 20 mg
Na4EDTAd 1 mg
NaHC03 200 mg
a. Add reagent-grade chemicals in the order listed, to 890 mL distilled
or deionized water. Each chemical must be dissolved before another
is added.
b. The final desired test salinity is attained by dilution at the time
of use. If the above solution is diluted to one liter, the salinity
should be 34 + 0.5 ppt and the pH 8.0 + 0.2.
c. From Kester ejt a±. (1967), Zaroogian et_ jil_. (1973), and Zillioux et
al- (1973).
d. Tetrasodium ethylenediaminetetraacetate. This compound should be
omitted when toxicity tests are conducted with metals. When tests
are conducted with plankton or larvae, the EDTA should be omitted and
the medium should be stripped of trace metals (Davey et^ jTL, 1970).
24
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SECTION 7
EFFLUENT SAMPLING AND HANDLING
EFFLUENT SAMPLING
Ordinarily, the effluent sampling point is the same as that specified
in the NPDES discharge permit (USEPA, 1979c), which is usually the point of
discharge (outfall). Conditions for exception would be: (1) if there is
better access to a sampling point between the final treatment and the
discharge point; (2) if the processed waste is chlorinated prior to
discharge to the receiving waters, the sampling point may be located prior
to contact with chlorine if the purpose of the test is to determine
toxicity levels of the unchlorinated effluent; or.(3) if there is a desire
to evaluate separate wastewater streams prior to their being combined with
other wastewater streams, or noncontact cooling water. Some regulatory
authories measure the toxicity of the effluent prior to chlorination, after
chlorination, and after dechlorination with sodium thiosulfate.
At times it may be necessary to filter the effluent through glass wool
or similar material to remove solids which would clog the orifices of
solenoid valves and capillary tubes used in flow-through systems. Aeration
during collection and transfer of effluents should be minimized to reduce
the loss of volatile chemicals.
The decision on whether to collect grab or composite samples is based
on the objectives of the test and an understanding of the short and
long-term operations and schedules of the discharger. If the toxicity of
the effluent is variable, grab samples collected during peaks of effluent
toxicity provide a measure of maximum effect. The compositing process has
an averaging effect which tends to dilute the toxicity peaks, and may
provide misleading results when testing for acute toxicity. Composited
samples, therefore, are more appropriate for chronic toxicity tests where
peak toxicity of short duration is of less concern (USEPA, 1984b).
SAMPLE TYPE
The advantages and disadvantages of grab and composite samples are
listed below:
1. Grab Samples
a. Advantages:
(1) Easy to collect; require a minimum of equipment and on-site time.
(2) Provide a measure of instantaneous toxicity. Toxicity spikes are
not masked by dilution.
25
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b. Disadvantages:
(1) Samples are collected over a very short period of time and on a
relatively infrequent basis. The chances of detecting a spike
in toxicity would depend on the frequency of sampling.
(2) A separate test must be conducted with each grab sample, if they
are to be useful.
2. Composite Samples:
a. Advantages:
(1) A single effluent sample is collected over a 24-h period, and
only one toxicity test is conducted.
(2) The sample is collected over a much longer period of time and
contains all toxicity spikes.
b. Disadvantages:
(1) Sampling equipment is more sophisticated and expensive, and must
be placed on-site for at least 24 h.
(2) Toxicity spikes may not be detected because they are masked by
dilution with less toxic wastes.
TEST TYPE
1. Static Tests
Sampling recommendations are discussed for two types of test --
nonrenewal and renewal. For nonrenewal tests, effluent samples are
collected only at the beginning of the test. For renewal tests, samples
are generally collected to renew the test solutions daily throughout the
test period.
The following effluent sampling methods are recommended for static tests:
a. Continuous Discharges
(1) If the facility discharge is continuous, but the calculated
retention time of a continuously discharged effluent is less than
14 days and the variability of the waste is unknown, one of the
following approaches is used:
(a) Nonrenewal tests: a minimum of four separate grab samples
are collected at evenly-spaced (6-h) intervals over the first
24-h period and used in four separate tests to determine the
variability in toxicity.
(b) Renewal tests: a minimum of four separate grab samples are
collected at evenly-spaced (6-h) intervals over the first
24-h period and used in four separate tests begun on the
first day and renewed daily with samples collected at the
appropriate time.
26
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(2) If the calculated retention time of a continuously discharged
effluent is greater than 14 days, or if it can be demonstrated
that the wastewater does not vary in chemical composition or
concentration regardless of holding time, one of the following
approaches is used:
(a) Nonrenewal tests: a minimum of one grab sample is collected
and used in a single test.
(b) Renewal tests: a minimum of one grab sample is collected each
day and used to renew the test solutions.
The retention time of the effluent in the wastewater treatment facility
may be estimated from calculations based on the volume of the retention
basin and rate of wastewater inflow. However, the calculated retention time
may be much greater than the actual time because of short-circuiting in the
holding basin. Where short circuiting is suspected, or sedimentation may
have reduced holding basin capacity, a more accurate estimate of the
retention time can be obtained by carrying out a dye study.
b. Intermittent Discharges
If the facility discharge is intermittent, one of the following
approaches is used:
(1) Where the effluent is continuously discharged during a single
eight-hour work shift or two successive eight-hour work shifts, a
minimum of one grab sample is collected midway during the
discharge period and used in a single nonrenewal test, or a grab
sample may be collected daily for a renewal test.
(2) Where the facility retains the wastewater during an eight-hour
work shift, then treats and releases the wastewater as a batch
discharge, a grab sample is collected for a single nonrenewal
test, or a grab sample is collected daily for a renewal test.
(3) Where the facility discharges wastewater to an estuary only
during an outgoing tide (usually during the four hours following
slack high tide), a grab sample is collected during a discharge
period for use in a single nonrenewal test, or a grab sample is
collected daily for a renewal test.
(4) At the end of the shift, clean up activities may result in the
discharge of a slug of toxic waste. It would be advisable,
therefore, to consider collecting a sample at that time and
conducting a separate toxicity test.
27
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2. Flow-through Tests
The following effluent sampling methods are recommended for flow-
through tests:
a. Continuous Discharges
If the facility discharges continuously, the effluent should be
pumped directly and continuously from the discharge line to the
diTutor system for the duration of the test. However, if the
effluent cannot be pumped directly and continuously to the diTutor
system, the following alternative methods are employed for
collection of the effluent:
(1) Where the calculated retention time of the effluent is less than
14 days, two grab samples are collected daily (e.g., 8:00 AM and
4:00 PM). The freshly collected effluent should not be combined
with the effluent remaining from the previously collected
sample. The remaining part of the previously collected sample
is discarded and the container is refilled with the fresh
effluent.
(2) Where the calculated retention time of the effluent is 14 days
or greater, a single grab sample of sufficient volume to supply
the dilutor for 24 h is collected daily. Here again, the volume
of sample remaining from the previous day is discarded and
replaced by the fresh sample.
b. Intermittent Discharges
If the facility discharge is intermittent, one of the following
procedures is used:
(1) Where a continuous discharge occurs during a single eight-hour
work shift or two successive eight-hour work shifts, at least
one grab sample of sufficient volume to supply the dilutor for
24 h is collected daily, midway during the discharge period.
(2) Where the facility retains the wastewater during an eight-hour
work shift, and then treats and releases it in a batch
discharge, a single grab sample of sufficient volume to last
24 h is collected daily during the test period.
(3) Where the facility discharges wastewater to an estuary only
during an outgoing tide (usually during the four hours following
slack high tide), a single grab sample of sufficient volume to
last 24 h is collected during one discharge period every 24 h
for the duration of the test. An alternate sampling method
would be to place the effluent sampling pump in the final waste
lagoon adjacent to the discharge pipe so that a continuous
source of effluent would be available for the test.
28
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EFFLUENT HANDLING
1. Samples Used in On-site Tests
a. Flow-through tests
Effluent grab samples used in on-site, flow-through toxicity tests
should be stored in covered, unsealed containers. Stainless steel
or fiberglass tanks are preferred because they can be easily
decontaminated and reused with different effluents. Because of the
large volume of sample normally required for flow-through tests with
fish -- approximately 400 L (100 gal)/day -- the samples are usually
held outside the laboratory at ambient air temperatures. It is not
feasible to cool the sample prior to its transfer from the sample
holding tank through the temperature control equipment and to the
diTutor system. However, in cold weather, it may be necessary to
use heater tape wound around the incoming line, or agitation with a
submersible pump, to prevent freezing.
b. Static renewal and nonrenewal tests
Samples that are collected for on-site tests should be used within
24 h. Samples that are not used immediately should be iced or
refrigerated.
2. Samples Shipped to Off-Site Test Facility
Samples collected for off-site toxicity testing are to be immediately
placed on ice, and are shipped iced to the central laboratory, where they
are transferred to a refrigerater (4°C) until used. Every effort must
be made to initiate the test with an effluent sample on the day of arrival
in the laboratory. The maximum lapsed time from collection of a grab or
composite sample and the initiation of the test should not exceed 72 h.
However, if the persistence of the toxicity of the sample is unknown, the
maximum holding time should not exceed 36 h.
Nonrenewal static tests generally require only 4 to 20 L (one to five
gallons) of effluent, which can be shipped in 4-L (1-gal) glass jugs,
CUBITAINERSR, or plastic "milk" jugs. Glass jugs can be cleaned and
reused. CUBITAINERSR and plastic jugs are punctured and discarded after
use.
29
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SECTION 8
TOXICITY TEST PROCEDURES
RANGE-FINDING TEST
EPA Regional and state personnel generally have observed that it is not
necessary to conduct a range-finding test prior to initiating a static,
acute, definitive toxicity test. However, when flow-through tests are
used, it is advisable to conduct a preliminary range-finding test because
of the lack of flexibility in changing the concentration after the test is
underway. The range-finding test most often used is an abbreviated static
test in which groups of five organisms are exposed to three to five widely
spaced effluent dilutions, such as 1%, 10%, 50%, and a control, for eight
to 24 h (for information on other test conditions, see the Definitive Test
Section below).
It should be noted that the toxicity (LC50) observed in range-finding
tests may be significantly different from the toxicity observed in the
definitive test because: (1) the definitive test is longer; and (2) the
test is performed with an effluent sample collected at a different time,
and the characteristics of the effluent and the receiving water may vary
significantly within short periods of time.
SCREENING TEST
Some Regional and State effluent biomonitoring programs stipulate the
use of abbreviated tests with a single (100%) effluent concentration for
acute toxicity screening purposes (Weber and Peltier, 1981). The duration
of the screening test is usually 24 h. However, where effluents are
acutely toxic, mortality often occurs within the first few hours of
exposure. In such cases, the test can be terminated within six to eight
hours. If lethality is observed in the screening test, a definitive test
may be required.
Acute toxicity screening tests are carried out as follows:
1. Measure the pH and DO of the (100%) effluent and control water. If the
pH falls outside the range of 6.0 - 9.0, set up two parallel tests, using
pH-adjusted (pH = 7.0) and unadjusted 100% effluent. The effluent sample
is adjusted to pH 7 by adding 1.27 ml IN NaOH and 2.0 ml 1M KH2P04 per
litre (Marking and Dawson, 1973). If the DO is less than 40% of saturation
when using warm water species, or less than 60% when using cold water
species, the test solutions should be aerated before use.
2. For each test, a total of 20 organisms are distributed among one to four
test vessels containing 100% effluent.
30
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3. A total of 20 organisms are also immediately distributed among one to
four vessels containing control medium consisting of reconstituted water.
4. The test vessels are checked frequently for mortality during the first
few hours, and the following action is taken:
a. If the mortalilty of the organisms exposed to 100% effluent exceeds
10% at any time during the first few hours, and the mortality in the
control has not exceeded 10%, the effluent is considered to exhibit
acute toxicity. The screening test is immediately terminated, and a
definitive test is initiated.
b. If the mortalilty of the organisms in the control beakers exceeds 10%
at any time during the first few hours or at any time later in the
test, the preliminary screening test is immediately terminated and
repeated.
c. If the mortality in the effluent and control vessels is less than 10%
after the first few hours, the test is continued until 10% mortality
is exceeded in either the effluent or controls, or until the test
period reaches 24 h, whichever occurs first.
5. If the test runs for the full 24-h period, the results are interpreted
as follows:
a. If the mortality in the effluent sample was less than 10%, the
effluent sample is not considered to be acutely toxic, and no further
acute tests are conducted on that sample unless the mortality in the
controls exceeded 10% or the reference toxicant data were not
acceptable.
b. If the mortality exceeded 10% in the effluent sample, but was less
than 10% in the controls, the effluent sample is considered to
exhibit acute toxicity and a definitive test is conducted (see
below).
c. If the mortality exceeded 10% in the controls, or if the reference
toxicant data were not acceptable, the screening test must be
repeated.
DEFINITIVE TEST
"Definitive" tests are distinguished from "screening" tests primarily
by: (1) the greater length of the test period; and (2) the use of multiple
concentrations of effluent to obtain a point estimate of toxicity in terms
of a LC50. Flow-through tests have the advantage of detecting temporal
changes in effluent toxicity, and the longer exposure period of the
definitive test increases the probability that the test period will include
toxicity spikes, if they occur.
31
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1. Test Duration
See the Summary of Test Conditions for information on test duration. In
static tests, most of the mortality generally occurs during the first
24 to 48 h. Therefore, there may be little advantage to extending these
tests to 96 h. However, state water quality standards may require a 96-h
test period.
2. Preparation of Test Solutions
The definitive toxicity test includes a control and a series of effluent
concentrations. The effluent concentrations are commonly selected to
approximate a geometric series, such as 100%, 50%, 25%, 12.5%, and 6.25%.
a. Static Tests
An excess volume (e.g., 8 L) of each solution is prepared to provide
sufficient material for toxicity testing and chemical analysis. The
solutions are well mixed with a glass rod, TEFLON^ stir bar or other
means. The DO is checked, and the solutions are aerated if necessary
to raise the DO concentration to the required level. Aliquots of each
concentration are delivered to the test chambers, and the chambers are
arranged in random order. The chambers are placed in a water bath or
otherwise brought to the required temperature, and the test organisms
are added as described in Paragraph 4. The remaining volumes of each
effluent concentration can be used for the chemical analyses.
Saline dilution water can be prepared by adding dry salts to the
receiving water, or to distilled or de-ionized water, or a freshwater
such as well water or a suitable surface water to obtain the same
salinity as the effluent (see p. 21). If receiving water (saline
water) is used as the diluent, a salinity control must be prepared
using distilled or deionized water and dried sea salts to determine if
the addition of sea salts alone has an adverse effect on the test
organisms. As noted above, it may be desirable to conduct static
toxicity tests at several salinities.
If the effluent is freshwater and the test is to be conducted with a
salt water organism, depending on the objectives of the test and the
policy of the regulatory agency, the test solutions may be prepared by
adding dried ocean salts (see Section 6) to a sufficient quantity of
100% effluent to raise the salinity to the desired level (usually
20 ppt). After the addition of the dried salts, stir gently for 30 to
60 min, preferably with a magnetic stirrer, to ensure that the salts
are in solution. It is important to check the final salinity with a
refractometer or salinometer.
Addition of dry ocean salts to effluents and dilution water may change
the pH and affect the toxicity of the waste. The pH of the
salinity-adjusted solutions can be brought to the desired level by
addition of acid or base (IN HC1 or NaOH). However, it is recommended
32
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that a concurrent test be conducted with salinity-adjusted effluent in
which the pH has not been altered after adding the salt.
The volume of the effluent used must be sufficient to prepare all
percent concentrations of the effluent needed for the toxicity test
and for chemical analysis. For example, to conduct tests with Menidia,
the use of 500 ml of test solution in each of duplicate exposure
vessels and five concentrations of effluent (10 exposure vessels),
would require a total of 2 L of 100% effluent. However, to provide
sufficient volumes of test solutions for chemical analysis and for
toxicity testing with invertebrates, additional effluent would be
required.
Note: If after one hour, 100% mortality has occurred in the higher
effluent concentrations (100% and 50%), additional concentrations of
effluents, such as 3.1%, 1.5%, and 0.75%, are added to the test at the
lower end of the concentration series.
b. Flow-through Tests
Flow-through tests are usually performed with the same effluent
concentrations that are used for the static tests, except that where
the receiving water is saline and the effluent is not, 100% effluent
can not be tested with a marine organism.
The dilutor system should be operated 24 h prior to adding the test
organisms. During this period, the dilutor is calibrated and the
necessary adjustments can be made in the temperature, flow rate
through the test chambers, and aeration. The flow rate through the
proportional dilutor must provide for a minimum of five 90%
replacements of water volume in each test chamber every 24 h (see
Fig. 2). This replacement rate should provide sufficient flow to
maintain an adequate concentration of dissolved oxygen. The dilutor
should also be capable of maintaining the test concentration at each
dilution within 5% of the starting concentration for the duration of
the test. The calibration of the dilutor should be checked carefully
before the test begins. This check should determine the volume of
effluent and dilution water used in each portion of the effluent
delivery system and the flow rate through each test chamber. The
general operation of the dilutor should be checked at least at the
beginning and end of each day during the test.
3. Control
The control consists of the same dilution water, test conditions,
procedures, and organisms used in testing the effluent. In the event a test
is to be conducted with salt water organisms, where each effluent dilution
has a different salinity, a static control is prepared for the lowest (or
highest, in the case of high salinity, e.g. brine wastes) salinity level
used in the flow-through test to determine if salinity alone has any adverse
effects on the test organisms. For a valid test, control mortality must not
exceed 10%.
33
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100
a.
o
0.4 06
4 10
Volume of Water in Tank
Flow of Water Per Hour
40 60 100
Figure 2. Approximate times required to replace water in test chambers in
flow-through tests. For example: for a chamber containing 4 L,
with a flow of 2 L/h, the above graph indicates that 90% of the
water would be replaced every 4.8 h. The same time period
(such as hours) must be used on both axes, and the same unit of
volume (such as litres) must be used for both volume and flow.
(From: Sprague, 1969).
4. Number of Test Organisms
At least 20 organisms of a given species are exposed to each effluent
concentration (Jensen, 1972). More than one species may be used in the same
test chamber in a given test, if segregated. Small fish and invertebrates
are captured with 4- to 8-mm inside diameter pipettes. Organisms larger
than 5 mm are captured by dip net. In a typical toxicity test involving
five effluent concentrations and a control (six concentrations X 20
organisms/concentration), fish and other large test organisms are captured
from a common pool and distributed sequentially to the test chambers until
the required number of organisms are placed in each. Some small organisms,
such as daphnids, mysids, and larval fish, are first distributed to small
intermediary holding vessels, such as weighing boats, petri dishes, or small
beakers. The water in the intermediary holding vessels is then drawn down
to a small volume and the entire lot is transferred to a test chamber.
34
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5. Replicate Test Chambers
One or more test vessels are provided for each effluent concentration and
the control. In the past, it has been common practice in toxicity tests to
require duplicate test vessels for each effluent concentration and control.
However, these duplicates were usually not prepared independently, and thus
were not true replicates. Also, the data from duplicate chambers are
usually combined to determine the LC50 and confidence interval. Since it is
not essential, statistically, to maintain two or more replicate test
chambers at each effluent concentration to determine the LC50 and its
confidence interval, the practice of dividing the test population for each
effluent concentration between two or more replicate chambers must be
considered optional. The practice has several advantages, however, and is
encouraged because it: (1) permits easier viewing and counting of test
organisms; (2) more easily avoids possible violation of loading limits,
which might occur if all (20) of the test organisms are placed in a single
test vessel; and (3) ensures against the invalidation of the test which
might result from accidental loss of a test vessel, where all of the test
organisms for a given treatment are in a single chamber.
6. Loading of Test Organisms
A limit is placed on the loading (weight) of organisms per liter of test
solution to minimize the depletion of dissolved oxygen, the metabolic
conversion of significant amounts of effluent constituents, the accumulation
of injurious concentrations of metabolic waste products, and/or stress
induced by crowding, any of which could significantly affect the test
results.
For flow-through tests, the live weight of test organisms in the test
chambers must not exceed 5 g/L of test solution at temperatures of 20°C or
less and minimum exchange rate (see above), or 2.5 g/L of test solution at
temperatures above 20°C.
For both renewal and non-renewal static tests, loading in the test
chambers must not exceed 0.8 g/L of test solution at temperatures of 20°C
or less and 0.4 g/L of test solution at temperatures above 20°C.
7. Test Temperature
The temperature of the test solutions must be maintained within _+ 2.0°C
of the recommended temperature for the test organism (Table 1). This can be
accomplished for flow-through tests by passing the effluent and/or dilution
water through separate coils immersed in a heating or cooling water bath
prior to entering the dilutor system. Coils should be made from materials
recommended in Section 4. For static tests, the temperature can be
controlled by use of a water bath, room air conditioner, or environmental
chamber.
8. Stress
Minimize stress on test organisms by avoiding unnecessary disturbances.
35
-------
Correction Factors for Oxygen
Saturation at Various Altitude's
Alt
Fctt
0
330
ess
9»0
1310
1640
1970
2300
2630
2950
3219
3(10
3940
4270
4600
4930
5250
5580
5910
6240
6560
(900
7220
7550
7890
3200
tude
Metres
0
100
200
300
400
500
600
700
600
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2500
Pressure
760
750
741
732
723
714
70S
696
687
679
671
663
655
647
639
631
623
615
608
601
594
567
580
573
566
560
Factor
100
1 01
103
104
105
1.06
101
109
1 II
1 12
1.13
1 IS
I 16
1.17
1 19
1.20
1.22
124
125
1.26
1.28
1 30
1 31
133
134
136
0 5 10 15 20 25 30
Water temperatures °C
2 3 4 5 6 78 9 10 11 12 13 14 15 16 17
5678
Oxygen cc per liter
10 11
Figure 3. Rawson's nomogram for obtaining oxygen saturation values at
different temperatures at sea level. When a straightedge is used
to connect the water temperature on the upper scale and the
concentration on the lower scale, the percent saturation can be
read from the point of intersection on the diagonal scale. To
determine the percent saturation at locations above sea level,
factors are provided to convert oxygen concentrations measured at
various altitudes to sea level values in the table at the upper
left. For example, an oxygen concentration of 6.4 mg/L measured
in a body of water at an altitute of 1000 m at a temperature of
15°C would be equivalent to a concentration of 6.4 X 1.13, or
7.2 mg/L at sea level. To determine the percent saturation, a
straight edge is used to connect the point at 15°C on the
temperature scale with the point, 7.2 mg/L on the concentration
scale, and the percent saturation is read at the point of
intersection (68%) on the diagonal scale. (From Welch, 1948).
36
-------
9. Dissolved Oxygen Concentration
Aeration may alter the results of toxicity tests and should be used
only as a last resort to maintain the required DO. Aeration can reduce the
apparent toxicity of an effluent by stripping it of highly volatile toxic
substances, or increase its toxicity by altering the pH. However, the DO
in the test solution must not be permitted to fall below 40% of saturation
for warm water species and 60% of saturation for cold water species
(Fig. 3).
In most flow-through tests, DO depletion is not a problem in the test
chambers because aeration occurs as the liquids pass through the dilutor
system. If the DO decreases to a level that would be a source of
additional stress, the turnover rate of the solutions in the test chambers
must be increased sufficiently to maintain acceptable DO levels. If the
increased turnover rate does not maintain adequate DO levels, aerate the
dilution water prior to the addition of the effluent, and aerate all test
solutions. To reduce the potential for driving off volatile compounds in
the wastewater, aeration may be accomplished by bubbling air through a 1-mL
pipet at a rate of no more than 100 bubbles/min, using an air valve to
control the flow. The turbulence caused by aeration should not result in a
physical stress to the test organisms.
In static tests, low DOs commonly occur in the higher concentrations of
wastewater. Aeration is accomplished by bubbling air through a pipet as
described above. It is advisable to monitor the DO closely during the
first few hours of the test. Samples with a potential DO problem generally
show a downward trend in DO within 4 to 8 h after the test is started.
Unless aeration is initiated during the first 8 h of the test, the DO may
be exhausted during an unattended period, thereby invalidating the test.
Caution must be exercised to avoid excessive aeration. When aeration
is used, the methodology must be detailed in the report. For safety
reasons, pure oxygen should not be used to aerate test solutions.
10. Illumination
Light of the duration, quality, and intensity normally obtained in the
laboratory during working hours is adequate (see Summary of Test
Conditions, pp. 39-42).
11. Feeding
a. Static Tests
Problems caused by feeding, such as the possible alteration of the
toxicant concentration, the build-up of food and metabolic wastes and
resulting oxygen demand, are common in static test systems. Where
feeding is necessary, however, excess food should be removed daily by
aspirating with a pipette.
37
-------
b. Flow-through Tests
Feeding does not cause the above problems in flow-through systems.
However, it is advisable to remove excess food, fecal material, and
any solids that may have settled from the effluent, from the bottom
of the test vessels daily by aspirating with a pipette.
PERSISTENCE OF EFFLUENT TOXICITY
The persistence of the toxicity of an effluent, expressed as its
half-life, may be of interest in assessing the potential effects of the
effluent on the receiving water (p. 122, USEPA, 1973). A rough
approximation of the persistence of the toxicity can be obtained by
measuring the 24-h LC50 of a portion of the sample immediately (within
36 h) following collection, and repeating the test on the remaining portion
of the sample 96 h after the first test. The portion of the effluent set
aside for the second test should be held in an uncovered glass container at
room temperature, in direct sunlight if possible, and must be aerated.
If the results of the second test indicate that the toxicity of the
effluent has decreased to 50% or less of its previous value during the
storage period (i.e., if the LC50 increased from 10% to 20% or more), the
toxicity can be considered to be nonpersistent (having a half-life of less
than 96 h). If the toxicity has not decreased to 50% of its previous
value, the toxicity of the effluent can be considered to be persistent.
The method is limited to use with effluents having an initial LC50 of less
than 50%.
SUMMARY OF TEST CONDITIONS FOR COMMONLY USED TEST ORGANISMS
Summaries of the test conditions for the daphnids, Daphnia pulex and j).
magna, the mysid, Mysidopsis bahia, fathead minnows, Pimephales promelas,
and the silverside, MenidTaT are provided in Tables 5-8.
38
-------
TABLE 5. RECOMMENDED TEST CONDITIONS FOR DAPHNIDS
(DAPHNIA PULEXa AND D. MAGNA)
1. Temperature (°C):
2. Light quality:
3. Light intensity:
4. Photoperiod:
5. Size of test vessel:
6. Volume of test solution:
7. Age of test animals:
8. No. animals
per test vessel:
9. No. of replicate
test vessels per
concentration:
10. Total no. organisms
per concentration:
11. Feeding regime:
12. Aeration:
13. Dilution water:
14. Test duration:
15. Effect measured:
20 + 2°C
Ambient laboratory illumination
50-100 footcandles (ft c)b(ambient
laboratory levels)
8-16 h light/24 h
100 mL beaker
50 mL
1-24 h (neonates)
10
20
Feeding not required during first 48 h.
For longer tests, feed every other day
beginning on the third day (Appendix A).
None, unless DO concentration falls below
40% of saturation, at which time start
gentle, single-bubble, aeration.
Receiving water or other surface water,
ground water, or synthetic water: hard
water for Daphnia magna; moderately hard
or soft water for D_- pulex
Screening test - 24 h (Static Tests)
Definitive test - 48 h (Static Tests)
Mortality - no movement of body or
appendages on gentle prodding (LC50)
a Use of D_. pulex is preferred.
b ft c = foot candles.
39
-------
TABLE 6. RECOMMENDED TEST CONDITIONS FOR MYSIDS
(MYSIDOPSIS BAHIA)
1. Temperature (°C):
2. Light quality:
3. Light intensity:
4. Photoperiod:
5. Size of test vessel:
6. Volume of test solution:
7. Age of test animals:
8. No. animals per
test vessel (200 ml):
9. No. of replicate test
vessels per
concentration:
10. Total no. organisms
per concentration:
11. Feeding regime:
12. Aeration:
13. Dilution water:
14. Test duration:
15. Effect measured:
20 + 2°C
Ambient laboratory illumination
50-100 ft c (ambient laboratory levels)
8-16 h light/24 h
250 mL
200 mL
1-5 days
10
20
Two drops of concentrated brine shrimp
nauplii suspension twice daily (approx.
100 nauplii/mysid). See Appendix A.
None, unless dissolved oxygen
concentration falls below 40% of
saturation, at which time gentle
single-bubble aeration should be started,
Natural seawater, or synthetic salt
water adjusted to 20 ppt salinity.
Screening test - 24 h (Static tests)
Definitive test - 48 h (Static tests)
- 48 to 96 h (Flow-thru)
Mortality - no movement of body or
appendages on gentle prodding (LC50)
40
-------
TABLE 7. RECOMMENDED TEST CONDITIONS FOR THE FATHEAD MINNOW
(PIMEPHALES PROMELAS)
1. Temperature (°C):
2. Light quality:
3. Light intensity:
4. Photoperiod:
5. Size of test vessels:
6. volume of test solution:
7. Age of fish:
8. No. of fish/0.75 L:
9. No. of replicate
test vessels/cone.:
10. Total no. organisms
per concentration:
11. Feeding regime:
12. Aeration:
13. Dilution water:
14. Test duration:
15. Effect measured:
20 i 2°C
Ambient laboratory illumination
50-100 ft c (ambient laboratory levels)
8-16 h light/24 h
1 L
0.75 L
1-90 days
10 (Not to exceed loading limits)
20
Feeding not required first 96 h. For
longer tests, see Appendix A for feeding
instructions.
None, unless DO concentration falls
below 40% of saturation, at which time
gentle single-bubble aeration should be
started.
Receiving water, other surface water,
ground water, or soft synthetic water.
Screening test - 24 h (Static tests)
Definitive test - 48 h (Static tests)
- 48-96 h (Flow-thru)
Mortality - no movement (LC50)
41
-------
TABLE 8. RECOMMENDED TEST CONDITIONS FOR SILVERSIDES
(MENIDIA SPP.)
1. Temperature (°C):
2. Light quality:
3. Light intensity:
4. Photoperiod:
5. Size of test vessel:
6. Volume of test solution:
7. Age of test animals:
8. No. of test
fish/0.75 L:
9. No. of replicate
test vessels/cone.:
10. Total no. organisms
per concentration:
11. Feeding regime:
12. Aeration:
13. Dilution water:
14. Test duration:
15. Effect measured:
20 + 2°C (Northern latitudes)
25 + 2°C (Southern latitudes)
Ambient laboratory illumination
50-100 ft c (ambient laboratory levels)
8-14 h light/24 h
1 L
0.75 L
1 to 90 days
10 (Not to exceed loading limits)
20
Feeding not required first 96 h. For
longer tests, see Appendix A for feeding
instructions.
None, unless DO concentration falls below
40% of saturation, at which time gentle
single-bubble aeration should be started.
Natural seawater or reconstituted saltwater
adjusted to 25-30 ppt salinity.
Screening test - 24 h (Static tests)
Definitive test - 48 h (Static tests)
- 48-96 h (Flow-thru)
Mortality - No movement (LC50).
42
-------
SECTION 9
EFFLUENT FRACTIONATION PROCEDURE
(Prepared by Gerald Walsh)
Depending on the objectives of the study and the resources available,
the source of toxicity in an effluent can be characterized by subjecting it
to a series of fractionations using ion exchange resins (Fig. 4). In this
approach, liquid effluent that exhibits toxicity is filtered to remove the
particulate matter, the filtrate is passed through an XAD-4 resin to
separate the organic and inorganic fractions, and the toxicity tests are
conducted with the two fractions. If either fraction exhibits toxicity,
subfractionation may be carried out using ion exchange resins for the
inorganics and extraction under basic and acidic conditions for the
organics. Toxicity test may be conducted on the subfractions.
Identification of possible toxic substances may be made by inductively
coupled plasma (ICAP), high performance liquid chromatography (HPLC), and
gas chromatography mass spectrometry (GCMS) analyses. Such analyses of all
subfractions are informative because some organic molecules behave as ions
and may be found occasionally in the "inorganic" fraction. For a detailed
discussion of the fractionation and toxicity test procedure, see Walsh and
Garnas (1983).
43
-------
0) >,
• S- S_
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ro (/) i —
i- T3 OJ
^- C 4->
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4_> 03 rO
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-------
SECTION 10
TEST DATA
BIOLOGICAL DATA
Death is the "effect" used for determining toxicity to aquatic
organisms in acute toxicity tests. The criteria usually employed in
establishing death of fish are: (1) no movement, especially no gill
movement; and (2) no reaction to gentle prodding.
Death is not as easily determined for some invertebrates, such as
daphnids, and is defined as the lack of movement of the body or appendages
on gentle prodding. However, the death of some invertebrates, such as
mysids, is easily detected because of a change in pigmentation from
transparent or translucent to opaque. General observations of appearance
and behavior (see Table 9), such as erratic swimming, loss of reflex,
discoloration, excessive mucus production, hyperventilation, opaque eyes,
curved spine, hemorrhaging, molting, and cannibalism , should also be noted
in the daily log.
The number of surviving organisms in each test container should be
counted at one, two, four and eight hours after the beginning of a
screening test, and daily (Figs. 5 and 6) in definitive tests. When
recognizable, dead organisms should be removed during each observation
period.
The mean lengths and weights of test fish are obtained by sacrificing
10 animals from the original pool of test organisms at the beginning of the
test, or using the control organisms at the conclusion of the test. The
species, source, and age of the test organisms should also be recorded.
CHEMICAL AND PHYSICAL DATA
In nonrenewal static tests, at a minimum, pH, and salinity or
conductivity, and total residual chlorine are measured in the control and
in the highest effluent concentration at the beginning of the test. The DO
should be monitored closely (every 2 h) for the first 4 to 8 h as described
above, and measured daily thereafter in all effluent concentrations in
which there are surviving organisms. Temperature is measured at the
beginning of the test, and daily thereafter. It is recommended that total
alkalinity and total hardness be measured in the control and highest
effluent concentration at the beginning of the test.
45
-------
TABLE 9. DEFINITION OF FISH BEHAVIOR TERMS3
TERM
DEFINITION
GENEKAL
BEHAVIOR -
a. Quiescent:
b. Hyperexcitable:
c. Irritated:
d. Surfacing:
e. Sounding:
f. Twitching:
g. Tetanous:
h. Flaccid:
i. Normal:
SWIMMING -
a. Ceased:
b. Erratic:
c. Gyrating:
d. Skittering:
e. Inverted:
f. On side:
PIGMENTATION -
a. Light discolored:
b. Dark discolored:
c. Varidiscolored:
INTEGUMENT -
a. Mucus shedding:
b. Mucus coagulation:
c. Hemmorrhagic:
RESPIRATION -
a. Rapid:
b. Slow:
c. Irregular:
d. Ceased:
e. Gulping air:
f. Labored:
Observable responses of the test fish, individually or in groups, to
their environment.
Marked by a state of inactivity or abnormally low activity; motionless or
nearly so.
Reacting to stimuli with substantially greater intensity than control fish.
Exhibiting more or less continuous hyperactivity.
Rising and remaining unusually long at the surface.
Diving suddenly straight to the bottom; remaining unusually long at the
bottom.
Moving the body or parts of the body with sudden jerky movements.
In a state of tetany; marked by intermittent tonic spasms of the voluntary
muscles.
Lacking tone, resilience or firmness; weak and enfeebled; flabby.
Unaffected by or not exposed to a particular experimental treatment;
conforming to the usual behavioral characteristics of the species.
Progressive self-propulsion in water by coordinated movement of
tail, body, fins.
Broken off or tapered off to a stop.
Characterized by lack of consistency, regularity, or uniformity;
fluctuating, uneven; eccentric.
Revolving around a central point; moving spirally about an axis.
Skimming hurriedly along the surface with rapid body movements.
Turned upside down, or approximately so.
Turned 90° laterally, more or less, from the normal body orientation.
Color or skin due to deposition or distribution of pigment.
Color appearance lighter than usual for the species.
Color appearance darker than usual for the species.
Color appearance abnormally varied; mottled.
The skin.
Observably losing mucous skin coating to an abnormal degree.
Showing observable clumping or clotting of the mucous skin coating,
especially at the gills.
Visibly bleeding as from gills, eyes, anal opening.
Physical action of pumping water into mouth and out through gills, so as to
absorb oxygen.
Observably faster than normal to a significant degree.
Observably slower than normal to a significant degree.
Failing to occur at regular or normal intervals.
Broken off or tapered off to a stop.
Swimming at surface with mouth open and laboriously pumping surface water
and air through gills.
Performed with apparent abnormally great difficulty and effort.
From Brusick and Young, 1981.
46
-------
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In flow-through and static renewal tests, at a minimum, pH, salinity
or conductivity, total alkalinity, total hardness, and total residual
chlorine are measured daily in the highest effluent concentration. DO and
temperature are measured at the beginning of the test and daily thereafter
in the control and all all effluent concentrations containing surviving
test organisms.
The measurement of specific conductance is recommended because it is a
very useful parameter in detecting transient fluctuations in the chemical
characteristics of effluents, and will indicate errors in test dilutions.
Water samples collected for chemical analysis should be taken midway
between the top, bottom, and sides of the test containers, and should not
include any surface scum or material stirred up from the bottom or sides.
Methods used for chemical analysis should be those specified for
Section 304(h) of the CWA (USEPA 1979b, 1982). For salinity measurements,
a refractometer may be used if calibrated with a sample of known salinity.
Temperature should be recorded continuously in all tests in at least one
chamber.
49
-------
SECTION 11
TOXICITY DATA ANALYSIS
INTRODUCTION
Data from acute effluent toxicity tests are used to estimate the median
lethal effluent concentration, or LC50, which is the concentration that is
lethal to 50% of the organisms within the test period. Only rarely will a
given effluent concentration result in exactly a 50% mortality of the test
organisms. Therefore, some mathematical means, such as regression
analysis, is generally needed to obtain an estimate of the LC50 by
interpolation between two concentrations. Also, because of the normal
variation in sensitivity of individuals within a group of test organisms,
there is a degree of uncertainty regarding the "true" or "exact" value of
the LC50. This uncertainty is expressed as a confidence interval, or range
of values within which the "true" LC50 could occur. The confidence
interval is defined in terms of the level of probability (usually 0.95)
that the "true" LC50 is included.
When plotted on linear graph paper, mortality data from a toxicity test
which results in partial mortalities at several concentrations of the
toxicant typically form an "S"-shaped, or sigmoid, curve (Fig. 7). The
major portion of the curve will approximate a straight line if the
concentrations are converted (transformed) to logarithms, or if the data
are plotted on semi-log paper. Linearity in the relationship between
concentration and mortality is most closely achieved in the vicinity of
QC
O
o
cc
TOXICANT CONCENTRATION
Figure 7. Linear plot of toxicity data.
50
-------
the LC50, in the central region of the curve. The LC50, therefore, is the
statistic that can be estimated with the greatest certainty. Transform-
ations of the data permit more exact estimates of the LC50 and facilitate
the calculation of confidence intervals. The similarity in the shape of
toxicity data curves after transformation of the data to Probits, Logits,
and angles is shown in Fig. 8. Commonly used methods of estimating the
LC50 and its confidence interval include the graphical, Litchfield-
Wilcoxon, binomial, Probit, Logit, moving average, moving average-angle,
and Spearman-Karber methods (Finney, 1971; Stephan, 1977; Peltier,
1978a,b). Fortunately, the availability of inexpensive but powerful
scientific pocket calculators and computers has eliminated most of the
tedium previously associated with the use of some of these methods.
LOGIT PROBIT
I I I I I
1 I I I
6
PROBIT
LOGIT
6
90
80
70
60
50
40
30
20
10
0
10 20 30 40 50 60 70
PERCENTAGE
80 90 100
Figure 8. Comparison of angular, Logit, and Probit transformations
of toxicity data. (From Knudsen and Curtis, 1947.)
ALL-OR-NOTHING NATURE OF MORTALITY DATA FROM EFFLUENT TOXICITY TESTS
Unlike tests with pure compounds, which usually result in a regular
progression in percent mortality with increasing toxicant concentration,
toxicity tests with effluents commonly yield all-or-nothing results, with
no concentrations showing partial mortalities. In these cases, exposures
to one or more of the higher concentrations of effluent (such as 100% and
50% effluent) result in 100% mortality of the test organisms, whereas
exposures at lower concentrations of effluent (such as 25%, 12.5%, and
6.25%) all result in 100% survival. These results obviate the use of some
51
-------
methods of calculating the LC50, such as the Probit, Litchfield-Wilcoxon,
and Spearman-Karber analyses. Of 60 effluent toxicity tests conducted
during FY-83 by the Environmental Services Division, USEPA Region 4,
Athens, Georgia, in which test organism lethality occurred, only four
(7%) were amenable to Probit analysis.
VARIABILITY IN TOXICITY TEST RESULTS
As was mentioned earlier, the toxicity test results will depend upon
the species used, the strain or source of the test organisms, the
condition or health of the organisms, and test conditions such as
temperature, DO, food, water quality, etc. The repeatability or
precision of toxicity tests is also a function of the number of test
organisms used at each toxicant concentration. Jensen (1972) discussed
the relationship between sample size (numbers of fish) and the standard
error of the test, and considered 20 fish/concentration as optimum.
Test precision can be estimated by using the same strain of organisms
under the same test conditions, and employing a known toxicant, such as a
reference toxicant. The single laboratory and multi-laboratory precision
of acute toxicity tests with several common test species and reference
toxicants is listed in Tables 10, 11, and 12 below. The values for
single laboratory precision (expressed as coefficient of variation) from
92 reference toxicant tests with three species, as listed in Table 10,
ranged from 10% to 86%, and had a weighted mean of 38%. The values for
multi-laboratory precision (expressed as coefficient of variation) from
153 reference toxicant tests with six species, as listed in Table 11,
ranged from 22% to 167%, and had a weighted mean of 50%. The multi-
laboratory precision of toxicity tests with standard effluents
is also 30% to 50% (see Table 12 and Buikema, 1983).
52
-------
TABLE 10. SINGLE LABORATORY PRECISION OF STATIC ACUTE TOXICITY TESTS
WITH AQUATIC ORGANISMS, USING REFERENCE TOXICANTS^.b
TEST ORGANISM
REFERENCE TOXICANT
SDS
Pimephales promelas
(Fathead Minnow) (96 h)
Daphnia pulex (48 h)
Daphnia magna (48 h)
N
9
10
8
CV(%)
22
43
29
NAPCP
N CV(%)
12 40
14 36
13 10
N
9
9
8
CD
CV(%)
86
21
72
a. SDS = Sodium dodecyl (lauryl) sulfate
NAPCP = Sodium pentachlorophenate
CD = Cadmium
N = Number of tests
CV(%) = Coefficient of variation = (standard deviation x 100)/mean.
b. Data provided by Philip Lewis and James Dryer, Aquatic Biology
Section, EMSL-Cincinnati, and taken in part from Lewis and
Weber, 1985.
53
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TABLE 11. MULTI-LABORATORY PRECISION OF ACUTE TOXICITY TESTS
WITH AQUATIC ORGANISMS, USING REFERENCE TOXICANTS**
TEST ORGANISM
1.
2.
Pimephales promelas
96-h static test
96-h flow-through test
Salmo gairdneri
96-h static test
96-h flow-through test
SILVER
N
10
10
10
10
REFERENCE
NITRATE
CV(%)b
52
40
64
32
TOXICANT
ENDOSULFAN
N CV(%)
12 38
12 46
12 50
12 43
3. Daphnia magna
48-h static 8 71 12 51
4. Acartia tonsa
96-h static test 5 42 6 82
5. Mysidopsis bahia
96-h static test 6 27 5 62
96-h flow-through test (Nom) 6 22 6 58
96-h flow-through test (Meas) 6 58 5 167
6. Cyprinodon variegatus
96-h static test (Nom) 4 35 6 37
96-h flow-through test (Nom) 5 50 6 46
96-h flow-through test (Meas) 5 46 6 38
a. Data for Pimephales promelas (fathead minnow), Salmo gairdneri
(rainbow trout), and Daphnia magna were taken from Broderius, 1983.
Data for Acartia tonsa, Mysidopsis bahia, Cyprinodon variegatus
(sheepshead minnow) were taken from Schimmel, 1981. Six laboratories
participated in each study.
In the studies with the freshwater organisms, the water hardness for
five of the six laboratories ranged between 36 and 75 mg/L. However,
the water hardness for the sixth laboratory was 255 mg/L, resulting in
54
-------
TABLE 11. Footnotes (continued)
LC50 values for silver nitrate more than an order of magnitude larger
than for the other five. These values were rejected in calculating
the CV*.
N, the total number of LC50 values used in calculating the CV, varied
with organism and toxicant because some data were rejected due to
water hardness, and/or because some of the LC50s were not calculable.
In studies with the marine organisms, only one LC50 (presumably the
combined LC50 from duplicate tests) was reported for each toxicity
test, and N = 6 in each case. LC50s for the 96-h flow-through tests
with Mysidopsis bahia and Cyprinodon variegatus were calculated two
different ways — (1) on the basis of the nominal toxicant
concentrations (Norn), and (2) on the basis of measured (Meas) toxicant
concentrations.
b. CV(%) = Coefficient of variation = (standard deviation x 100)/mean.
55
-------
TABLE 12. MULTI-LABORATORY PRECISION OF 48-H ACUTE TOXICITY TESTS
WITH DAPHNIA MAGNA, USING A STANDARD EFFLUENT^.b
Laboratory
Industry
1
2
3
Government
1
2
3
Commercial
1
2
3
Mean
SO
CV(%)
LC50s From
Replicate Mean
Tests LC50
4.2
4.9 4.6
6.8
6.1
6.1 6.3
3.5
7.1 5.3
4.4
4.4
4.1 4.3
4.5
4.5 4.5
4.2
4.9
4.7 4.8
3.7
5.6 4.6
9.0
9.1
8.6 8.9
5.5
1.8
31.8
SDC CV(%)d
0.40 6.4
0.17 4.0
0.26 3.0
a. From Table 2, p. 191, Grothe and Kimerle, 1985.
b. LC50 expressed in percent effluent.
c. SD = Standard deviation; determined only for three
or more replicates.
d. CV(%) = (Standard deviation X 100)/Mean
56
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MANUAL METHODS FOR ESTIMATING THE LC50
1. Graphical Method
The first step in the evaluation of the toxicity test data should consist
of the preparation of a plot of the data using 2-cycle semi-log graph paper
(Fig. 9). This technique is commonly referred to as the Log-Concentration-
Versus-Percent-Mortality method. The logarithmic axis (y axis) is used for
percent effluent concentration, and the linear (x axis) is used for percent
mortality. The graph is useful in providing: (1) a rapid overview of the
distribution of the data that will aid in determining if there is a regular
progression of mortality with increasing concentration, especially in the
central region of the curve; and (2) a reasonably-accurate estimate of the
LC50. However, the method does not provide a confidence interval around the
estimate of the LC50.
General Procedure:
a. Plot the percent effluent volumes and the corresponding percent mortality
on semi-logarithmic graph paper as described above.
b. Locate the two points on the graph which are separated by the 50%
mortality line, and connect them with a straight line (see Fig. 9).
However, if one of the points is obviously irregular, the mortality value
for the next lower or higher percent effluent volume is used.
c. On the scale for percent effluent volume, read the value of the point
where the diagonal line and the 50% mortality line intersect. This value
is the LC50 for the test, expressed as a percent effluent volume.
Example:
Step 1. The percent effluent volumes and the corresponding percent mortality
values listed below are plotted in Fig. 9.
Effluent
Concentration
(%)
100.0
50.0
25.0
12.5
6.25
Percent
Mortality
100
100
0
0
0
Step 2. The two adjacent points which are separated by the 50% mortality
line T50% effluent and 25% effluent) are located and connected with a
diagonal straight line.
Step 3. An LC50 of 35% effluent was derived for the test by determining the
effluent concentration at the point where the diagonal line drawn between
100% and 0% mortality intersected the 50% mortality line.
57
-------
=3
O
o
cc
100-
90-
80-
70-
SO-
SO-
40'
30
20
15
10
9
8
?•
6
5
4
0 10 20 30 40 50 60 70 80 90 100
PERCENT MORTALITY
Figure 9. Plotted data and fitted line for log-concentration-versus
percent-mortality method of determining LC50.
58
-------
2. Moving Average-angle Method
The moving average-angle method is recommended for use in calculating
the LC50 and its confidence interval for effluent toxicity data for two
important reasons:
a. It permits the calculation of the 95% confidence interval for the LC50
when no partial mortalities occur in a test, which is a common
occurrence in effluent bioassays, and
b. The method lends itself readily to the use of inexpensive, scientific
pocket calculators or pocket computers, that have log, antilog, and
inverse sine or inverse tangent functions.
The use of the method, however, is limited to those data sets in which
there are two effluent concentrations above the LC50.
Example of Calculation of the LC50 and 95% Confidence Interval:
An example of an LC50 calculation with the moving average-angle method
is provided below, based on the data listed on p. 57 and shown in Fig. 9:
Step 1. Prepare a table using the data from an effluent toxicity test and
carry out the calculations as illustrated below.
TABLE 13. SAMPLE DATA FOR MOVING AVERAGE-ANGLE METHOD
Effluent
Concentration
(%)
(a)
100.0
50.0
25.0
12.5
6.25
Transformed
Effluent
Concentration
(Logic)
(b)
2.0000
1.6990
1.3979
1.0969
0.7959
Response
Proportion
(RP)
(c)
1.0
1.0
0.0
0.0
0.0
Transformed
RP
(Degrees)
(d)
83.5807
83.5807
6.4193
6.4193
6.4193
Moving
Average-Angle
(Degrees)
(e)
57.8602
32.1398
*
*
No calculation required (see Step 5, below).
Step 2. List the effluent concentrations (as percent effluent) and
°f the concentration of each treatment level (see columns "a" and "b" in
the table above).
Step 3. Calculate the response proportion (RP) at each effluent
concentration (see column "c" in the table above), where:
RP = (number of dead or "affected" organisms)/(initial number).
59
-------
Step 4. Transform each RP to an angle, using Arcsin (Sin~l), as follows
(see column "d" in the table above):
a. For RP = 0.01 to 0.99: (Eq. 1)
Angle = Arcsin (RP)0.5
b. For RP = 0 (no mortality) or 1 (100% mortality), the following
adjustments are made:
(1) For RP = 0 (no mortality): (Eq. 2)
Angle = Arcsin (1/4N)0-5
Where: N = Number of animals/treatment
Example: If 20 animals are used,
Angle = Arcsin (1/80)0-5
= Arcsin 0.1118
= 6.4193 degrees
(2) For RP = 1.0 (100% mortality):
Angle = 90.0 - (angle for RP = 0) (Eq. 3)
Example: Using above value
Angle = 90.0 - 6.4193
= 83.5807 degrees
Step 5. Calculate the moving average-angles as follows, until two average
angles are obtained that bracket 45° (see column "e" in the table above):
a. Sum the largest three angles and divide by 3:
(83.5807 + 83.5807 + 6.4193)73 = 57.8602
b. Sum the next largest three angles and divide by 3:
(83.5807 + 6.4193 + 6.4193)73 = 32.1398
c. These two angles bracket 45°. No further averaging is required.
d. Proceed to Step 6 (LC50 calculation).
60
-------
Step 6. Calculate the LC50:
a. Calculate A:
. 45.0 - LMAA _ 45.0 - 32.1398 _ 12.8602 _ n ,nnn
M " UMAA - LMAA " 57.8602 - 32.1398 " 25.7204 " u'ouuu
Where: 45° = Position of the LC50 (percent effluent)
LMAA = (Lower Moving Average-Angle) The angle less than
but closest to 45° (32.1398 degrees in the above
example).
UMAA = (Upper Moving Average-Angle) The angle greater
than, but closest to 45° (57.8602 in the above
example).
b. Using A above, calculate the LC50:
(1) Log10 LC50 = LLC + (LUC - LLC)(A) (Eq. 5)
= LOG(25) + ((LOG(50)-LOG(25))(A)
= 1.3979 + (1.6990 - 1.3979)(0.5000)
= 1.5485
Where: LLC = Log Lower Concentration, or logiQ of the effluent
concentration just below, but closest to, RP = 0.5
(1.3979 in above example)
LUC = Log jJpper Concentration, or log]Q of the effluent
concentration just above but closest to, RP = 0.5
(1.6990 in above example)
(2) LC50 = lo1'5485 = 35.35% Effluent
Step 7. Calculate the 95% confidence limits for the LC50:
a. Calculate G:
sa . 1641.4d.86)2 - . - 6306,6 -
(N)(IC)(UMAA - LMAAr (20)(9) (25.7209T
Where: K = Number of angles averaged (3)
N = Number of organisms per treatment (20)
2(90/Tr)2 = 1641.4
aFor further reference to "G," see Appendix E.
61
-------
b. Calculate AG:
AG = A - G/2 _ 0.5000 - (0.05295/2) _ Q Q (Eq. 7)
1 - G 1 - 0.05295
c. Calculate AU and AL:
(1) AU = AG + (GO-5/(l - G))((A - 0.5)2 + 5/4(1 - G))0.5
= 0.5000 + (0.2301/0.9470)((.5000-0.5)2 + 5/4(.9470))0.5
= 0.5000 + (0.2430)(1.088)
= 0.5000 + 0.2644 = 0.7644
(2) AL = AG - (G0.5/(i - G))((A - 0.5)2 + 5/4(1 - 6))0.5
= 0.5000 - 0.2644 = 0.2356
d. Calculate the 95% confidence limits of LC50:
(1) Upper confidence limit (UCL): (Eq. 8)
(a) Log10 UCL = LLC + (LUC - LLC)(AU)
= 1.3979 + (1.6990 - 1.3979)(.7644)
= 1.3979 + 0.2302 = 1.6281
(b) UCL = TO1'6281 = 42.47%
(2) Lower confidence limit (LCL):
(a) Log10 LCL = LLC + (LUC - LLC)(AL) (Eq. 9)
= 1.3979 + (1.6990 - 1.3979)( 0.2356)
= 1.4688
(b) LCL = 101'4688 = 29.43% effluent
Step 8. Results:
a. LC50 = 35.35% effluent
b. The 95% confidence interval = 29.4% - 42.5% effluent
62
-------
3. Litchfield - Wilcoxon Abbreviated Method^
This method cannot be used unless partial mortalities occur in the test.
General Procedure:
a. Tabulate the data showing the percent-effluent volumes used, the total
number of organisms exposed to each percent-effluent volume, the number
of affected organisms, and the observed percent-effluent organisms (see
Table 14). Do not list more than two consecutive 100% affects at the
higher percent effluent volumes or more than two consecutive 0% affects
at the lower percent-effluent volumes.
b. Plot the percent-affected organisms against the percent-effluent volume
on two-cycle, logarithmic probability paper (Fig. 10), except for 0% or
100% affect values. With a straight edge, fit a temporary line through
the points, particularly those in the region of 40% to 60% affects.
c. Using the line drawn through the points, read and list an "expected"
percent effect for each percent-effluent volume tested. Disregard the
"expected" percent value for any of the percent volumes less than 0.01 or
greater than 99.99. Using the expected-percent-affect, calculate from
Table 15 a "corrected" value for each 0% or 100% affect obtained in the
test. (Since the expected values in the table are whole numbers, it will
be necessary to obtain intermediate values by interpolation.) Plot these
values on the logarithmic probability paper (Fig. 10) used in Step 2 and
inspect the fit on the line to the completely plotted data. If after
plotting the corrected expected values for 0% and 100% affected, the fit
is obviously unsatisfactory, redraw the line and obtain a new set of
expected values.
d. List the difference between each observed (or corrected) value and the
corresponding expected value. Using each difference and the
corresponding expected value, read and list the contributions to
Chi-square (Chi2) from Fig. 11. (A straight edge connecting a value on
the Expected-Percent-Affected scale with a value on the
Observed-Minus-Expected scale, will indicate at the point of intersection
of the Chi2 scale, the contribution to Chi2). Sum the contributions
to Chi2 and multiply the.total by the average number of organisms per
effluent volume, i.e., the number of organisms used in K concentrations
divided by K, where K is the number of percent-affected organism values
plotted. The product is the "calculated" Chi2 of the line. The
degrees of freedom (N) are 2 less than the number of points plotted,
i.e., N = K-2. If the calculated Chi2 is less than the Chi2 given in
Table 16 for N degrees of freedom, the data are non-heterogeneous and the
line is a good fit. However, if the calculated Chi2 is greater than
the Chi2 given in Table 16 for N degrees of freedom, the data are
heterogeneous and the line is not a good fit. In the even a line cannot
be fitted (the calculated Chi2 is greater than the tabular Chi2), the
data can not be used to calculate an LC50 or EC50. Litchfield and
^Taken from Peltier, 1978a,b.
63
-------
Wilcqxon provided an alternate method for calculating the 95%
confidence limits under these circumstances. However, the toxicity
test should be repeated.
e. Determine the 95% confidence limits of the LC50.
(1) Read from the fitted line (Fig. 10), the percent effluent volumes
for the corresponding 16%, 50%, and 84% affects (LC16, LC50, and
LC84).
(2) Calculate the slope function, S, as:
S = LC84/LC50 + LC50/LC16
2
(3) From the tabulation of the data determine N1, which is defined as
the total number of test organisms used within the
percent-affected- organism interval of 16% and 84%. Calculate the
exponent (2.77/\/Tr) for the slope function and the factor,
f[_C50» used to establish the confidence limits for the LC50 (or
EC50).
f _ .(2.77//N7)
TLC50 " *
The f|_C50 can be obtained directly from the nomogram in Fig. 12
by laying a straight-edge across the appropriate base and exponent
values and reading the resultant "f" value.
(4) Calculate the 95% confidence limits of the LC50 as follows:
(a) Upper limit = LC50 X f[_C50
(b) Lower limit = LC50/f|_c50
Example:
Steps 1-4. The data are tabulated, as in Table 14 (Step 1), plotted
(Step 2, Fig. 10), and the expected values are read from the graph
(Step 3). Chi2 is calculated (Step 4) as follows:
a. Mean number of organisms used in 'K1 (K = 6) concentrations = 120/6 = 20
(Note that the data for the 10% effluent volume were aberrent and were
not used. Therefore, K = 6, and the total number of organisms = 120)
b. Calculate Chi2 = 20 x 0.057 = 1.14
c. Degrees of Freedom (N)=K-2=6-2=4
d. From Table 16, the Chi2 for 4 degrees of freedom = 9.49
e. The calculated Chi2 is less than the tabular Chi2. Therefore, it
is assumed the line is a good fit, and the data are non-heterogeneous.
64
-------
TABLE 14. SAMPLE DATA FOR LITCHFIELD-WILCOXON ABBREVIATED METHOD
Percent
Effluent
Volume
3.2
5.6
10.0
18.0
32.0
56.0
100.0
STEP
Number of
Organisms
20
20
20
20
20
20
20
ONE
Number of
Affected
Organisms
0
1
11
7
12
18
20
Observed %
Affected
Organisms
0 (0.2)a
5
55
35
60
90
100 (99.0)a
STEP THREE
Expected %
(Fig. 10)
0.6
3.5
(14.5)b
38.0
67.0
87.5
97.0
STEP FOUR
Observed
Minus
Expected
0.4
1.5
Aberrant
3.0
7.0
2.5
2.0
TOTAL
Chi^
0.003
0.006
Value
0.004
0.024
0.006
0.014
0.057
a. Step 3 "Corrected" affected values from Table .15.
b. Percent-affected organisms at the 10% effluent volume is obviously an
aberrant value and should be omitted when fitting the line in Step 2.
Step 5. Determine the LC50 and 95% confidence limits.
a. From the fitted line in Fig. 10, determine the (percent) effluent
concentrations corresponding to the 16%, 50%, and 84% affected organism
values:
(1) LC84 effluent concentration = 50.0%
(2) LC50 " " = 23.0%
(3) LC16 " " = 10.5%
b. Calculate the slope function, 'S1, as:
S = LC84/LC50 + LC50/LC16 = 50.0/23/0 + 23.0/10.5
2 2
= 2.17 + 2.19 = 4.36 = 2.18
c. Determine N': N1 = 40 (From Fig. 10).
d. Calculate the exponent (2.111 N1) and factor, fLC50:
<2-77/ 40) = (2. 77/6.32)
fLC50 = S- = 2.18
= 2.18(°-438> = 1.41
e. Calculate the 95% confidence limits of the LC50:
(1) Upper limit = LC50 X fLC50 = 23.0 x 1.41 = 32.4%
(2) Lower limit = LC50/f|_C50 = 23.0/1.41 = 16.3%
65
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TABLE 15. CORRECTED VALUES FOR 0% OR 100% EFFECT
Expected
Value 0
0
10
20
30
40
50
60
70
80
90
3.2
6.0
8.3
9.9
__
90.1
91.7
94.0
96.8
1
0.3
3.5
6.2
8.4
10.0
89.5
90.2
91.9
94.3
97.1
2
0.7
3.8
6.5
8.6
10.1
89.6
90.4
92.2
94.5
97.4
3
1.0
4.1
6.7
8.8
10.2
89.6
90.5
92.4
94.8
97.7
Corrected Value
456
1.3
4.4
7.0
9.0
10.3
89.6
90.7
92.6
95.1
98.0
1.6
4.7
7.2
9.2
10.4
89.7
90.8
92.8
95.3
98.4
2.0
4.9
7.4
9.3
10.4
89.7
91.0
93.0
95.6
98.7
7
2.3
5.2
7.0
9.4
10.4
89.8
91.2
93.3
95.9
99.0
8
2.6
5.5
7.8
9.6
10.4
89.9
91.4
93.5
96.2
99.3
9
2.9
5.7
8.1
9.8
10.5
90.0
91.6
93.8
96.5
99.7
TABLE 16. VALUES OF Chi? (P = 0.05)
Degrees
of Freedom (N)
1
2
3
4
5
6
7
8
9
10
Chi2
3.84
5.99
7.82
9.49
11.1
12.6
14.1
15.5
16.9
18.8
66
-------
% EFFLUENT VOLUME
Figure 10. Line fitted to data, and LC16, LC50, and LC84, as read
from the line (Steps 2, 3, and 4).
67
-------
EXPECTED
%
AFFECTED
Chi
50
70
80
90
95
96- —4
97-J— 3
98
994—1.0
99.5
99.6
99.7-
99.8
99.J
99.95
99.96-
9 ft 97-
99.98-
— 50
— 30
— 20
t—10
— 5
- .5
-.4
-.3
.2
05
.04
.03
.02
OBSERVED MINUS
EXPECTED
— 50
— 40
— 30
— 20
10
5
4
3
— 2
.5
.4
- -.3
- -.2
- -.1
- -.05
— 2.0
— 1.0
— .50
— .40
— .30
— .20
— .10
— .05
— .04
— J03
— .02
— .01
— .005
— .004
— .003
— .002
—.001
Figure 11. Nomograph for obtaining Chi? from expected-percent-affected
and observed-minus-expected values (Step 4).
68
-------
B/
10 —
-
SX5-
4X> —
3.0 —
-
2.0—
-
1.30 —
1.40—
1.30—
-
-
1.20—
-
-
1.10—
kSE S -p
!L
EXPONE
.C50
- 100
— 50
— 10.0
e f\
O.U
— 4.0
— 3.0
-3.0
— 1.5
- 1.4
-1.3
— 1.2
— 1.10
- 1.05
— 1.04
- 1.03
- 1.02
:NT
- 4.0
-
— 3.0
-
— 2.0
-1.5
-
— 1.0
— .90
— .80
— .70
— .60
— .50
-
— .40
— .30
~
.20
Figure 12. Nomograph for raising Base S to a fractional exponent.
69
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4. Probit Method
Probit analysis consists of a group of statistical methods used to
analyze data from concentration-response experiments, and provides an
estimate of the LC50 and the precision of this estimate. In Probit
analysis, the percentages of affected organisms are converted to Probits
(Probability Units), and the effluent concentrations are converted to
logarithms. The relationship between the Probits and the logarithmic
values of the concentrations is approximately linear. A Probit regression
line drawn through the data points is used to estimate the LC50 and its
precision. NOTE: TO USE PROBIT ANALYSIS, AT LEAST TWO PARTIAL MORTALITIES
MUST BE OBTAINED IN THE TOXICITY TEST.The following steps are involved in
the analysis:
Step 1. Prepare a table (Table 17) with the following headings:
a. Effluent concentrations used in the test (%).
b. LogiQ of the effluent concentration (x).
c. Number of organisms at each concentration (N).
d. Observed mortality at each concentration (r).
e. Percent mortality at each concentration (P).
f. Empirical (observed) Probit (EP)
g. Expected (calculated) Probit (Y)
Step 2. Items "a-e" consist of data from the experiment.
Step 3. The EP is obtained from Table 18 by entering P.
Step 4. A graph (Fig. 13) is prepared using the logio concentrations (x)
and the EPs. A regression line is then drawn through the points by eye.
Step 5. From the regression line, determine the logio concentration (m),
or logio LC50, corresponding to a Probit of 5.0. In our example, m =
1.72.
Step 6. From the regression line, determine "s", the rate of increase in
log concentration (x) per unit increase in the empirical probit (EP) as
follows: from the points at x = 0.80 and x = 2.00 we obtain probits of
3.30 and 5.55 respectively. The slope is (2.00 - 0.80)7(5.55 - 3.30),
which equals 1.20/2.25, or 0.533, the value of "s".
Step 7. The values of Y can now be calculated from Y = 5 + (x - m)/s, and
entered in Table 17.
Step 8. To test the hypothesis of no association between effluent concen-
tration and Probit response, we construct Table 19. The quotients in the
last column are summed, and the sum is a Chi2 statistic with degrees of
freedom equal to two less than the number of effluent levels used in the
probit analysis (5-2=3). A significant Chi2 value implies that the
70
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TABLE 17. TOXICITY DATA FOR PROBIT ANALYSIS
Effl
Cone
(%)
100
50
25
12.5
6.25
Logio
Cone
(x)
2.00
1.70
1.40
1.10
0.80
Number
of
Organisms
(N)
20
20
20
20
20
Observed
Mortality
(r)
15
9
5
2
1
%
Mortality
(P)
75
45
25
10
5
Empirical
Probit
(EP)
5.67
4.87
4.33
3.72
3.36
Calculated
Probit
(Y)
5.55
4.96
4.38
3.79
3.30
TABLE 18. EMPIRICAL (OBSERVED) PROBIT PERCENT MORTALITY RELATIONSHIP
% 0
0
10
20
30
40
50
60
70
80
90
%
3
4
4
4
5
5
5
5
6
0
-
.72
.16
.48
.75
.00
.25
.52
.84
.28
.0
2.67
3.77
4.19
4.50
4.77
5.03
5.28
5.55
5.88
6.34
0.1
2.95
3.82
4.23
4.53
4.80
5.05
5.31
5.58
5.92
6.41
0.2
3.12
3.87
4.26
4.56
4.82
5.08
5.33
5.61
5.95
6.48
0.3
3.25
3.92
4.29
4.59
4.85
5.10
5.36
5.64
5.99
6.55
0.4
3.36
3.96
4.33
4.61
4.87
5.13
5.39
5.67
6.04
6.64
0.5
3.45
4.01
4.36
4.64
4.90
5.15
5.41
5.71
6.08
6.75
0.6
3.52
4.05
4.39
4.67
4.92
5.18
5.44
5.74
6.13
6.88
0.7
3.59
4.08
4.42
4.69
4.95
5.20
5.47
5.77
6.18
7.05
0.8
3.66
4.12
4.45
4.72
4.97
5.23
5.50
5.81
6.23
7.33
0.9
7.33 7.37 7.41 7.46 7.51 7.58 7.65 7.75 7.88 8.09
a. Values between 99.0 and 99.9
71
-------
6.0 -
55-
5.0 -
00
o
cc
4.5 -
4.0 -
3.5 -
3.0 -
/
! I I I I I I I I I I I I I I
0.6 0.7 08 0.9 10 1.1 1.2 1.3 14 15 1.6 1.7 1.8 1.9 2.0
LOG,o EFFLUENT CONCENTRATION
Figure 13. Plot of toxicity test data for Probit analysis.
72
-------
data are not adequately represented by the regression line. In our
example, the Chi-square value equals 0.321 (Table 19), which is not
significant.
Step 9. To determine the standard error for the logio LC50 (m) estimate
obtained above from the regression line, we construct Table 20, with the
headings as follows:
a. Logio of the effluent concentration (x).
b. Number of organisms at each concentration (N).
c. Expected, or calculated, probit (Y).
d. Weighting factor (w), obtained by entering Table 21 with the expected
probit (Y).
e. Products (Nw), (Nwx), and (Nwx2) are obtained as the last three
columns. The sums of these columns have the prefix "S", (SNw, SNwx,
and SNwx2).
An approximate value for the standard error (SE) of logio LC50 is
calculated as follows using "s", the rate of increase in log
concentration (x) per unit increase in the empirical probit (EP) computed
above, where s = 0.533 and SNw = 46.62:
SE (logio LC50) =
0.553
(SNw)
0.5
(46.62)
0.5
= 0.0781
f. Using the standard error of the Logio LC50 derived in Paragraph "a"
above, the approximate value for SE LC50 can be obtained by raising 10
to the power of m (which is 10^-^2 = 52.5), multiplying by Ioge10
(which is 2.3026) and then by SE logio LC50 (which is 0.0781).
Thus:
SE LC50 = 52.5 X 2.3026 X 0.0781 = 9.43
g. A more accurate estimate of SE logio LC50 is obtained from the
information in Table 17 as follows.
SE Iog10 LC50 =
( SNw
(m -
SNw(x - x)'
0.5
Where (from Table 20):
x = 71.21/46.62 = 1.527
m = 1.72
SNw = 46.62
SNwx = 71.72
SNwx2 = 115.40
SNw(x-x)2 = SNwx2 - (SNwx)2/SNw
= 115.40 - (71.21)2/46.62
= 6.630.
73
-------
c£
o n
er
I/I
I
CM||O-
i
O O
o *> —I
CU
o
UJ
UJ
o
o-
(/>
I
I—I
z
o
r—
Si
I. §
CU •—
T, 2
cu 10
-------
TABLE 20. STANDARD ERROR OF THE LOG-|0 LC50 (M).
Logic
Cone
(x)
2.00
1.70
1.40
1.10
0.80
Number
of
Organisms
(N)
20
20
20
20
20
Calculated
Probit
(Y)
5.55
4.96
4.38
3.79
3.30
SUM ("S1
Weight
(w)
0.569
0.635
0.552
0.367
0.208
Product
(Nw)
11.38
12.70
11.04
7.34
4.16
46.62
Product
(Nwx)
22.76
21.59
15.46
8.07
3.33
71.21
Product
(Nwx!)
45.52
36.70
21.64
8.88
2.66
115.40
a. See paragraph 9(e), above.
TABLE 21. WEIGHTING FACTOR (W) FOR THE CALCULATED PROBIT (Y).
Y 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
1 0.001 0.001 0.001 0.002 0.002 0.003 0.005 0.006 0.008 0.011
2 0.015 0.019 0.025 0.031 0.040 0.050 0.062 0.076 0.092 0.110
3 0.131 0.154 0.180 0.208 0.238 0.269 0.302 0.336 0.370 0.405
4 0.439 0.471 0.503 0.532 0.558 0.581 0.601 0.616 0.627 0.634
5 0.637 0.634 0.627 0.616 0.601 0.581 0.558 0.532 0.503 0.471
6 0.439 0.405 0.370 0.336 0.302 0.269 0.238 0.208 0.180 0.154
7 0.131 0.110 0.092 0.076 0.062 0.050 0.040 0.031 0.025 0.019
8 0.015 0.011 0.008 0.006 0.005 0 003 0.002 0.002 0.001 0.001
75
-------
Thus:
SE Logio LC50 =
(0.533)'
(1.72 - 1.527)'
67630
0.5
= (0.00764)0.5 = 0.0874.
This result gives rise to a more accurate approximate value for
SE LC50, by substituting the more accurate value of SE log-|Q
LC50 in the previous expression. In our example, SE LC50 =
(52.5) (2.3026) (0.0874) = 10.55.
The LC50 + SE = 52.5% + 10.55.
76
-------
COMPUTER CALCULATION OF LC50s and CONFIDENCE INTERVALS
Computer programs are available from EMSL-Cincinnati for the following
analyses:
1. Binomial
2. Moving average
3. Moving average-angle
4. Probit
5. Trimmed Spearman-Karber
The results obtained from these programs differ only slightly in most
cases, as shown in Table 22, and the LC50s are similar to those obtained
with the graphical method.
Listings for the above programs, together with instructions and sample
data for their use, are included in Appendix E.
These programs also reside on the EPA IBM 370/168 at Research Triangle
Park (NCC). Instructions for the direct transfer of machine readable
copies of the programs to other computers (such as PDP-ll/70s) may be
obtained by contacting the Aquatic Biology Section, EMSL-Cincinnati.
77
-------
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78
-------
SECTION 12
ESTIMATING THE POTENTIAL FOR ACUTE AND CHRONIC
TOXICITY OF WASTE IN RECEIVING WATERS
BASED ON THE LC50 FROM ACUTE TOXICITY (LETHALITY) TESTS
(Prepared by Lee B. Tebo)
To avoid the potential for toxicity in receiving waters, the following
relationship must hold:
IWC = NOEC (Formula 1)
Where: NOEC = No observed effect concentration, expressed as percent
waste.
IWC = Instream (receiving water) waste concentration,
expressed in the same unit.
Several critical considerations are embedded in Formula 1 above:
1. Is the NOEC to be expressed in terms of acute toxicity (lethality) or
chronic toxicity, or both?
2. If the LC50s from acute toxicity (lethality) tests described in this
manual are to be used to estimate the acute or chronic NOECs in
receiving waters, there must be known relationships between the LC50 and
the acutely lethal or chronically toxic concentrations. These
relationships may be expressed in the state water quality standards.
3. A margin of safety, based on varying species sensitivity, should be
provided. The need for a margin of safety may be minimized by testing a
diverse representation of vertebrate and invertebrate species and using
the LC50 of the most sensitive species.
4. What design flow (7Q10, 30Q2, etc.) is to be used in calculating the IWC
in rivers and streams? (7Q10 is lowest average daily flow during any
consecutive seven days in any ten-year period; 30Q2 is the lowest
average daily flow for consecutive 30 days in any given two-year period).
In the following discussion of the principles involved in the use of
the LC50s from acute toxicity (lethality) tests to estimate the acute or
chronic NOECs in receiving waters, the application factors, design flows,
and margins of safety employed are used only as examples, and do not
necessarily represent current Agency policy or guidance. The values used
for specific discharges should be based on the existing water quality
standards, Agency policy and/or guidance (USEPA, 1984b), where applicable,
and current scientific knowledge, and should be applied on a case-by-case
basis.
79
-------
ESTIMATING THE ACUTE NOEC
To employ the acute LC50 to protect the receiving water against acutely
toxic (lethal) concentrations of waste, the use of an (acutely lethal
concentration)/(acute LC50) ratio of 1:3 has been suggested (Hart et al.,
1945; Tebo, 1985). If this ratio is used, Formula 1 becomes:
IWC = u;p (Formula 2)
and 3(IWC) = LC50 (Formula 3)
The generalized form of Formula 3 is:
^p— = LC50 (Formula 4)
Where: AF = An application factor which is the ratio of the acute
or chronic toxicity (NOEC) to the acute LC50, as
described above.
For rivers and streams:
« • TQ^QFT x '°° • iW) (Fo™uU 5)
Where: Qw = Waste flow in a standard unit, such as mgd.
Qr = Receiving water flow (design stream flow) in the same
standard unit.
To protect rivers and streams from acute lethality (acute NOEC), using
the example of an acutely lethal concentration/acute LC50 ratio of 1:3,
Formula 3 above becomes:
T|2IQ»1T < LCSO (Formula 6)
The design stream flow (Qr) for the above determination may be specified
in the state water quality standards or may be based on a determination of
the frequency that the regulatory agency is willing to allow acutely toxic
(lethal) conditions in the receiving waters. For a first approximation, the
7Q10 is frequently used, and indeed this is the design flow presently
specified in many state water quality standards.
The remaining consideration concerns the margin of safety to be provided
to account for varying species sensitivity. If the LC50 is based on the most
sensitive of two species tested (an invertebrate and vertebrate), a safety
margin of one order of magnitude is suggested as a reasonable approximation.
80
-------
Thus, after including a margin of safety of one order of magnitude for
variations in species sensitivity, Formula 6 becomes:
= LC50 (Formula 7)
(Qw + QrJ
ESTIMATING THE CHRONIC NOEC
The IWC for protecting rivers and streams from chronic toxicity
(chronic NOEC) is derived using Formulas 2 - 7 in the same manner as
described above for the acute NOEC. For protection against chronically
toxic concentrations of waste in receiving waters, the National Academy of
Sciences (USEPA, 1973) proposed the use of (chronically toxic
concentration)/(acute LC50) ratios of 1:20 for nonpersistent wastes and
1:100 for persistent wastes. If these chronic ratios are used, depending
on the nature of the waste, Formula 2 becomes:
< LC50 or> IWC. < LC50
and Formula 7 becomes:
20.000(Qw) < LC5Q 100,000(Qw)
(Qw + Qr) LLbU °r' (Qw + Qr)
It is important to recognize that if sampling is inadequate to account
for short term (diel) and long term (annual) variations in waste toxicity,
an appropriate safety factor must be applied to the formulas disucssed
above (USEPA, 19845).
LAKES, IMPOUNDMENTS, AND ESTUARIES
Formula 4 may also be used where a mixing zone is specified. In the
case of lakes, impoundments, and estuaries, a mixing zone must be provided
or receiving water toxicity calculations cannot be performed. By
definition, a mixing zone includes a boundary around the outfall within
which there should be no acutely lethal conditions and outside of which the
waste should not exhibit acute or chronic toxicity. Where a mixing zone is
to be used, there are three steps involved in establishing safe receiving
water concentrations:
1. Establish mixing zone boundaries according to state water quality
standards.
2. Determine the waste concentration at designated mixing zone boundaries.
This can be determined using an appropriate model at design flow
conditions, or determined empirically by conducting a dye study.
3. Calculate the potential for acute or chronic toxicity using Formula 4.
The IWC in Formula 4 is the waste concentration at the appropriate
mixing zone boundary.
If no mixing zone is permitted, the IWC in Formula 4 is equivalent to
100% effluent (effluent at the end of the pipe).
81
-------
SECTION 13
REPORT PREPARATION AND DATA UTILIZATION
The report should contain the following types of information. The
specific content and format of the report, however, may vary on a
case-by-case basis.
1. Introduction - Objectives, how the advance arrangements were made for
the test, persons contacted (state, industry, etc.),
names of investigators, plant location and NPDES permit
number, the name of the receiving water body, and the
date the test was conducted.
2. Plant Operations - Type of plant, operating schedule, final products,
schematic of waste treatment, discharge information,
flows, etc.
3. Effluent and Dilution Water - Sources and Sampling Methods -
a. A detailed description of the effluent, including its source, date
and time of collection, composition, known physical and chemical
properties, and variability.
b. The source of the dilution water, the date and time of its
collection, its chemical characteristics, and a description of any
pretreatment.
4. Test Methods - Description of methods and any deviations from
established methods
a. A description of the test procedure, the definition of the adverse
effect (death, immobility, etc.) used in the test, a description of
the test chambers, including the depth and volume of solution, the
way the test was begun, the number of organisms per treatment, and
the loading. For the flow-through system, indicate the water volume
changes per 24 h in each test chamber.
b. Detailed information about the test organisms, including scientific
name, mean length and weight (where appropriate), age, life stage,
source, history, observed diseases, treatments, and acclimation
procedure.
82
-------
c. The methods used for, and the results of, all chemical analyses.
d. The mean and range of the acclimation temperature and the test
temperature.
5. Results - Should include the following:
a. The results of all chemical analyses of the effluent and dilution
water.
b. A daily record of the number and percentage of organisms in each
test chamber (including the control chambers) that died or showed
the selected "effect."
c. A summary of general observations of other effects or symptoms of
toxicity observed during the test.
d. An LC50 value for the test organisms for the period of interest.
If 100% effluent did not kill or affect 50% or more of the test
organisms, report the percentage of the test organisms killed or
affected by various concentrations of the effluent.
e. When possible, the 95% confidence interval for the LC50, and the
method used to calculate it.
f. Any deviation from the standard test methods.
g. Any other relevant information.
6. Discussion and Recommendations -
a. Prediction of potential acute and chronic toxicity in the receiving
water, based on the IWC method described in Section 12.
83
-------
SELECTED REFERENCES
Abram, F.S.H. 1973. Apparatus for control of poison concentration
in toxicity studies with fish. Water Res. 7:1875-1879.
Bartlett, M.S. 1936. Square root transformation in analysis of
variance. Suppl. J. Royal. Statist. Soc. 3:68-78.
Bengtsson, B.E. 1972. A simple principle for dosing apparatus in
aquatic systems. Arch. Hydrobiol. 70:413-415.
Bennett, B.M. 1952. Estimation of LD50 by moving average angles.
J. Hygiene 50:157-164.
Benoit, D.A., V.R. Mattson, and D.L. Olson. 1982. A continuous-flow
mini-dilutor system for toxicity testing. Water Res. 16:457-464.
Bishop, W.E., R.D. Cardwell, and B.B. Heidolph, eds. 1983. Aquatic
toxicology and hazard assessment. Proceedings of the sixth annual
symposium on aquatic toxicology. ASTM STP 802, American Society for
Testing and Materials, Philadelphia, Pennsylvania.
Branson, D.R., and K.L. Dickson, eds. 1981. Aquatic toxicology and hazard
assessment. Proceedings of the fourth annual symposium on aquatic
toxicology. ASTM STP 737, American Society for Testing and Materials,
Philadelphia, Pennsylvania.
Broderius, S.O. 1983. Analysis of an interlaboratory comparative study of
acute toxicity tests with freshwater aquatic organisms. Environmental
Research Laboratory, U. S. Environmental Protection Agency, Duluth,
, Minnesota. 54 pp.
Brungs, W., and A. Lemke. 1978. Manual for construction and operation
of toxicity-testing proportional diTutors. Environmental Research
Laboratory, U.S. Environmental Protection Agency, Duluth, Mn PB-287-
606-8BE.
Brungs, W.A., and D.I. Mount. 1967. A device for continuous
treatment of fish in holding chambers. Trans. Amer. Fish. Soc. 96:55-57.
Brusick, D.J., and R. R. Young. 1981. IERL-RTP procedures manual: Level 1
environmental assessment biological tests. Industrial Environmental
Research Laboratory, U. S. Environmental Protection Agency, Research
Triange Park, N. Carolina. EPA-600/8-81-024.
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-------
Buikema, A.L. 1983. Inter- and intralaboratory variation in conducting
static acute toxicity tests with Daphnia magna exposed to effluents and
reference toxicants. American Petroleum Institute, API Publ. 4362,
Washington, D.C.
Buikema, A.L., and J. Cairns, Jr., eds. 1980. Aquatic invertebrate
bioassays. ASTM STP 715, American Society for Testing and Materials,
Philadelphia, Pennsylvania.
Buikema, A.L., D.R. Lee, and J. Cairnes, Jr. 1976. A screening bioassay
using Daphnia pulex for refinery wastes discharged into freshwater.
J. Test. Eva!.4(27:119-125
Buikema, A.L., Jr., B.R. Niederlehner, and J. Cairns, Jr. 1982.
Biological monitoring. Part IV - toxicity testing. Water Res. 16:239-262.
Cairns, J, Jr., K.L. Dickson, and A. W. Maki, eds. 1978. Estimating
the hazard of chemical substances to aquatic life. ASTM STP 657,
American Society for Testing and Materials, Philadelphia, Pennsylvania.
Cline, T.F., and G. Post. 1972. Therapy for trout eggs infected
with Saprolegnia. Prog. Fish-Cult. 34:148-151.
Davey, E.W., J.H. Gentile, S.J. Erickson, and P. Betzer. 1970.
Removal of trace metals from marine culture media. Limnol. Oceanogr.
15:486-488.
Davis, H.S. 1953. Culture and diseases of game fishes. University of
California Press, Berkeley. 332 pp.
DeFoe, D.L. 1975. Multichannel toxicant injection system for flow-
through bioassays. J. Fish. Res. Bd. Can. 32:544-546.
DeWoskin, R.S. 1984. Good laboratory practice regulations: a comparison.
Research Triangle Institute, Research Triangle Park, N. Carolina. 63 pp.
Eaton, J. G., R. R. Parrish, and A. C. Hendricks, eds. 1980. Aquatic
toxicology. Proceedings of the third annual symposium on aquatic
toxicology. ASTM STP 707, American Society for Testing and Materials,
Philadelphia, Pennsylvania.
Esenhart, C. 1947. Inverse sine transformation. Techn. Stat.
Analysis, Chapt. 16, McGraw-Hill, New York, New York.
FDA. 1978. Good laboratory practices for nonclinical laboratory studies.
Part 58, Fed. Reg. 43(247):60013-60020, December 22, 1978.
Finney, D.J. 1964. Statistical method in biological assay. 2nd ed.
Hafner Publ. Company, New York, New York. 668 pp.
Finney, D.J. 1971. Probit analysis. 3rd ed. Cambridge University Press,
London. 333 pp.
85
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Finney, D.J. 1978. Statistical method in biological assay. 3rd ed.
Charles Griffin & Co. Ltd, London. 508 pp.
Freeman, R.A. 1971. A constant flow delivery device for chronic
bioassay. Trans. Amer. Fish. Soc. 100:135-136.
Granmo, A., and S.O. Kollberg. 1972. A new simple water flow system
for accurate continuous flow tests. Water Res. 6:1597-1599.
Grothe, D.R., and R.A. Kimerle. 1985. Inter- and intra-laboratory
variability in Daphnia magna effluent toxicity test results. Environm.
Toxicol. Chem. 4(2):189-192.
Harris, E.K. 1959. Confidence limits for the LD50 using the moving
average angle method. Biometrics 15(3):424-432.
Hart, W.B, P. Douderoff, and J. Greenbank. 1945. The evaluation of the
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APPENDICES
Appendices 91
A. Distribution, Life Cycle, Taxonomy, and Culture Methods . . 92
Daphnia (j). magna, D. pulex,) 92
Mysids (Mysidopsis ¥ahia) . 104
Fathead Minnows (Pimephales promelas)
(by Donald J. Klein]~ 112
Silversides (Menldia) (by Douglas P. Middaugh) .... 126
Brine Shrimp 138
B. Dilutor Systems 141
Solenoid and Vacuum Siphon Dilutor Systems 141
Solenoid System Equipment List 145
Vacuum System Equipment List 148
Dilutor Control Panel Equipment List 153
C. Bioassay Mobile Laboratory Plans 154
Tandem-axle Trailer 154
Fifth-wheel Trailer 157
D. Check Lists and Information Sheets 158
Bioassay Field Equipment List 158
Information Check List for On-site Industrial and
Municipal Waste Toxicity Tests 160
Daily Events Log 166
Dilutor Calibration Form 167
Daily Dilutor Calibration Check 168
E. Computer Programs for Calculating the LC50 and
95% Confidence Interval (by James Dryer) 169
Moving Average-angle Program 170
Probit Program 182
Trimmed Spearman-Karber Program 192
Program for Statistical Comparison of LC50s 202
TOXDAT Multi-method Program (Binomial,
Moving Average, and Probit Methods) 205
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APPENDIX A
DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS
1. DAPHNIA (£. MAGNA AND D. PULEX)]
GEOGRAPHICAL AND SEASONAL DISTRIBUTION
J). magna is principally a lake dweller and is restricted to waters in
northern and western North America exceeding a hardness of 150 mg/L (as
CaC03) (Pennak, 1978). 0. pulex (Fig. 14) is found over most of the
North American continent. It is principally a pond dweller, but also is
found in lakes.
Daphnia populations generally are sparce in winter and early spring,
but as the water temperature reaches 6° to 12°C, they increase in
abundance and subsequently may reach population densities as high as 200
to 500 individuals/L (Pennak, 1978). Populations in ponds decline during
the summer months to very low numbers. In the autumn there may be a
second population pulse, followed by a decline to winter lows.
During most of the year populations of Daphnia consist almost
exclusively of females, the males being abundant only in spring or
autumn. Males are distinguished from females by their smaller size,
larger antennules, modified postabdomen, and first legs, which are armed
with a stout hook used in clasping. Production of males appears to be
induced principally by high population densities and subsequent
accumulations of excretory products, and/or a decrease in available
food. These conditions may induce the appearance of sexual (resting)
eggs in cases or ephippia (Fig. 15), which are cast off during the next
molt. It appears that the shift towards male and sexual egg production
is related to the metabolic rate of the parent. Any factor, in addition
to those above, which tends to lower metabolism may be responsible. As a
rule, males and ephippia will not be observed in the lab unless stock
cultures are neglected, or the culture experiences some type of
environmental stress.
LIFE CYCLE
The life span of Daphnia, from the release of the egg into the brood
chamber until the death of the adult, is highly variable depending on the
species and environmental conditions (Pennak, 1978). Generally the life
span increases as temperature decreases, due to lowered metabolic
activity. The average life span of J). magna is about 40 days at 25°C,
and about 56 days at 20°C. The average life span of Daphnia pulex at
20°C is approximately 50 days. Four distinct periods may be recognized
in the life history of Daphnia: (1) egg, (2) juvenile, (3) adolescent,
and (4) adult (Pennak, 1978). Typically, a clutch of 6 to 10 eggs is
released into the brood chamber. The eggs hatch in the brood chamber and
^Taken from Weber and Peltier, 1981.
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the juveniles, which are already similar in form to the adults,are released
in approximately two days when the female molts (casts off her exoskeleton
or carapace). The time required to reach maturity (produce their first
offspring) varies from six to 10 days and also appears to be dependent on
body size. The growth rate of the organism is greatest during its juvenile
stages (early instars), and the body size may double during each of these
stages. D. pulex has three to four juvenile instars, whereas £. magna has
three to Tive instars. Each instar stage is terminated by a molt. Growth
occurs immediately after each molt while the new carapace is still
elastic.
Following the juvenile stages, the adolescent period is very short, and
consists of a single instar. It is during the adolescent instar that the
first clutch of eggs reaches full development in the ovary. Generally,
eggs are deposited in the brood chamber within minutes after molting, and
the young which develop are released just before the next molt.
JJ. magna usually has six to 22 adult instars, and JJ. pulex has 18 to
25. In general, the duration of instars increases with age, but also
depends on environmental conditions. A given instar generally lasts
approximately two days under favorable conditions, but when conditions are
unfavorable, it may last as long as a week.
Four events take place in a matter of a few minutes at the end of each
adult instar: (1) release of young from the brood chamber to the outside,
(2) molting, (3) increase in size, and (4) release of a new clutch of eggs
into the brood chamber. The number of young per brood is highly variable
for Daphnia, depending primarily on food availability and environmental
conditions. D. magna and £. pulex may both produce as many as 30 young
during each aFult instar, but more commonly the number is six to 10. The
number of young released during the adult instars of FJ. pulex reaches a
maximum at the tenth instar, after which there is a gradual decrease
(Anderson and Zupancic, 1937). The maximum number of young produced by
D. magna occurs at the fifth adult instar, after which it decreases
TAnderson and Jenkins, 1942).
MORPHOLOGY AND TAXONOMY
JJ. pulex attains a maximum length of approximately 3.5 mm, whereas JJ.
magna are much larger, and attain a length of 5.0 or 6.0 mm. However,
these two species can be differentiated with certainty only by determining
the size and number of spines on the postabdomenal claws (Fig. 16), using a
dissecting or compound microscope (Pennak, 1978).
CULTURING METHODS
1. Sources of Organisms
Daphnia are available from the Environmental Monitoring & Support
Laboratory-Cincinnati, from other EPA, state or private laboratories in
which Daphnia are used in toxicity tests, and from commercial biological
supply houses. Only a small number of organisms (20-30) are needed to
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Figure 14. Anatomy of female Daphnia pulex (De Geer), X70; A, antenna; BC,
brood chamber; H, heart; INT, intestine; L, legs; 0V, ovary; P,
postabdomen; PC, postabdominal claw. (From Pennak, 1978).
Figure 15. Daphnia maqna with
ephippium (sexual egg
case). (From Doma, 1979)
Figure 16. Daphnia postabdomin.
Daphnia pulex: A, postabdomen;
B, postabdominal claw. Daphnia
magna: C, postabdomen;
D, postabdominal claw.
(From Pennak, 1978)
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start a culture. [).. pulex is preferred over J3. magna by some biologists
because it is more widely distributed than JJ. magna, and is easier to
culture. However, the neonates of j). magna are larger and, therefore,
somewhat easier to use in toxicity tests. Guidance on the source and
species of Daphnia to be used by a permittee for effluent toxicity tests
should be obtained from the permitting authority, and the species used
should be verified by a taxonomic authority.
2. Culture Media
Although Daphnia stock cultures can be successfully maintained in some
tap waters, well waters, and surface waters, use of synthetic water as the
culture medium is recommended because (1) it is easily prepared, (2) it is
of known quality, (3) it produces predictable results, and (4) allows
adequate growth and reproduction. Reconstituted hard water (hardness: 160
to 180 mg/L CaC03) is recommended for D. magna, whereas moderately hard
reconstituted water (hardness: 80 to 9TJ mg/L CaC03) is recommended for
JJ. pulex .
The preparation of the media is as follows:
a. Hard reconstituted water medium (for ID. magna)
Reagent Concentration (mg/L)
NaHC03 192.0
CaS04 • 2H20 120.0
MgS04 120.0
KC1 8.0
b. Moderately-hard reconstituted water medium (for D_. pulex)
Reagent Concentration (mg/L)
NaHC03 96.0
CaS04 • 2H20 60.0
MgS04 60.0
KC1 4.0
The compounds are dissolved in distilled or deionized water and the
media are vigorously aerated for several hours before using. The initial
pH of the media is approximately 8.0, but it will rise as much as 0.5 unit
as the Daphnia population increases. Although Daphnia can survive over a
wide pH range, the optimum range is 7.0 to 8.6 (Lewis and Weber, 1985).
Since the pH usually remains within this range, there is no need to monitor
or adjust it.
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To make up media as needed (usually once a week for each set of five
stock cultures), prepare stock solutions of KCL, MgSCty and NaHCOa as
follows (these solutions will provide a six to 12 months supply):
KCL Stock Solution:
a. Place 8 g of crystalline, reagent grade KCL in a 1-L volumetric
flask.
b. Bring the volume to 1 L with distilled water.
c. Store in a 1-L polyethylene bottle.
MgS04 Stock Solution:
a. Place 120 g of reagent grade, anhydrous MgSCty powder in a 1-L
volumetric flask.
b. Bring the volume to 1 L with distilled water.
c. Store in a 1-L polyethylene bottle.
NaHC03 stock solution:
a. Place 96 g reagent grade NaHCO^ powder in a 1-L volumetric flask.
b. Bring the volume to 1 L with distilled water.
c. Store in a 1-L polyethylene bottle.
To make hard reconstituted water for Daphnia magna:
a. Place 18.2 L of deionized or distilled water in a 20-L carboy that
has been thoroughly cleaned (see Section 4 on cleaning glassware).
b. Add 2.4 g of CaSC>4 • 2^0 to 1 L of deionized or distilled
water in a 2-L beaker, and stir until dissolved. Add this solution
to the carboy and mix well.
c. Add each of the following stock solutions to the carboy and mix well
after each addition:
(1) 20 mL KCL stock solution
(2) 20 mL MgS04 stock solution
(3) 40 mL NaHCOs stock solution.
d. Vigorously aerate the medium in the carboy for 2 h.
To make moderately-hard reconstituted water for Daphnia pulex:
a. Place 18.6 L of deionized or distilled water in a 20-L carboy which
has been thoroughly cleaned (according to instructions in Section 4).
b. Add 1.2 g of CaS04 • 21^0 to 1 L of deionized or distilled
water in a 2-L beaker and stir until dissolved. Add this solution to
the carboy.
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c. Add each of the following stock solutions to the carboy and mix well
after each addition:
(1) 10 ml KCL stock solution
(2) 10 nt MgS04 stock solution
(3) 20 ml NaHC03 stock solution
d. Vigorously aerate the medium in the carboy 2 h.
3. Feeding: Quantity and Frequency
Food preparation and feeding are of great importance in Daphnia
culturing. Daphnia can be cultured using algae or a prepared food
consisting of a suspension of a trout chow, alfalfa, and yeast. The latter
diet is easily prepared and provides adequate nutrition for organisms used
in acute toxicity tests (Winner et_ aT_., 1977).
Trout chow, alfalfa, and yeast:
The food is prepared as follows:
a. Place 6.3 g of trout chow pellets, 2.6 g of dried yeast, and 0.5 g
of dried alfalfa in a blender.
NOTE: The trout chow must conform to Fish & Wildlife Service
Specification PR(ll)-78, and can be obtained through livestock
feed stores. Dried yeast, such as Fleischmann's dried yeast,
can be obtained at any grocery store. Dried alfalfa can be
obtained at health food stores.
b. Add 500 ml of distilled or deionized water.
c. Mix at high speed for 5 min.
d. Place in a refrigerator and allow to settle for 1 h.
e. Decant the top 300 mL and save; discard the remainder.
f. Place 30 to 50 mL aliquots in small (50 to 100 mL) polyethylene
bottles with screw caps and freeze.
g. Thaw portions as needed. After thawing, keep in refrigerator for a
maximum of one week, then discard.
Feed 1.5 mL of prepared food per 1000 mL of medium, three times per
week (i.e. Monday, Wednesday, Friday). There may be some excess food in
the medium at this rate of feeding, but if the medium is aerated
continuously and replaced each week, as discussed below, this should cause
no problems.
An alternate method3 of feeding Daphnia involves the use of
Selenastrum capricornutum Printz, the green alga commonly employed in the
aBased on information provided by Dr. Thomas Waller and James Lazorchak,
Environmental Sciences Program, University of Texas at Dallas, P.O. Box
688, MS BE 22, Richardson, Texas 75080 (214-690-2966 or 2970).
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algal growth potential test. The algal cultures are maintained as
described in Miller £t jTL (1978). Algal stock cultures are started each
Monday, Wednesday, Friday and Sunday. The objectives of the culture
procedure are to produce seven-day old cultures that contain four to five
million algal cells per mL, and two to four day old cultures that contain
one to three million cells per ml. The algal food is prepared and fed
Monday, Wednesday, and Friday, as described below:
a. Volumes of seven-day-old and three-day-old algal cultures are
combined in a ratio of two volumes to one, respectively (for
example, 1000 ml seven-day-old culture and 500 mL of three-day-old
culture.
b. The algae are sedimented by centrifugation and resuspended in a
volume of moderately hard or hard reconstituted water (such as used
for the Daphnia cultures) equal to the original volume of
seven-day-old algal cultures from which the algae were concentrated.
c. The resuspended algae should number three to four million cells
per mL. A sufficient volume of the cell suspension is added to the
Daphnia stock cultures every day to provide approximately 100,000
algal cells per mL of Daphnia culture. This would involve the
addition of approximately 25 mL of cell suspension to 1 L of Daphnia
stock culture.
4. Culture Temperature
Daphnia can be cultured successfully over a wide range of temperatures,
but should be protected from sudden changes in temperature, which may cause
death. The optimum temperature is approximately 20°C, and if ambient
laboratory temperatures remain in the range of 18 to 26°C, normal growth
and reproduction of Daphnia can be maintained without special temperature
control equipment.
5. Illumination
The variations in ambient light intensities (50 to 100 ft c) and
prevailing day/night cycles in most laboratories do not seem to affect
Daphnia growth and reproduction significantly. However, a minimum of 16 h
of illumination should be provided each day.
6. Culture Vessels
Culture vessels of clear glass are recommended since they allow easy
observation of the Daphnia. A practical culture vessel is an ordinary 3-L
glass beaker, which can be filled with approximtely 2.75 L of medium
(reconstituted water). Maintain several (at least five) culture vessels,
rather than only one. This will ensure back-up cultures so that in the
event of a population "crash" in one or several chambers, the entire
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Daphnia population will not be lost. If a 3-L vessel is stocked with
30 Daphnia, it will provide approximately 300 young each week.
Initially, all culture vessels should be washed well (see Section 5).
After the culture is established, clean each chamber weekly with distilled
or deionized water and wipe with a clean sponge to rid the vessel of
accumulated food and dead Daphnia (see section on culture maintenance
below). Once per month wash each vessel with detergent during medium
replacement. Rinse three times with tap water and then with culture medium
to remove all traces of detergent.
7. Aeration
Daphnia can survive when the dissolved oxygen concentration is as low
as 3 mg/L, but do best when the level is above 6 mg/L. Each culture vessel
should be continuously and gently aerated. This is best accomplished by
using air stones. The air can come from either an aquarium air pump or
from a general laboratory compressed air supply. If the laboratory air
supply is used, first pass it through a flask full of cotton batting to
filter out oil or other contaminants.
8. Weekly Culture Media Replacement
Careful culture maintenance is essential. The medium in each stock
culture vessel should be replaced each week with fresh medium. This is
best accomplished as follows:
a. Siphon the old medium out, using plastic tubing (6 mm) covered with
fine mesh netting around the open end.
b. Retain 1/10 (300 ml) of the orginial medium containing the Daphnia
population.
c. Pour this (old) medium containing the Daphnia into a temporary
holding vessel.
d. Clean the culture vessel as described above.
e. Fill the newly-cleaned vessel with fresh medium. Gently transfer
(by pouring) the contents of the temporary holding vessel (old
medium and Daphnia) into the vessel containing the fresh medium.
If the medium is not replaced weekly, waste products will accumulate,
which could cause a population crash or the production of males and/or
sexual eggs.
Daphnia populations should be thinned weekly to about 30 adults per
stock vessel to prevent over-crowding, which may cause a population crash,
or the production of males and/or ephippia. A good time to thin the
populations is during medium replacement. To transfer Daphnia use a 15-cm
disposable, jumbo bulb pipette, or 10-mL "serum" pipette that has had the
delivery end cut off and fire polished. The diameter of the opening should
be approximately 5 mm. When using a serum pipette, a pipette bulb, such as
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a PropipetteR, or (MopetR) portable, motorized pipetter, provides the
controlled suction needed when selectively collecting Daphnia.
Liquid containing adult £. pulex and D. magna can be poured from one
container to another without risk of air becoming trapped under their
carapaces. However, the very young Daphnia are much more susceptible to
air entrapment and for this reason should be transfered from one container
to another using a pipette. The tip of the pipette should be kept under
the surface of the liquid when the Daphnia are released.
Each culture vessel should be covered with a clear plastic sheet or
glass plate to exclude dust and dirt and minimize evaporation.
TEST ORGANISMS
Daphnia magna or Daphnia pulex 24 h or less in age (neonates, or first
instars) are to be used in the tests. To obtain the necessary number of
young for a test, remove adult females bearing embryos in their brood
pouches from the stock cultures 24 h preceding the initiation of the test,
and place them in 400 ml beakers containing 300 ml of medium and 0.5 mL of
prepared food (see culturing method above). The young that are found in
the beakers the following day are used for the toxicity test. Five
beakers, each containing 10 adults, usually will supply enough first
instars for one toxicity test.
Since the appearance of ephippia in cultures generally is indicative of
unfavorable conditions, Daphnia used for toxicity tests should not be taken
from cultures that are producing ephippia.
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SELECTED REFERENCES
Abel, P.O. 1974. Toxicity of synthetic detergents to fish and aquatic
invertebrates. 0. Fish Biol. 6:279-298.
Adema, D.M.M. 1978. Daphnia magna as a test animal in acute and chronic
toxicity tests. Hydrobiol. 59(2):125-134.
Alibone, M.R., and P. Fair. 1981. The effects of low pH on the
respiration of Daphnia magna Straus. Hydrobiol. 85:185-188.
Anderson, B.G., and J.C. Jenkins. 1942. A time study of the events in the
life span of Daphnia pulex. Biol. Bull. 83:260-272.
Anderson, B.G., and L.J. Zupancic, Jr. 1937. Growth and variability in
Daphnia pulex. Biol. Bull. 89:444-463.
Attar, E.N., and E.J. Maly.-1982. Acute toxicity of cadmium, zinc and
cadmium-zinc mixtures to Daphnia magna. Arch. Environ. Contain. Toxicol.
11:291-296.
Bellavere, C., and J. Gorbi. 1981. A comparative analysis of acute
toxicity of chromium, copper and cadmium to Daphnia magna, Biomphalaria
glabrata, and Brachydanio rerio." Environ. Technol. Letters 2:119-128.
Berge, W. F. Ten. 1978. Breeding Daphnia magna. Hydrobiol.
59(2):121-123.
Biesinger, K.E., and G.M. Christensen. 1972. Effects of various metals on
survival, growth, reproduction, and metabolism of Daphnia magna. J. Fish.
Res. Bd. Can. 29:1691-1700.
Buikema, A.L., Jr., J.G. Geiger, and D.R. Lee. 1980. Daphnia toxicity
tests. In: A.L. Buikema, Jr., and John Cairns, Jr., eds., Aquatic
Invertebrate Bioassays, ASTM STP 715, American Society for Testing and
Materials, Philadelphia, Pennsylvania, pp. 48-69.
Buikema, A.L., Jr., D.R. Lee, and J. Cairns, Jr. 1976. A screening
bioassay using Daphnia pulex for refinery wastes discharged into
freshwater. J. Testing Eval. 4(2):119-125.
Canton, J.H., and D.M.M. Adema. 1978. Reproducibility of short-term and
reproduction toxicity experiments with Daphnia magna and comparison of
the sensitivity of Daphnia magna with Daphnia pulex and Daphnia cucullata
in short-term experiments. Hydrobiol. 59(2):135-140.
Carlson, R.M., and R. Caple. 1977. Chemical/biological implications of
using chlorine and ozone for disinfection. U.S. Environmental Protection
Agency, Environmental Research Laboratory, Duluth, Minnesota 55804.
EPA-600/3-77-066. 87 pp.
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Carvalho, G.R., R.N. Hughes. 1983. The effect of food availability, female
culture-density and photoperiod on ephippia production in Daphnia magna
Straus (Crustacea: Cladocera). Freshw. Biol. 13:37-46.
Davis, P., and G.W. Ozburn. 1969. The pH tolerance of Daphnia pulex
(Leydig, emend., Richard). Can. J. Zool. 47:1173-1175.
Doma, S. 1979. Ephippia of Daphnia magna Straus - A technique for their
mass production and quick revival. Hydrobiol. 67(2):183-188.
France, R.L. 1982. Comment on Daphnia respiration in Low pH water.
Hydrobiol. 94:195-198.
Geiger, J. G., A.L. Buikema, Jr., and J. Cairns, Jr., J. 1980.
A tentative seven-day test for predicting effects of stress on
populations of Daphnia pulex. In: J.G. Eaton, P.R. Parrish, and A.C.
Hendricks, eds., ASTM STP 707, American Society for Testing and
Materials, Philadelphia, Pennsylvania, pp. 13-26.
Gophen, M., and Gold, B. 1981. The use of inorganic substances to
stimulate gut evacuation in Daphnia magna." Hydrobiol. 80:43-45.
Havas, M. 1981. Physiological response of aquatic animals to low pH.
In: R. Singer, ed., Effects of Acidic Precipitation on Benthos, North
American Benthological Society, Box 878, Springfield, Illinois 62705).
pp. 49-65.
How, M. J. 1980. The application and conduct of ring tests in aquatic
toxicology. Water Res. 14:293-296.
Ingersoll, C.G., and R.W. Winner. 1982. Effect on Daphnia pulex (De Geer)
of daily pulse exposure to copper or cadmium. Environ. Toxicol. Chem.
1:321-327.
LeBlanc, G. A. 1982. Laboratory investigation into the development of
resistance of Daphnia magna (Straus) to environmental pollutants.
Environ. Pollut. (Ser. A) 27:309-322.
Lee, D. R., and A.L. Buikema, Jr. 1979. Molt-Related sensitivity of
Daphnia pulex in toxicity testing. J. Fish. Res. Bd. Can. 36:1129-1133.
Leonhard, S. L., and S.C. Lawrence. 1981. Daphnia magna (Straus), Daphnia
pulex (Leydig) Richard." In: S. G. Lawrence, ed., Manual for the
Culture of Selected Freshwater Invertebrates, Can. Spec. Publ. Fish.
Aquat. Sci. 54:31-50.
Lewis, P.A., and C.I. Weber. 1985. A study of the reliability of Daphnia
acute toxicity tests. Proc. Seventh Annual Symposium on Aquatic
Toxicology, ASTM STP 854, American Society for Testing and Materials,
Philadelphia, Pennsylvania, pp. 73-86.
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Maki, A.M., and W.E. Bishop. 1979. Acute toxicity studies of surfactants
to Daphnia magna and Daphnia pulex. Arch. Environ. Contam. Tox.
8:599-622.
Miller, W.E., J.C. Greene, and T. Shiroyama. 1978. The Selenastrum
capricornutum Printz, algal assay bottle test. U. S. Environmental
Protection Agency, Environmental Research Laboratory, Corvallis, Oregon.
EPA-600/9-78-018. 126 pp.
Nebeker, A. V. 1982. Evaluation of a Daphnia magna renewal life-cycle
test method with silver and endosulfan. Water Res. 16:739-744.
Parent, S., and R.D. Cheetham. 1980. Effects of acid precipitation on
Daphnia magna. Bull. Environ. Contam. Toxicol. 25:298-304.
Peltier, W.H. 1978. Methods for measuring the acute toxicity of effluents
to aquatic organisms. 2nd ed. Environmental Monitoring and Support
Laboratory, U. S. Environmental Protection Agency, Cincinnati, Ohio.
July, 1978. EPA-600/4-78-012.
Pennak, R.W. 1978. Fresh-water invertebrates of the United States. 2nd ed.
John Wiley & Sons, New York, NY.
Pucke, S.C. 1981. Development and standardization of Daphnia culturing
and bioassays. M.S. Thesis, University of Cincinnati.
Schultz, T.W., S.R. Freeman, and N.N. Dumont. 1980. Uptake, depuration
and distribution of selenium in Daphnia and its effects on survival and
ultrastructure. Arch. Environ. Contam. Toxicol. 9:23-40.
USEPA. 1975. Methods for acute toxicity tests with fish,
macroinvertebrates, and amphibians. U.S. Environmental Protection
Agency, Environmental Research Laboratory, Duluth, Minnesota.
EPA-660/3-75-009. 61 pp.
Walton, W.E., S.M. Compton, J.D. Allan, and R.E. Daniels. 1982. The
effect of acid stress on survivorship and reproduction of Daphnia pulex
(Crustacea:Cladocera). Can. J. Zool. 60:573-579.
Weber, C.I., and W.H. Peltier. 1981. Effluent toxicity screening test
using Daphnia and mysid shrimp. Environmental Monitoring and Support
Laboratory, U. S. Environmental Protection Agency, Cincinnati, Ohio.
Winner, R.W. 1984. Selenium effects on antennal integrity and chronic
copper toxicity in Daphnia pulex (deGreer). Bull. Environ. Contam.
Toxicol. 33:605-611.
Winner,. R.W., T. Keeling, R. Yeager, and M.P. Parrel!. 1977. Effect of
food type on the acute and chronic toxicity of copper to Daphnia magna.
Freshw. Biol. 7:343-349.
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APPENDIX A
DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS
2. MYSIDS (MYSIDOPSIS BAHIA)1
DISTRIBUTION
Mysids (Fig. 17) are primarily coastal water organisms and occur in
estuaries along the Atlantic, Gulf and Pacific coasts. They are an
important link in estuarine and marine food chains and serve as the primary
food for many species of fish (Molenock, 1969; Shaefer, 1970; Jacobs and
Grant, 1974; Nimmo et jil_., 1977; Price, 1982). Mysidopsis almyra, M.
bahia, fl. bigelowi, Metamysidopsis elongata, and Neomysis americana have
been used extensively in toxicity tests (Nimmo and Hamaker, 1982). The
mysids have been called "opossum shrimp" because the female carries its
young in a brood pouch while they develop.
LIFE CYCLE
In laboratory studies, it has been observed that females of ^. bahia
reach sexual maturity in 12 to 20 days, depending on water temperature and
diet (Nimmo et^ &]_., 1977). Unlike Daphnia, the eggs will not develop
unless fertilized. Brood pouches form when the female reaches the age of
13 to 15 days, and young are released in 17 to 20 days. The number of eggs
produced is a direct function of body length. Mature females may produce
as many as 20 young per brood, but average five to seven. A new brood is
produced every four to seven days. The juveniles are planktonic for the
first 24 h and then settle to the bottom, orient to the current, and
actively pursue food organisms such as Artemia. Carr et aj_. (1980)
reported that the stage in the life cycle of M_. almyra most sensitive to
drilling mud was the first juvenile molt, which occurs between 24 and 48 h
after release from the brood pouch.
MORPHOLOGY AND TAXONOMY
Adults of j^. bahia range in length from 4.4 mm to 9.4 mm (Molenock,
1969), measured from the anterior margin of carapace to the end of
uropods. Living organisms are usually transparent, but may be tinted
yellow, brown or black. Mysidopsis bahia differs from the other two
species of Mysidopsis (ML almyra and fl. bigelowi) by the armature of the
telson, the spines on the uropod, and an unsegmented antennal scale
(Molenock, 1969)(Figs. 17 and 18).
aTaken from Weber and Peltier, 1981.
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antennal scale
dorsal process
8 thoracic iimb pleopoas
loracic segments dorsal process
Figure 17. Lateral and dorsal view of a typical mysid.(From Stuck et al.,
1979).
Figure 18. Morphological characteristics used in mysid identification
(Mysidopsis bahia). A, antenna 1, ventral male; B, antenna 2;
C, telson; D, right uropod, dorsal. Scale lines 0.5 mm in
length. (From Molenock, 1969).
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CULTURE METHODS
1. Sources of Organisms
Mysids can be obtained from EPA laboratories, or commercial sources,
or can be collected from estuaries. Since many species of mysids may be
present at a given collection site, the identification of the organisms
selected for culture should be verified by an experienced taxonomist.
The permittee should consult the permitting authority for guidance on the
source of test organisms.
2. Culture Vessels
Stock cultures are maintained in a recirculating static system in
200-L aquaria (Ward, 1984). Each aquarium is equipped with a
commercially-available, under-gravel filter which is covered with two to
five cm of small, smooth, crushed oyster-shell fragments (four to 15 mm
in length) or dolomite. Both are commercially available and should be
washed and autoclaved before use.
The culture medium (natural or synthetic sea water) is added to the
aquarium containing the shells and conditioned (aged) for two to three
weeks before mysids are introduced. During this period the water is
circulated through the under-gravel filter.
The tops of the two bubble-up standpipes from the filters are placed
immediately above the water surface. The rate of water flow through the
filters should be sufficient to establish a moderate current in the
aquarium. This will be evident in the orientation of the mysids, which
commonly align themselves with the current.
3. Culture Media
Use filtered (0.45 urn pore diameter) natural sea water or
reconstituted sea water prepared at 20 to 25 ppt by adding artificial
marine salts, such as Instant OceanR, or equivalent, to distilled or
deionized water. It should be noted that mysids are sensitive to
nitrite, and will not survive or reproduce in water containing more than
0.05 mg nitrite/L. The water can be checked for this ion by using an
inexpensive (color comparison) test kit that is available at most
aquarium supply stores.
The culture media should be aged to allow the build-up of bacteria in
the substrate. To expedite the aging process, add 5 mL of a concentrated
suspension of 48-h old Artemia daily. After seven days, the suitability
of the new medium is checked by adding 20 mysids. If the organisms
survive for 96 h, the culture medium is ready for stocking.
4. Culture Conditions
Maintain the temperature within a range of 24°C to 26°C for
reproduction. Provide 12 to 16 h illumination daily at 50 to 100 ft c.
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Maintain dissolved oxygen above 60% saturation by vigorous aeration with
an air stone.
5. Feeding
Frequent feeding of live food is necessary to prevent cannibalism of
the young by the adults. Newly hatched brine shrimp (Artemia )are fed to
mysids twice daily. Tropical fish food flakes (such as Tetra-MinR
flakes) may also be used to supplement the brine shrimp diet.
6. Preparation of Brine Shrimp (Artemia)
For methods of preparing brine shrimp, see Appendix A.5.
7. Culture Maintenance
To avoid excessive build up of algal growths, periodically scrape the
walls of the aquaria and standpipes, and turn over the shell substrate to
cover the existing algae. In the static culture system, the mysid
population tends to build to a peak and then undergoes a dramatic die off
(crash) every several months. Population crashes can be prevented by
removing 10% of the population every two weeks, or by transferring the
brood stock to new medium in clean tanks every two months. If a decline
in the brood population is observed before the scheduled date of transfer
to new media, the stock should be transfered as soon as possible.
8. Production Level
At least four aquaria should be maintained to ensure a sufficient
number of organisms on a continuing basis. If each aquarium is initially
stocked with 200 adults, they will provide several thousand test
organisms per month.
TEST ORGANISMS
Juvenile Mysidopsis almyra, hi. bahia, M_. bigelowi, or Neomysis
americana, one to five days old, are used in the tests. To obtain the
necessary number of young for a test, remove about 200 adult females
(bearing embryos in their brood pouch) from the stock cultures (one adult
female for each juvenile required), and place them in a large (10 cm
X 15 cm), standard, fish transfer net that is partially submerged in an
8-L aquarium containing 4 L of culture medium removed from a brood stock
tank. As they are released from the brood pouches, the juveniles will
drop through the fish net into the aquarium, thus preventing canibalism
by the adults. The adults and juveniles in the aquarium must be fed
twice daily with Artemia. The adults are allowed to remain in the net
for 48 h, and are then returned to the stock tanks. The juveniles that
are produced in the small tank may be used in toxicity tests over a
five-day period.
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SELECTED REFERENCES
Anderson, J.W., J.M. Neff, B.A. Cox, H.E. Tatem, and G.H. Hightower. 1974.
Characteristics of dispersions and water-soluble extracts of crude and
refined oils and their toxicity to estuarine crustaceans and fish.
Mar. Biol. 27:75-88.
Anonymous. 1979. Test 6: Mysidopsis bahia life cycle. Fed. Reg.
44(53):16291.
Astthorsson, O.S., R. Ralph. 1984. Growth and moulting of Neomysis
integer (Crustacea: Mysidacea). Mar. Biol. 79:55-61.
Bahner, L.H., C.D. Craft, and D.R. Nimmo. 1975. A saltwater
flow-through bioassay method with controlled temperature and salinity.
Progr. Fish-Cult. 37:126-129.
Bahner, L.H., A.J. Wilson, Jr., J.M. Sheppard, J.M. Patrick, Jr., L.R.
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93:189-194.
Buikema, A.L., Jr., B.R. Niederlehner, and J. Cairns, Jr. 1981. The
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Toxicol. 10:231-240.
Carr, R.S., L.A. Reitsema, and J.M. Neff. 1980. In: Proceedings of
Research on Environmental Fate and Effects of Drilling Fluids and
Cuttings. Vol. II, Amer. Petrol. Inst. pp. 944-960.
Cripe, G.M., D.R. Nimmo, and T.L. Hamaker. 1981. Effects of two
organophosphate pesticides on swimming stamina of the mysid Mysidopsis
bahia. In: Vernberg, F.J., A. Calabrese, F.P. Thurberg, and W.B.
Vernberg, eds., Biological Monitoring of Marine Pollutants. Academic
Press, New York, pp 21-36.
Farrell, D.H. 1979. Guide to the shallow-water mysids from Florida.
Fla. Dept. Environ. Reg., Techn. Ser. 4(l):l-69.
Fotheringham, N., and S.L. Brunenmeister. 1975. Common marine
invertebrates of the northwestern Gulf coast. Gulf Publ. Co., Houston,
Texas.
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Gentile, S.M., J.H. Gentile, J. Walker, and J.F. Heltshe. 1982.
Chronic effects of cadmium on two species of mysid shrimp: Mysidopsis
bahia and M. bigelowi. Hydrobiol. 93:195-204.
Heard, R.W. 1982. Guide to the common tidal marsh invertebrates of the
northeastern Gulf of Mexico. Publ. No. MASGP-79-004, Mississippi-
Alabama Sea Grant Consortium, Ocean Springs, Mississippi.
Jacobs, F., and G.C. Grant. 1974. Acute toxicity of unbleached kraft
mill effluent (UKME) to the Oppossum Shrimp, Neomysis americana Smith.
Water Res. 8:439-445.
Jensen, J.P. 1958. The relation between body size and number of eggs in
marine malacostrakes. Meddr. Danm. Fisk.-og Havunders 2:1-25.
Johns, D.M., W.J. Berry, and W. Walton. 1981. International study on
Artemia. XVI. Survival, growth and reproductive potential of the mysid
Mysidopsis bahia Molenock fed various geographical strains of the brine
lysi
shri
shrimp Artemia. J. Exp. Mar. Biol. Ecol. 53:20-9-219.
Lawler, A.R., and S.L. Shepard. 1978. Procedures for eradication of
hydrozoan pests in closed-system mysid culture. Gulf Res. Rept.
6:177-178.
Mauchline, J. 1980. The biology of mysids. Adv. Mar. Biol. 18:3-369.
Molenock, J. 1969. Mysidopsis bahia, a new species of mysid (Crustacea:
Mysidacea) from Galveston Bay, Texas. Tulane Stud. Zool. Bot.
Morgan, M.D. 1982. The ecology of Mysidacea. Developments in
hydrobiology 10. W. Junk, Publ., The Hague, Netherlands. 232 pp.
Nimmo, D.R., L.H. Banner, R.A. Rigby, J.M. Sheppard, and A.J.
Wilson, Jr. 1977. Mysidopsis bahia: an estuarine species suitable for
life-cycle toxicity tests to determine the effects of a pollutant. In:
F.L. Mayer and J.L. Hamelin, eds., Aquatic Toxicology and Hazard
Evaluation, ASTM STP 634, American Society for Testing and Materials,
Philadelphia, Pennsylvania, pp. 109-111.
Nimmo, D.R., and T.L. Hamaker. 1982. Mysids in toxicity testing - a
review. Hydrobiol. 93:171-178.
Nimmo, D.R., T.L. Hamaker, J.C. Moore, and C.A. Sommers. 1979. Effect
of diflubenzuron on an estuarine crustacean. Bull. Environm. Contam.
Toxicol. (22):767-770.
Nimmo, D.R., T.L. Hamaker, E. Matthews, and J.C. Moore. 1981. An
overview of the acute and chronic effects of first and second
generation pesticides on an estuarine mysid. In: Vernberg, F.J., A.
Calabrese, F.P. Thurberg, and W.B. Vernberg, eds., Biological
Monitoring of Marine Pollutants. Academic Press, New York. pp. 3-19.
109
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Nimmo, D.R., T.L. Hamaker, E. Matthews, and W.T. Young. 1982. The long-
term effects of suspended participates on survival and reproduction of
the mysid shrimp, Mysidopsis bahia, in the laboratory. In: G.F. Mayer,
ed., Ecological Stress and the New York Bight: Science and Management.
Estuarine Res. Found., Columbia, S. Carolina, pp. 41-50.
Nimmo, D.R., T.L. Hamaker, J.C. Moore, and R.A. Wood. 1980. Acute
and chronic effects of Dimilin on survival and reproduction of
Mysidopsis bahia. In: J.6. Eaton, P.R. Parrish, and A.C. Hendricks,
eds., ASTM STP 707, American Society for Testing and Materials,
Philadelphia, Pennsylvania, pp. 366-376.
Nimmo, D.R., T.L. Hamaker, and C.A. Sommers. 1978a. Culturing the mysid
(Mysidopsis bahia) in flowing sea water or a static system. In:
Bioassay Procedures for the Ocean Disposal Permit Program, U.S.
Environmental Protection Agency, Environmental Research Laboratory,
Gulf Breeze, Florida. EPA-600/9-78-010. pp. 59-60.
Nimmo, D.R., T.L. Hamaker, and C.A. Sommers. 1978b. Entire life cycle
toxicity test using mysids (Mysidopsis bahia) in flowing water. In:
Bioassay Procedures for the Ocean Disposal Permit Program, U.S.
Environmental Protection Agency, Environmental Research Laboratory,
Gulf Breeze, Florida. EPA-600/9-78-010. pp. 64-68.
Nimmo, D.R., and E.S. Hey, Jr. 1982 Culturing and chronic toxicity of
Mysidopsis bahia using artificial seawater. Office of Toxic Substances,
U.S. Environmental Protection Agency, Washington, DC., Publ. PA 902.
Nimmo, D.R., R. A. Rigby, L.H. Bahner, and J.M. Sheppard. 1978. The
acute and chronic effects of cadmium on the estuarine mysid, Mysidopsis
bahia. Bull. Environm. Contam. Toxicol. 19(l):80-84
Peltier, W.H. 1978. Methods for measuring the acute toxicity of
effluents to aquatic organisms. 2nd ed. Environmental Monitoring and
Support Laboratory, U. S. Environmental Protection Agency, Cincinnati,
Ohio. July 1978, EPA-600/4-78-012.
Price, W.W. 1982. Key to the shallow water Mysidacea of the Texas coast
with notes on their ecology. Hydrobiol. 93(1/2) :9-21.
Reitsema, L.A. 1981. The growth, respiration, and energetics of
Mysidopsis almyra (Crustacea; Mysidacea) in relation to temperature,
salinity, and hydrocarbon exposure. Ph.D. thesis, Texas A & M
University, College Station, Texas.
Reitsema, L.A., and J.M. Neff. 1980. A recirculating artificial
seawater system for the laboratory culture of Mysidopsis almyra
(Crustacea; Pericaridea). Estuaries 3:321-323.
Salazar, M.H., S.C. U'ren, and S.A. Steinert. 1980. Sediment bioassays
for Navsta San Diego dredging project. Naval Oceans Systems Center,
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Shaefer, R.C. 1970. Feeding habits of striped bass from the surf waters
of Long Island. N.Y. Fish Game J. 17:1-17.
Shuba, P.O., H.E. Tatem, and J.H. Carroll. 1978. Biological assessment
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material. Techn. Rept. D-78-50. U.S. Army Engineer Waterways Experiment
Station, Vicksburg, Mississippi. 77 pp.
Stuck, K.C., H.M. Perry, and R.W. Heard. 1979a. An annotated key to the
Mysidacea ofthe North Central Gulf of Mexico. Gulf Res. Rept.
6(3):225-238.
Stuck, K.C., H.M. Perry, and R.W. Heard. 1979b. Records and range
extensions of Mysidacea from coastal and shelf water of the Eastern
Gulf of Mexico. Gulf Res. Rept. 6(3):239-248.
Tattersall, W.M., and O.S. Tattersall. 1951. The British Mysidacea.
Royal Soc. London. 460 pp.
USEPA. 1981. Acephate, aldicarb, carbophenothion, DEF, EPN, ethoprop,
methyl parathion, and phorate: their acute and chronic toxicity,
bioconcentration potential, and persistence as related to marine
environments. EPA-600/4-81-041.
USEPA. 1981. Acute toxicity test standard using mysid shrimp in static
and flow-through systems. Toxic Substances Control Act, Section 4.
U.S. Environmental Protection Agency, Office of Toxic Substances,
Health and Environmental Review Division, Washington, DC. 17 pp.
USEPA. 1981. Chronic toxicity test standard using mysid shrimp in a
flow-through system. Toxic Substances Control Act, Section 4. U.S.
Environmental Protection Agency, Office of Toxic Substances, Health and
Environmental Review Division, Washington, DC. 19 pp.
USEPA. 1981. Technical support document for using mysid shrimp in acute
and chronic toxicity tests. Toxic Substances Control Act, Section 4.
U.S. Environmental Protection Agency, Office of Toxic Substances,
Health and Environmental Review Division, Washington, DC. 43 pp.
Ward, S.H. 1984. A system for laboratory rearing of the mysid,
Mysidopsis bahia Molenock. Progr. Fish-Cult. 46(3) :170-175.
Weber, C.I., and W.H. Peltier. 1981. Effluent toxicity screening test
using Daphnia and mysid shrimp. Environmental Monitoring and Support
Laboratory, U. S. Environmental Protection Agency, Cincinnati, Ohio.
Ill
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APPENDIX A
DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS
3. FATHEAD MINNOW (PIMEPHALES PROMELAS)
(Prepared by Donald J. Klemm)
DISTRIBUTION
The fathead minnow is widely distributed in North America (Fig.
19). It is a popular bait fish, and the ease with which it is propagated
has led to its widespread introduction both within and outside the native
range of the species. The species has been so widely distributed in the
eastern and southwestern United States by bait transportation that it is
difficult to determine its original range. The presumed native
distribution (Vandermeer, 1966; Scott and Grossman, 1973; Lee, _et al.
1980) extended from the Great Slave Lake in the northwest to New
Brunswick, in eastern Canada, southward throughout the Mississippi valley
in the United States, to southern Chihuahua in Mexico. Distribution
records for this species also now include Oregon (Andreasen, 1975), and
the Central Valley (Kimsey and Fisk, 1964) and other locations in
California (Andreasen, 1975), but there are no records for British
Columbia.
The fathead minnow is found in a wide range of habitats. This
species is most abundant in brooks, small streams, creeks, ponds, and
small lakes. Trautman (1957) and Scott and Crossman (1973) reported that
species associated with the fathead minnow seem to vary greatly
throughout its range. The fathead minnow is uncommon or absent in
streams of moderate and high gradients and in most of the larger and
deeper impoundments. It is tolerant of high temperature and turbidity,
and low oxygen concentrations.
The fathead minnow is primarily omnivorous although Coyle (1930)
reported finding algae to be one of its main foods in Ohio. Elsewhere in
the United States young fish have been reported to feed on organic
detritus from bottom deposits and unicellular and filamentous algae and
planktonic organisms. Adults feed on aquatic insects, worms, small
crustaceans, and other animals. Scott and Crossman (1973) and others
regard the fathead minnow as a highly desirable forage fish, providing
food for other fishes and birds.
LIFE CYCLE
The natural history and spawning behavior (Markus, 1934; Flickinger,
1973; Andrews and Flickinger, 1974; and others) of the fathead minnow are
well known because of the early interest in raising the fish for bait and
for feeding other pond fish, such as black bass. Male and female fathead
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; - ' *% rv - ,-*•>. -
:-, VT^.^V- '-rt^
^ /r Si-. ^.;-^--v
'y •< £'*\?'^
-1 j : V- . ; , •—
:^c*^r^
Figure 19. Map showing the distribution of the fathead
minnow in North America. Open circles represent
transplanted populations. Most Atlantic slope
records are probably transplanted populations.
(From Lee et al., 1980).
minnows show sexual dimorphism at maturity. The breeding males develop a
conspicuous, narrow, elongated, gray, fleshy pad of spongy tubercles on
the back, anterior to the dorsal fin, and two or three rows of strong
nuptial tubercles across the snout. The sides of the body of breeding
males becomes almost black except for two wide vertical bars which are
light in color. The females remain quite drab.
In the wild, adult fathead minnows spawn in the spring when the water
temperature reaches 16° C to 18° C, and they continue to spawn
throughout most of the summer. Carlander (1969) stated that the minimum
temperature for spawning is 16°C.
Markus (1934) reported that fathead minnows always spawn at night,
and Isaak (1961) indicated they spawned at night and sometimes during the
day. Gale and Buynak (1982) and others reported that spawning often
began before dawn and was usually completed before noon.
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The males first select sites for spawning, such as a log or branch,
rock, board, tin can, or almost any other solid inanimate object, usually
in water from 7 cm to 1 m in depth. An interested female is sought out
and herded into position below the nest site. After circling below the
nesting site the female is nudged and lifted on the male's back until she
lies on her side immediately below the undersurface of the spawning
substrate.
The spawning female releases a small number of eggs (usually 100 to
150) at a time. The eggs are adhesive and attach to the underside of the
spawning substrate. The females have an ovipositor (urogenital
structure) to help deposit the eggs on the under side of objects.
Flickinger (1966) indicated that the ovipositor is noticable at least a
month prior to spawning. The reported size of the eggs varies from
1.15 mm (Markus, 1934) to 1.3 mm in diameter (Wynne-Edwards, 1932).
Immediately after the eggs are laid, they are fertilized by the male,
and the female is driven off. Once eggs are deposited in the nest, the
male becomes very aggressive and will use the large tubercles on his
snout to help drive off all intruding small fishes. In addition to
fertilizing and guarding the eggs, the male agitates the water around the
eggs, which ventilates them and keeps them free of detritus. Some males
will spawn with a number of females on the same substrate, so that the
nest may contain eggs in various stages of development. The number of
eggs per nest will vary from as few as nine or 10 to as many as 12,000.
The ovaries of the females contain eggs in all stages of development,
and they spawn repeatedly as the eggs mature. A female may deposit eggs
in more than one nest. Although the average number of eggs per spawn per
female is generally 100 to 150, large females may lay 400 to 500 eggs per
spawn.
Gale and Buynak (1982), in a study using five captive pairs of
fathead minnows in separate outdoor pools, stated that each pair produced
16 to 26 clutches of eggs between May and August. The time between
spawns, which ranged from two to 16 days, was affected by water
temperature. As temperature increased, the intervals between spawning
sessions become shorter and more uniform. In their study, from nine to
1,136 (mean of 414) eggs were spawned per clutch. The average number of
eggs spawned per session by individual females ranged from 371 to 480,
and the total number of eggs spawned per female ranged from 6,803 to
10,164 (mean of 8,604). The length of the spawning period during a given
season also varied greatly with individual females. They suggested that
the fecundity of fathead minnows is much higher than has generally been
recognized, but they noted that fecundity of fish in the natural
environment, where conditions might be more or less favorable, might
differ from that of captive fish.
The hatching time depends on temperature. The average hatching time
required at 25° C is 4.5 to 6 days. The newly hatched young (fry) are
about 5 mm long, white in color, with large black eyes. In warm,
food-rich water, growth is rapid. Markus (1934) stated that fish hatched
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in May in Iowa reached adult size and were spawning by late July. Hubbs
and Cooper (1935) and others noted that such rapid growth is unlikely in
more northerly waters, and that the young do not spawn the first year. In
cooler water the adult size is probably not reached until the second year.
The males generally grow faster than the females, a characteristic of
minnow species.
The fathead minnow is short lived, and rarely survives to the third
year. However, Scott and Grossman (1973) stated that the age has been
found to vary throughout the geographic range of the species. Several
authors indicate that postspawning mortality in fathead minnows is often
great, but Gale and Buynak (1982) stated that postspawning mortality did
not occur in their study. However, they indicated that in nature, where
males have to fight off intruders to defend their territory, and where they
may become weakened by a lack of food over a prolonged period, their
resistance to disease may be lowered. Also, at this time many waters are
warm and somewhat stagnate, and conditions favor the spread of parasites
and disease.
TAXONOMY
The specific name (Pimephales promelas) appears to be incorrectly
applied to this fish because the fathead minnow does not fit the
description originally given by Rafinesque (1820) (Lee et al., 1980).
Common names include "northern fathead minnow", and "blackfiead minnow" in
addition to fathead minnow. The holotype was collected near Lexington,
Kentucky.
Some geographic variations have been noted in the morphology of the
fathead minnow. Vandermeer (1966) indicated that the introduction of this
species outside its native range may have resulted in some local deviations
from broad patterns of geographic variation in taxonomic characters. Some
populations have been designated as subspecifically distinct: Pimephales
promelas promelas, the northern form; P_. £. harveyensis, the Harvey Lake
form, from Isle Royal in Lake Superior and P_. JD. confertus, the southern
form (Hubbs and Lagler, 1949, 1964). However, Taylor (1954), Vandermeer
(1966), and others expressed doubt concerning the validity of assigning
subspecific status to the variants and recommended against their
recognition. Vandermeer (1966), in a statistical analysis of the
geographic variations taxonomic characters, stated that two of the three
described subspecies intergrade clinally. Of the eight characters
measured, two showed a north-south trend; (1) eye diameter, with the
northern fish having smaller eyes, and (2) completeness of the lateral
line, with the northern fish having the least complete lateral line.
However, Scott and Grossman (1973), indicated that some Canadian
populations exhibit a nearly complete lateral line. The American Fisheries
Society (1980), recognizes only one species.
1. Morphology and Identification (General characters)
Fathead minows vary greatly in many characteristics throughout their
wide geographic range. The morphology and characters for identification
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are taken from Trautman (1957), Clay (1962), Hubbs and Lagler (1964),
Eddy and Hodson (1961), and Scott and Grossman (1973). They are small
fish, being typically 43 mm to 102 mm, or an average about 50 mm, in
total length (Fig. 20). The first rudimentary ray of the dorsal fin is
more or less thickened and distinctly separated from the first
well-developed ray by a membrane. The lateral line is usually
incomplete, but may be complete in specimens from some geographic areas.
The scales are cycloid and moderate in size. Andrews (1970), reporting
on fish collected in Colorado, noted that no scales were found on fish
smaller than 14 mm, and the average length for first scale formation was
16.3 mm. The scales in the lateral series number 41 to 54.
The mouth is terminal. The snout does not extend beyond the upper
lip and is decidedly oblique. Nuptial tubercles occur on mature males
only, are large and well-developed on the snout, and rarely extend beyond
the nostrils. They occur in three main rows, with a few on the lower
jaw. In addition to nuptial tubercles, there is an elongate fleshy or
spongy pad extending in a narrow band from the nape to the dorsal fin.
Wide anteriorly, the pad narrows to engulf the first dorsal ray. A dark
spot is usually present in front of the dorsal fin in mature males.
The peritoneum is brownish-black, and the intestine is long and
coiled one or more times. A dusky band is usually absent on the snout
and opercules except in some young; a lateral band across the body is
distinct only in some young and in adults from clear and weedy waters.
The band may be absent in breeding males, or if present, it becomes very
diffuse anteriorly. This band is usually most apparent on preserved
specimens. Dymond (1926), Trautman (1957), and others described the
saddle-like pattern often associated with breeding males in which a light
area develops just behind the head and another beneath the dorsal fin,
the areas between producing a saddle affect. A narrow, dark, vertical
bar or spot is present at the base of the caudal fin, but often is not
very distinct. The standard lengths are usually less than four and
one-half times the body depth.
Figure 20. Fathead minnow: adult female (left) and breeding male
(right). (From Eddy and Hodson, 1961).
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2. Hybridization:
Trautman (1957) stated that under some conditions the fathead minnow
may hybridize with the bluntnose minnow, Pimephales notatus
(Rafinesque). He also indicated that fathead and bluntnose minnows were
competitors, and that the fathead occurred in greater population
densities only where the bluntnose was absent or comparatively few in
number.
CULTURE METHODS
1. Sources of Organisms
When test fish are secured from an outside source, there is no
guarantee that they will be of the age, size, quality or condition needed
for performing bioassays. Additionally, because of the possibility of
occurrence of disease in fish brought into the laboratory, it may be
necessary to give them a prophylactic treatment for disease to prevent
and/or eliminate infections. Such treatment places a stress on the test
fish and requires a quarantine period of seven days.
To avoid the problems associated with using fish of unknown condition
and age, an inhouse laboratory culture facility can be developed to
provide a continuous supply of eggs (embryos) and/or other developmental
stages of known age, which are free of disease or other stress, for use
in toxicity tests.
2. Laboratory Culture Facility
Fathead minnows can be cultured in either a static or flow-through
system. Flow-through systems require large volumes of water and may not
be feasible in some laboratories. The culture facility consists of the
following components:
0 Water supply.
0 Spawning tanks in which eggs are produced by sexually mature
adults.
0 Holding tanks in which replacement spawners are held.
° Egg incubation units in which fertilized eggs are placed to
hatch.
0 Rearing tanks in which the hatched young fish are held until
used in toxicity tests.
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a. Water Supply
(1) Water Quality
Synthetic water or dechlorinated tap water can be used, but
untreated well water is preferred. If a static system is used,
it is necessary to equip each aquarium with a carbon filter
system (similar to those sold for tropical fish hobbyists) to
control the accumulation of metabolic wastes in the water.
(2) Dissolved oxygen
The dissolved oxygen concentration in the culture tanks should be
maintained near the 100% saturation level, using air stones if
necessary. Brungs (1971) in a carefully controlled long-term
study, found that the growth of fathead minnows was reduced
significantly at all dissolved oxygen concentrations below
7.9 mg/L. Soderberg (1982) presented an analytical approach to
the reaeration of flowing water for culture systems.
(3) Maintenance
Adequate procedures for culture maintenance must be followed
consistently to avoid poor water quality of the culture system.
Tanks are cleaned monthly or more often as required. They should
be kept free of debris (excess food, detritus, waste, etc.) by
siphoning the accumulated materials (such as dead brine shrimp
nauplii or cysts) from the bottom of the tanks daily with a
pipette. To avoid excessive build-up of algal growth,
periodically scrape the wall of aquaria. Activated charcoal and
filter pads in the aquaria filtration systems should be changed
weekly. Culture water may be maintained by preparation of
synthetic water or use of dechlorinated tap water. Distilled or
deionized water is added as needed to compensate for evaporation.
b. Spawning Tanks
The spawning unit is designed to simulate conditions in nature
conducive to spawning. Fathead minnows spawn in spring and summer,
and the lab photoperiod is maintained to mimic the natural spawning
conditions. For breeding tanks, it is convenient to use 76 L (20
gal) aquaria. Spawning tanks must be held at a temperature of
25 +_ 2°C. Each aquarium is equipped with a heater, continuous
filtering unit, and spawning substrates. The photoperiod for the
spawning tanks must be rigidly controlled and maintained at 16 h
light and 8 h dark (5:00 AM to 9:00 PM is a convenient photoperiod).
An illumination level of 50 to 100 ft c is adequate. The breeding
fish are fed all the frozen brine shrimp and tropical fish flake food
or dry commercial fish food that they can eat twice daily (8:00 AM
and 4:00 PM) during the week and once a day on weekends. If properly
maintained, each breeding tank will produce a spawn of 100 to 200
eggs approximately every four days.
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In nature, the female deposits her adhesive eggs on the underside of
sticks, rocks, or boards, and the male then fertilizes, guards, and
cleans the eggs. It is necessary, therefore, to provide a surface
for the laying and fertilizing of eggs, as well as a territory for
the male. The number of substrates placed in each tank depends on
the size of the tank and the number of males. Two substrates are
used for each male. The substrates should be placed equi-distant
from each other and staggered from each other on the bottom of the
tanks so that the spawning territories of the males do not overlap.
The recommended spawning substrates consist of inverted 15-cm (6-in.)
sections of 10-cm (4-in.) diameter clay tile or PVC cut in half
longitudinally. Four substrates are used in each 76-L (20 gal)
spawning tank.
The number of fish in each aquarium, as well as the ratio of females
to males, depends on the size of the aquarium. Five to eight females
and two males are placed in each breeding tank.
Sexing of the fish to ensure a correct female/male ratio in each tank
can be a problem. However, the task usually becomes easier as
experience is gained. Sexually mature females usually have large
bellies and a tapered snout. The sexually mature males are usually
distinguished by their larger overall size, dark vertical color
bands, and the spongy nuptial tubercles on the snout. Unless the
males exhibit these secondary breeding characteristics, no reliable
method has been found to distinguish them from females. However,
using the coloration of the males and the presence of an enlarged
urogenital structures and other characteristics of the females, the
correct selection of the sexes can usually be achieved by trial and
error.
If by accident, more than two males are placed in a tank, the less
aggressive males will not mature, and may continue to be confused
with females. Sexually immature males are usually recognized by
their aggressive behavior and partial banding. These undeveloped
males must be removed because they will eat the eggs and constantly
harass the mature males, tiring them and reducing the fecundity of
the breeding unit. Therefore, the fish in the spawning tanks must be
carefully checked periodically for extra males.
The female contains eggs in all stages of development, and spawns
repeatedly as the eggs mature until she has spawned her entire egg
complement. Therefore, spawning females must be replaced
periodically (every two to three months) with other mature, egg-laden
females to ensure a continuous supply of eggs.
c. Replacement Spawners
Young males removed from spawning tanks and other surplus mature
males are placed in all-male holding tanks for future use as
spawners. Similarly, young and surplus mature females are held in
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all-female holding tanks until needed as spawners. Tanks holding
replacement spawners need not be temperature-controlled, but for ease
of transfer to the spawning tanks, temperatures close to those of the
spawning tanks should be maintaned.
d. Spawning, Egg Collection, and Incubation
Once spawning conditions are right, eggs are produced. Fathead
minnows spawn in the early morning hours. They should not be
disturbed except for a morning feeding (8:00 AM) and daily
examination of substrates for eggs between 9:30 AM and 10:30 AM. In
nature, the male protects, cleans, and aerates the eggs until they
hatch. In the laboratory, however, it is necessary to remove the
eggs from the tanks to prevent them from being eaten by the adults,
and for ease of handling for purposes of recording egg count and
hatchability, and use in embryo-larval and larval tests.
The substrates are each lifted carefully and checked for newly
spawned eggs. If eggs are observed on a tile, remove it immediately
from the spawning tank, stand on end in a small (1- to 2-L) tank, and
place an active air stone at the bottom of the tank near the egg
mass. Vigorous aeration inhibits fungal growth better than weak
aeration.
Alternately, the eggs can be removed from the substrate with a razor
blade or with a rolling action of the index finger, placed in a 1-L
jar or beaker, and aerated with sufficient vigor to keep them in
suspension.
If fungal growth is a problem, it can be controlled for the entire
incubation period by adding 3 ml of 1% methylene blue stock solution
per liter of water in the incubation vessels (see Herwig, 1979, pp.
160-161).
During the incubation period, the eggs are examined daily for
viability and fungal growth, until they hatch. Unfertilized eggs,
and eggs that have become infected by fungus, should be removed with
forceps using a table top magnifier illuminator. Non-viable eggs
become milky and opaque, and are easily recognized. The non-viable
eggs are very suseptible to fungal infection, which may then spread
throughout the egg mass. Removal of fungus should be done quickly,
and the substrates should be returned to the incubation tanks as
rapidly as possible so that the good eggs are not damaged by
desiccation.
Hatching takes four to five days at an optimal temperature of
25°C. Hatching can be delayed several (two to four) days by
incubating at 10 to 15°C.
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e. Rearing Tanks
(1) Transfer and feeding of newly-hatched fry.
Newly-hatched fish are transferred daily from the egg incubation
tanks to small (8-L) rearing tanks, using a large bore pipette,
until the hatch is complete. New rearing tanks are set up on a
weekly basis to separate fish by age group. A density of 150 fry
per liter is suitable for the first four weeks. The rearing
tanks are allowed to follow ambient laboratory temperatures of 20
to 23°C, but sudden, extreme variations in temperature must be
avoided.
Fathead minnow fry are fed freshly-hatched brine shrimp (Artemia)
nauplii twice daily until they are four weeks old. Utilization
of older (larger) brine shrimp nauplii will result in starvation
of the young fish because they are unable to ingest the larger
food organisms.
See Appendix A.5 for instructions on the preparation of brine
shrimp nauplii.
(2) Feeding and stocking density of fish older than four weeks.
Fish older than four weeks are fed frozen brine shrimp and
commercial fish starter (#1 and #2), which is ground fish meal
enriched with vitamins. As the fish grow, larger pellet sizes
are used, as appropriate.
3. Disease Control
Bacterial or fungal infections are the most common diseases
encountered. However, if normal precautions are taken, disease outbreaks
will rarely, if ever, occur. If disease occurs, treat with 0.6 ml
Procaine Penicillin-G per 76-L tank (see Table 2). Hoffman and Mitchell
(1980) have put together a list of some chemicals that have been used
commonly for fish diseases and pests.
In aquatic culture systems where filtration is utilized, the
application of certain antibacterial agents should be used with caution.
A treatment with a single dose of antibacterial drugs can interrupt
nitrate reduction and stop nitrification for various periods of time,
resulting in changes in pH, and in ammonia, nitrite and nitrate
concentrations (Collins et a/L, 1976). These changes could cause the
death of the culture organisms.
Do not transfer equipment from one tank to another without first
disinfecting it. If an outbreak of disease occurs, any equipment, such
as nets, airlines, tanks, etc., which has been exposed to diseased fish
should be disinfected with sodium hypochlorite.
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TEST ORGANISMS
Fish one to 90 days old are used in the toxicity test. Before the
fish are removed, most of the water in the holding tank containing the
fish should be siphoned off. The fish are then transferred to finger
bowls, using a large-bore, fire-polished glass tube (6 mm to 9 mm I.D. X
30 cm long) equipped with a rubber bulb. It is important to note that
larvae should not be handled with a dip net. Dipping small fish such as
these with a net will result in very high mortality. The same
large-bore, fire-polished glass tube discussed above should be used to
transfer the fish from the finger bowl to the test vessels. The fish are
counted as they are caught and are placed gently into the test vessels.
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SELECTED REFERENCES
American Fisheries Society. 1980. A list of common and scientific names
of fishes from the United States and Canada. 4th ed. American
Fisheries Society, Committee on Names of Fishes. 174 pp.
Andreasen, J.K. 1975. Occurrence of the fathead minnow, Pimephales
promelas, in Oregon. Calif. Fish Game 6(3):155-156.
Andrews, A.K. 1970. Squamation chronology of the fathead minnow,
Pimephales promelas. Trans. Amer. Fish. Soc. 99(2):429-432.
Andrews, A., and S. Flickinger. 1974. Spawning requirements and
characteristics of the fathead minnow. Proc. Ann. Conf. Southeastern
Assoc. Game Fish Comm. 27:759-766.
Brungs, W.A. 1971. Chronic effects of low dissolved oxygen
concentrations on fathead minnows (Pimephales promelas). J. Fish. Res.
Bd. Can. 28:1119-1123.
Carlander, K. 1969. Handbook of freshwater fishery biology, Vol. 1.
Iowa State Univ. Press, Ames, Iowa.
Clay, W. 1962. The Fishes of Kentucky. Kentucky Dept. Fish and
Wildlife Res., Frankfort, Kentucky.
Collins, M.T., J.B. Gratzer, D.L. Dawe, and T.G. Nemetz. 1976.
Effects of antibacterial agents on nitrification in aquatic
recirculating systems. J. Fish. Res. Bd. Can. 33:215-218.
Coyle, E.E. 1930. The algal food of Pimephales promelas (fathead
minnow). Ohio J. Sci. 30(l):23-35.
Cross, F.B. 1967. Handbook of fishes of Kansas. Univ. Kansas Mus.
Natur. Hist. Misc. Publ. 45:1-357.
Dymond, 1926. The Fishes of Lake Nipigon. Univ. Toronto Stud. Biol.
Ser. 27 Publ. Ont. Fish. Res. Lab 27:1-108.
Eddy, S., and A.C. Hodson. 1961. Taxonomic keys to the common animals of
the north central states. Burgess Publ. Co., Minneapolis, Minnesota
55415.
Flickinger, S.A. 1966. Determination of sexes in the fathead minnow.
Trans. Amer. Fish. Soc. 98(3):526-527.
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Flickinger, S.A. 1973. Investigation of pond spawning methods for
fathead minnows. Proc. Ann. Conf. Southeast. Assoc. Game and Fish
Commiss. 26: 376-391.
Gale, W.F., and G.L. Buynak. 1982. Fecundity and spawning frequency of
the fathead minnow—A fractional spawner. Trans. Amer. Fish. Soc.
111:35-40.
Hedges, S., and R. Ball. 1953. Production and harvest of bait fishes in
ponds. IrK Michigan Dept. Conservation. Miscel. Publ. 6, Lansing,
Michigan, pp. 1-30.
Herwig, N. 1979. Handbook of drugs and chemicals used in the treatment
of fish diseases. Charles C. Thomas, Publ., Springfield, Illinois. 272
pp.
Hoffman, G.L., and A.J. Mitchell. 1980. Some chemicals that have been
used for fish diseases and pests. Fish Farming Exp. Sta., Stuttgart,
Arkansas 72160. 8 pp.
Hubbs, C.L., and G.P. Cooper. 1935. Age and growth of the long eared and
the green sunfishes in Michigan. Pap. Mich. Acad. Sci. Arts. Letts.
20:669-696.
Hubbs, C.L, and K.F. Lagler. 1949. Fishes of Isle Royale, Lake Superior,
Michigan. Pap. Mich. Acad. Sci. Arts. Letts. 33:73-133.
Hubbs, C.L., and K.F. Lagler. 1964. Fishes of the Great Lakes Region.
Univ. Mich. Press, Ann Arbor, Michigan.
Isaak, D. 1961. The ecological life history of the fathead minnow,
(Pimephales promelas Rafinesque). Doctorial dissertation, Univ.
Minnesota. Microfilm 6104598, Univ. Microfilms International. Ann
Arbor, Michigan. 150 pp.
Kimsey, J.B., and L.O. Fisk. 1964. Freshwater nongame fishes of
California. Calif. Dept. Fish and Game., Sacramento, California.
Lee, D.S., C.R. Gilbert, C.H. Hocutt, R.E. Jenkins, D.E. McAllister, and
R. Stauffer, Jr. 1980. Atlas of North American freshwater fishes.
Publ. 1980-12, N. Carolina State Museum Nat. Hist., Raleigh, N.
Carolina. 27611.
Markus, H. 1934. Life history of the blackhead minnow (Pimephales
promelas). Copeia 1934:116-122.
Rafinesque, C.S. 1820. Ichthyologia Ohiensis, or natural history of the
fishes inhabiting the river Ohio and its tributary streams, preceded by
a physical description of the Ohio and its branches. Lexington,
Kentucky. 90 pp.
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Scott, W., and E. Grossman. 1973. Freshwater fishes of Canada.
Fish. Res. Bd. Can. Bull. 184. 966 pp.
Soderberg, R.W. 1982. Aeration of water supplies for fish culture in
flowing water. Prog. Fish-Cult. 44(2):89-93.
Taylor, W.R. 1954. Records of fishes in the John N. Lowe collection
from the Upper Peninsula of Michigan. Misc. Publ. Mus. Zool. Univ.
Michigan, 87. 50 pp.
Trautman, M.B. 1957. The fishes of Ohio. Ohio State Univ. Press.,
Columbus, Ohio. 683 pp.
Vandermeer, J.H. 1966. Statistical analysis of geographic variation of
thefathead minnow, Pimephales promelas. Copeia 1966(3) :457-466.
Westman, J. 1938. Studies on the reproduction and growth of the
bluntnose minnow Hydorhynchus notatus (Rafinesque). Copeia 1938: 57-61.
Wynne-Edwards, V.C. 1932. The breeding habits of the black-headed minnow
(Pimephales promelas Raf.). Trans. Amer. Fish. Soc. 62:382-383.
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APPENDIX A
DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS
4. SILVERSIDES (MENIDIA)
(Prepared by Douglas Middaugh)
DISTRIBUTION
Silversides occur in estuaries along the Atlantic and Gulf coasts.
The Atlantic silverside, Menidia menidia, is a resident of estuaries from
Maine to northern Florida. It occurs at intermediate to high salinities,
typically of 12 to 30 parts per thousand (ppt), and remains in Atlantic
estuaries throughout most of the year (De Sylva et al., 1962; Dahlberg,
1972). Recent evidence indicates an offshore migration at northern
latitudes in the fall and reappearance of adults in estuaries in late
spring (Conover and Kynard, 1981). This species is an important
component in estuarine ecosystems, serving as forage fish for
commercially and recreationally valued species such as striped bass,
bluefish and spotted seatrout (Merriman, 1941; Bayliff, 1950; Middaugh,
1981).
Although the culturing methods described in this section were written
primarily for Menidia menidia, they are also suitable for the inland
silverside, M. beryll'ina, and the tidewater silverside, ^. peninsulae.
The staff of the Environmental Research Laboratory, Gulf Breeze, Florida,
is currently developing procedures for spawning, culturing, and testing
of other fishes, including the California grunion, Leuresthes tenuis, and
the Pacific surf smelt, Hypomesus pretiosus. The availability of these
fishes as test organisms will permit the use of indigenous fish in
toxicity tests of wastes discharged along the entire coast line of the
contiguous United States and Alaska.
LIFE CYCLE
The Atlantic silverside spawns during spring and summer. Spawning
runs generally occur during April - June or July at northern latitudes,
and March through July or August at southern latitudes (Bayliff, 1950;
Hildebrand and Schroeder, 1928; Middaugh and Lempesis, 1976). Spawning
occurs in the upper intertidal zone during daytime high tides (Middaugh,
1981). Eggs are deposited on a variety of substrates which provide
protection from thermal stress and desiccation (Middaugh et al., 1981;
Conover and Kynard, 1981). Females typically release 200 to 800 eggs,
1.0 to 1.2 mm diameter, as they spawn. Individuals may spawn up to five
or six times, at two week intervals, during the reproductive season. The
life span is generally 12 to 15 months, although year class-2 fish are
occasionally found (Beck, 1979).
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MORPHOLOGY AND TAXONOMY
Adult Atlantic silversides attain a total length of up to 117 mm
(Fig. 21A). Females in general are slightly larger than males. The
first dorsal fin has three to seven, usually four or five spines. The
second dorsal fin has one spine and eight or nine rays; the anal fin has
one spine and 19 to 29, usually 21 to 26, rays; and the pectoral fin has
12 to 16, usually 14 or 15, rays (Robbins, 1969). Atlantic silverside
embryos are easily distinguished from those of the closely related inland
silverside, Menidia beryllina. The former have a bundle of elastic
filaments attached to the chorion at one small area of insertion (Fig.
218, C). These filaments, typically longer than the diameter of the egg,
are all the same diameter. In contrast, inland silverside eggs posses
one or two thick, elongated filaments, up to 50 mm long and four to nine
shorter, thinner filaments (Fig. 21D, E).
CULTURING METHODS
1. Sources of Organisms
a. Field Collections
The optimal time for collecting ripe fl. menidia is just prior to
daytime high tides between 8:00 AM and noon (usually one to four days
after the occurrence of a new or full moon), when prespawning schools
move into the upper intertidal zone (Middaugh, 1981; Middaugh et a_1_.,
1981). Since the Atlantic silverside prefers relatively high
salinities, it is recommended that collections be made in areas with
salinities of 20 ppt or greater. Sandy beaches, bordering open but
protected estuarine bays, are suitable for collecting adults. A
1 X 10-m bag seine with knotless 5-mm mesh is ideal for collecting.
Since Atlantic silversides typically reside in shallow water, 1.5 m
deep, they are easily captured by seining close to shore. It is
important to avoid total beaching of the bag seine when collecting NL
menidia. These fragile fish will quickly die if removed from water
and, more importantly, ripe females often abort their eggs if
stranded. Ideally, the bag portion of the seine, containing captured
adults, should remain in water 5 to 15 cm deep (Middaugh and
Lempesis, 1976).
It is possible to transport the spawn (fertilized eggs) or adults to
the laboratory. The following procedure is recommended for
stripping, fertilizing and transporting eggs from the field to the
laboratory:
(1) Immediately after seining (while still on the beach) three to
five ripe females should be dipped into a bucket of seawater to
remove sand and detritus.
(2) Eggs are stripped into a glass culture dish containing seawater
or onto a nylon screen (0.45 to 1.0 mm mesh) (Fig. 22), which is
then gently lowered into a culture dish of seawater with the eggs
on the upper surface of the screen (Barkman and Beck, 1976). If
127
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Figure 21. Silverside (Menidia): (A-C) f4. menidia, (Atlantic silverside);
(A) adult, (B) unfertilized, (C) developing embryo; note that
filaments are all equal in diameter; (D-E) ) M. beryllina
(inland silverside), (D) unfertilized egg, and" (E) developing
embryos; note one thick filament and several thin filaments.
128
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HAND STRIPPING EGGS
HAND STRIPPING MALES
ONTO 500/U NYUON SCREEN
IMMERSED IN SEA WATER
SPERM SUSPENSION
IN SEA WATER
FERTILIZED EGGS
ADHERED TO SCREEN
ADD SPERM SUSPENSION TO
TRAY CONTAINING EGGS
AERATION
15 MINUTES
FILTERED
SEA WATER (FSW)
HATCHED LARVAE
= 11 DAYS AT 20° C
'SCREEN WITH
EGGS
400/1
SCREENED
DISCHARGE
EGG INCUBATION JAR
CONSTANT
LEVEL SIPHON
LARVAE HARVESTED
WITH NET WITH
PLASTIC FILM
BOTTOM
USE
FOOD: BRINE
SHRIMP NAUPLII
TRANSFER TO
VAE
STANDP1PE- SCREENED
LARVAL REARING
TANK- FILLED WITH SEA WATER
CONTINUOUS FLOW
SCREENED
OUTFLOW
Figure 22. Techniques for collection of silverside eggs in the field and
production of larvae in the laboratory (From Beck, 1979).
129
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excessive pressure is required to strip eggs, the female should
be discarded. Mature eggs, 1.0 to 1.2 mm in diameter, are clear,
with an amber hue.
(3) Milt from several males can then be stripped into the culture
dish and mixed with the eggs by gently tilting the dish from side
to side.
Upon contact with seawater, adhesive threads on mature eggs
uncoil, making enumeration and separation difficult. If eggs are
stripped directly into the culture dish, one end of a nylon
string may be dipped into the dish and gently rolled so the
embryos adhere (Middaugh and Lempesis, 1976). The Barkman and
Beck (1976) technique for attaching the eggs to nylon screening
minimizes the natural clumping tendency due to entanglement of
the filaments on IM. menidia eggs.
(4) Strings of embryos or embryos on screens may be transported to
the laboratory by placing them in an insulated glass container
filled with seawater at the approximate temperature and salinity
of fertilization.
If gravid fish are transported to the laboratory for subsequent
spawning, care must be taken to avoid overcrowding of fish in
transport containers. Continuous, vigorous aeration is required
and any increase in container water temperature should be
minimized (Beck, 1979).
A mass culture system for incubating the screen-adhered eggs and
collecting the hatched larvae in a flowing seawater system (Fig.
22) was described in detail by Beck (1979). A similar procedure
utilizing a recirculating system was described by Middaugh and
Lempesis (1976).
b. Laboratory year-round Spawning
Atlantic, inland, and tidewater silversides may be spawned in the
laboratory on a year-round basis. Procedures described by Middaugh and
Takita (1983), and Middaugh and Hemmer (1984), provide for maintenance of
a brood stock of 30 to 50 fish, sex ratio 1:1, in 1.3 m diameter,
circular holding tanks which are part of a recirculating seawater system
(Fig. 23). The photoperiod should be adjusted to 14 L:10 D (lights on at
5:00 AM and off at 7:00 PM, intensity 50-100 ft c), with the water
temperature maintained at 18 to 20°C for fish from northern latitudes,
and 20 to 25°C for southern latitudes. Suitable salinities for the
culture units would be 25 to 30 ppt for the Atlantic and tidewater
silversides, and 7 ppt for the inland silverside. Fish are fed 8 g
Tetramin® each morning and afternoon, and concentrated Artemia nauplii
(hatch obtained from approximately 15 ml of eggs after 48 h of incubation
at 25°C) in mid-afternoon (see section on Artemia culture). Excess
food should be siphoned from the holding tanks weekly. Filter media
(activated charcoal) located in a reservoir tray should be changed
130
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weekly, immediately after cleaning the holding tanks. To induce spawning
by the Atlantic silverside, the circulation current velocity in the
holding tanks should be reduced to zero (from 8 to 0 cm/sec) twice daily
by turning off the seawater circulation pump from midnight to 1:00 AM,
and from noon to 1:00 PM. Atlantic silversides will spawn in response to
interrupted current velocities during daytime (noon to 1:00 PM).
Spawning of the tidewater silverside also is enhanced by reducing the
current velocity twice daily, but spawns primarily during nighttime. No
interruption in current is necessary to enhance spawning by the inland
silverside.
A suitable spawning substrate can be made by cutting enough 25 cm lengths
of No. 18 nylon string to form a small bundle, and tying a string around
the middle of the bundle to form a "mop." The mop is suspended just
below the surface of the water, in contact with the side of the holding
tanks. Spawning fish will deposit eggs on this substrate. The mops are
removed from the holding tanks daily and suspended in incubation
vessels. Typical egg production ranges from 300 to 1200 per spawn. Fish
generally can be expected to spawn three to four days each week.
It is essential that light-tight curtains surround the holding tanks.
These curtains should remain closed except during periodic feedings,
tanks cleaning, and during removal and replacement of spawning substrates.
2. Culture Vessels
Embryos attached to nylon screening or nylon string may be suspended in a
culture system such as shown in Fig. 22. The culture chambers for
embryos should be constructed of glass. Upon hatching, larvae may be
transferred from the collection container to a 90-cm diameter glass or
fiberglass tank with a volume of 350 L. Tanks receive a continuous flow
of seawater at 2 L/min. Water is introduced at the tank periphery
causing a gentle current sufficient to induce orientation to water
movement and normal schooling behavior. Water is discharged from the
tank by two automatic siphons. Siphon openings are protected by a 400 urn
nylon screen to prevent escape of larvae. An inverted funnel is used at
the siphon to decrease the velocity of discharge water, thus preventing
impingement of larvae.
Embryos can also be incubated in small (4- to 10-L) glass aquaria, by
placing the nylon screening or strings just below the surface of the
water. Gentle aeration should be provided by an airstone positioned near
the bottom of the holding aquaria.
3. Culture Media
Use natural seawater if it is available and unpolluted. Otherwise use
synthetic seawater preDared by adding artificial marine salts, such as
Instant Ocean*** or Rila® Marine Mix, to distilled or deionized water.
If synthetic seawater is used, it should be aged for a least one week
before being utilized in culture aquaria.
131
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Figure 23. Holding and spawning system utilized in the culture of
silversides (Menidia). (A) 1.3 m diameter tanks, (B)
circulation pump, (C) reservoir, (D) seawater distribution
system, (E) by-pass line, (F) seawater return line, and (G)
reservoir filter system. (From Middaugh and Hemmer, 1984).
132
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4. Culture Conditions
The salinity maintained during incubation should be similar to that
of the water from which the adults were taken, if collected in the field,
or at which the adults are being maintained in the laboratory, if the
embryos originate from laboratory brood stock. Water temperature should
be maintained at 20 to 25°C depending upon the latitude where fish are
collected. Provide a photoperiod of 12 to 14 h of illumination daily at
50-100 ft c (12 h minimum light/24 h). Embryos will hatch in seven to 14
days, depending upon the incubation temperature and salinity (Middaugh
and Lempesis, 1976).
5. Feeding and Stocking Density
Upon hatching, Menidia larvae should be fed immediately. Newly
hatched brine shrimp (Artemia) nauplii (less than eight hours old) are
fed larvae twice daily. It is essential to feed newly hatched Menidia
larvae with newly-hatched brine shrimp. Utilization of older, larger,
brine shrimp nauplii will result in starvation of the larvae since they
are unable to ingest the larger food organisms. Three to four days after
hatching, the fish are able to consume older (larger) brine shrimp
nauplii. Methods for culturing brine shrimp are discussed in Appendix
A.5. A stocking density of about 300 larvae is suitable in an 76-L
aquarium.
6. Culture Maintenance
To avoid excessive build up of algal growths, periodically scrape the
walls of aquaria. Activated charcoal in the aquarium filtration systems
should be changed weekly and detritus (dead brine shrimp nauplii or
cysts) siphoned from the bottom of holding aquaria each week. Salinity
may be maintained at the proper level by addition of distilled or
deionized water to compensate for evaporation.
TEST ORGANISMS
Fish one to 90 days old are used in tests. Most of the water in the
holding aquarium should be siphoned off before removal of larvae. Larvae
can then be siphoned from the holding tanks into a holding vessel. It is
essential that larvae not be handled with a dip net, because it will
result in very high mortality. A large-bore, fire-polished glass tube,
6mm I.D. x 500mm long (1/4" ID X 18" long), equipped with a rubber
squeeze bulb should be used to transfer the larvae from the holding
vessel to the test vessels. It is more convenient to first transfer five
fish to each of several small beakers containing 20 ml of saline dilution
water. The appropriate number of fish (multiples of five) can then be
added to test vessels.
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SELECTED REFERENCES
Anderson, W.D., J.K. Dias, R.K. Dias, D.M. Cupka, and N.A. Chamberlain.
1977. The macrofauna of the surf zone off Folly Beach, S. Carolina.
NOAA Tech. Rept. NMFS SSRF-704. 23 pp.
Barkman, R.C., and A.D. Beck. 1976. Incubating eggs of the Atlantic
silverside on nylon screen. Prog. Fish-Cult. 38:148-150.
Bayliff, W.H. 1950. The life history of the silverside, Menidia menidia
(Linnaeus). Contr. Chesapeake Biol. Lab., Publ. 90:1-27.
Beck, A.D. 1979. Laboratory culture and feeding of the Atlantic
silverside, Menidia menidia. Conference on Aquaculture and Cultivation
of fish fry and its live food. Polish Hydrobiological Soc. Syzmbark,
Poland, September, 1977. Spec. Publ. No. 4, European Mar. Soc. pp.
63-85.
Bigelow, H.B., and W.C. Schroeder. 1953. Fishes of the Gulf of Maine.
U.S. Fish Wildl. Serv. Fish. Bull. 53:1-577.
Briggs, P.T. 1975. Shore-zone fishes in the vicinity of Fire Island
Inlet, Great South Bay, New York. N.Y. Fish Game 22:1-12.
Chernoff, B., J.V. Conner, and C.F. Bryan. 1981. Systematics of the
Menidia beryllina complex (Pices:Atherinidae) from the Gulf of Mexico
and its tributaries. Copeia 2:319-335.
Chesmore, A.P., D.J. Brown, and R.D. Anderson. 1973. A study of the
marine resources of Essex Bay. Mass. Div. Mar. Fish. Monogr. Ser. No.
13. 38 pp.
Clark, F.N. 1925. The life history of Leuresthes tenuis, an atherine
fish with tide controlled spawning habits. Calif. Fish. Game Comm.
Bull. 10:1-51.
Conover, D.O. 1979. Density, growth, production and fecundity of the
Atlantic silverside, Menidia menidia (Linnaeus), in a central New
England estuary. M.S. Thesis, Univ. Massachuetts, Amherst. 59 pp.
Conover, D.O., and B.E. Kynard. 1981. Environmental sex determination:
Interaction of temperature and genotype in a fish. Science 213:577-579.
Conover, D.O., and B.E. Kynard. 1984. Field and laboratory observations
of spawning periodicity and behavior of a northern population of the
Atlantic silverside, Menidia menidia (Pices: Atherinidae). Environm.
Biol. Fish. 11(3):161-171.
Conover, D.O., and S. Murawski. 1982. Offshore winter migration of the
Atlantic silversides, Menidia menidia. Fish. Bull. 80:145-149.
134
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Dahlberg, M.D. 1972. An ecological study of Georgia coastal fishes.
U.S. Fish Wild!. Serv. Fish. Bull. 70(2):323-353.
DeSylva, D.P., F.A. Kalber, Jr., and C.N. Schuster. 1962. Fishes and
ecological conditions in the shore zone of the Delaware River Estuary,
with notes on other species collected in deeper water. Univ. Delaware
Mar. Labs., Information Serv., Publ. No. 5., 164 pp.
Elston, R., and B. Bachen. 1976. Diel feeding cycle and some effects of
light on feeding intensity of the Mississippi silverside, Menidia
audens, in Clear Lakes, California. Trans. Amer. Fish. Soc. 105:84-88.
Gettor, C.D. 1981. Ecology and survival of the key silverside, Menidia
conchorum, an atherinid fish endemic to the Florida keys. Ph.D. Thesis,
University of Miami, Coral Gables, Florida. 128 pp.
Goodman, L.R., D.P. Middaugh, D.J. Hansen, P.K. Higdon, and G.M. Cripe.
1983. Early life-stage toxicity test with tidewater silversides
(Menidia peninsulae) and chlorine-produced oxidants. Environm. Toxicol.
Chem. 2:337-342.
Gosline, W.A. 1948. Speciation in the fishes of the genus Menidia.
Evol. 2:306-313.
Hildebrand, A.E. 1922. Notes on habits and development of eggs and
larvae of the silversides Menidia menidia and Menidia beryllina. Bull.
U. S. Bur. Fish. 38:113-120.
Hildebrand, S.F., and W.C. Schroeder. 1928. Fishes of Chesapeake Bay.
Bull. U.S. Bur. Fish. 43(1):366 pp.
Hillman, R.E., N.W. Davis, and J. Wennemer. 1977. Abundance, diversity
and stability in shore zone fish communities in an area of Long Island
Sound affected by the thermal discharge of a nuclear power station.
Estuarine Coastal Mar. Sci. 5:355-381.
Oohnson, M.S. 1975. Biochemical systematics of the atherinid genus
Menidia. Copeia 1975:662-691.
Kendall, W.C. 1902. Notes on the silversides of the genus Menidia of the
east coast of the United States, with descriptions of two new
subspecies. Rept. U. S. Comm. Fish and Fisheries of 1901, pp. 241-267.
Koltes, K.H. 1984. Temporal patterns in three-dimensional structure and
activcity of schools of the Atlantic silverside Menidia menidia. Mar.
Biol. 78:113-122.
Loosanoff, V.L. 1937. The spawning run of the Pacific surf smelt,
Hypomesus pretiosus (Girard). Intern. Rev. ges. Hydrobiol. Hydrogr.
36:170-183.
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McMullen, D.M. 1982. The effect of temperature and food density on
growth and survival of larval Menidia peninsulae. M.S. Thesis, Univ.
West Florida. 33 pp.
Merriman, D. 1941. Studies of the striped bass (Roccus saxatilis) of
the Atlantic coast. U.S. Fish Wild!. Serv. Fish Bull. 35:1-77.
Middaugh, D.P. 1981. Reproductive ecology and spawning periodicity of
the Atlantic silverside, Menidia menidia (Pisces: Atherinidae).
Copeia. 4:766-776.
Middaugh, D.P., R.G. Domey, and G.I. Scott. 1984. Reproductive
rhythmicity of the Atlantic silverside. Trans. Amer. Fish. Soc.
113:472-478.
Middaugh, D.P., and M.J. Hemmer. 1984. Spawning of the tidewater
silverside, Menidia peninsulae (Goode and Bean) in response to tidal
and lighting schedules in the laboratory. Estuaries 7(2):(In press).
Middaugh, D.P., H.W. Kohn III, and I.E. Burnett. 1983. Concurrent
measurement of intertidal environmental variables and embryo survival
for the California grunion, Leuresthes tenuis, and Atlantic silverside,
Menidia menidia (Pices:Atherinidae).Calif. Fish and Game. 69(2):89-96.
Middaugh, D.P., and P.W. Lempesis. 1976. Laboratory spawning and rearing
of a marine fish, the silverside, Menidia menidia. Mar. Biol.
35:295-300.
Middaugh, D.P., G.I. Scott, and J.M. Dean. 1981. Reproductive behavior
of the Atlantic silverside, Mendia menidia (Pisces, Atherinidae).
Environ. Biol. Fish. 6(3/4):269-276.
Middaugh, D.P., and T. Takita. 1983. Tidal and diurnal spawning cues in
the Atlantic silverside, Menidia menidia. Environ. Biol. Fish.
8(2):97-104.
Moore, C.J. 1980. Spawning of Menidia menidia (Pices:Atherinidae).
Copeia 1980:886-887.
Mulkana, M.S. 1966. The growth and feeding habits of juvenile fishes in
two Rhode Island estuaries. Gulf Res. Rep. 2:97-168.
Penttila, D. 1977. Studies of the surf smelt (Hypomesus pretiosus) in
Puget Sound. State of Washington, Dept. Fish., Techn. Rept. No. 42. 47
pp.
Richards, C.E., and M. Castagna. 1970. Marine fishes of Virginia's
eastern shore (Inlet and Marsh, seaside waters). Chesapeake Sci.
11:235-248.
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Robbins, T.W. 1969. A systematic study of the silverside Membras
Ronaparte and Menidia (Linnaeus) (Atherinidae, TeleosteiJ^PhTD.
Dissertation, Cornell University. 282 pp.
Rubinoff, I. 1958. Raising the atherinid fish, Menidia menidia, in the
laboratory. Copeia 1958(2):146-147.
Rubinoff, I., and E. Shaw. 1960. Hybridization in two sympatric species
of atherinid fishes, Menidia menidia (Linnaeus) and Menidia beryllina
(Cope). Amer. Mus. Nov. 1999:1-13.
Ryder, O.A. 1883. On the thread-bearing eggs of the silverside, Menidia.
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Thomson, D.A., and K.A. Muench. 1976. Influence of tides and waves on
the spawning behavior of the Gulf of California grunion, Leuresthes
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75:198-203.
Thompson, W.F., and O.B. Thompson. 1919. The spawning of the grunion.
Calif. Fish and Game Comm. Bull. 3:1-29.
Walker, B.W. 1949. Periodicity of spawning by the grunion, Leuresthes
tenuis, an atherine fish. Ph.D. Dissertation, Univ. California, Los
Angeles. 166 pp.
Walker, B.W. 1952. A guide to the grunion. Calif. Fish and Game
38:409-420.
137
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APPENDIX A
DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS
5. BRINE SHRIMP
SOURCES OF BRINE SHRIMP EGGS
Although there are many commercial sources of brine shrimp eggs, the
Brazilian strain is preferred because it has low concentrations of
chemical residues (one source is: Aquarium Products, 180 L Penrod Ct,
Glen Burnie, MD, 21061). Reference Artemia cysts are available in
limited quantity from the Artemia Reference Center, State University of
Ghent, Belgium, J. Plafeousfruar 22, B 9000, Ghent, Belgium. In the
United States, contact the Environmental Research Laboratory, USEPA,
South Ferry Road, Narragansett, Rhode Island, 02882, for information on
reference materials and commercial sources of good quality Artemia eggs.
INCUBATION CHAMBER AND PROCEDURE
A 2000 mL separatory funnel makes a convenient brine shrimp hatching
vessel. A satisfactory but less expensive apparatus can be prepared by
cutting the bottom out of a 2-L plastic soft drink bottle and inserting a
rubber stopper with a flexible tube and pinch cock. Add approximately
1800 mL dechlorinated water and four tablespoons of non-iodized salt to
the hatching vessel and shake until the salt dissolves (or use synthetic
or filtered natural seawater). Add the desired quantity of eggs (usually
15 to 20 mL) to the vessel and mix well. The quantity of eggs used
depends on feeding requirements. For example, approximately 15 mL of
eggs will provide enough brine shrimp nauplii to feed three large stock
cultures of mysids in 76-L aquaria, or 1000 to 1500 newly hatched fish in
four to six 8-L tanks.
After the appropriate volume of eggs is added to the hatching vessel,
air is vigorously bubbled through a 1-mL pipet which is lowered through
the neck of the funnel so that the tip rests at the bottom. Aeration
will keep the eggs and newly-hatched nauplii from settling on the bottom,
where the dissolved oxygen quickly would be depleted.
The eggs will hatch in 24 h at a temperature of 27°C. Hatching
time varies with incubation temperature and the geographic strain of
Artemia used.
HARVESTING THE NAUPLII
The nauplii can be easily harvested in the following manner:
1. After 24 h at 27°C, remove the pipet supplying air, allow the
nauplii to settle to the bottom of the separatory vessel. The empty
egg shells will float to the top.
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2. After approximately five minutes, using the stopcock, drain off the
nauplii into a 250-ml beaker.
3. After another five minutes, again drain the nauplii into the beaker.
4. The nauplii are further concentrated by pouring the suspension into a
small cylinder which has one end closed with #20 plankton netting.
5. The concentrate is resuspended in 50 ml of appropriate culture water,
mixed well, and dispensed with a pipette. (Mysids require
approximately 100 nauplii/mysid/day.)
6. Discard the remaining contents of the hatching vessel and wash the
vessel with hot soap and water.
7. Prepare fresh salt water for each new hatch.
To have a fresh supply of Artemia nauplii daily, several hatching
vessels must be set up and harvested on alternate days.
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SELECTED REFERENCES:
Beck, A.D., and D.A. Bengtson. 1982. International study on Artemia
XXII: Nutrition in aquatic toxicology - Diet quality of geographical
strains of the brine shrimp, Artemia. In: Pearson, J.G., R.B. Foster,
and W.E. Bishop, eds., ASTM STP 766, American Society for Testing and
Materials, Philadelphia, Pennsylvania, pp. 161-169.
Beck, A.D., D.A. Bengtson, and W.H. Howell. 1980. International study on
Artemia. V. Nutritional value of five geographical strains of Artemia:
Effects of survival and growth of larval Atlantic silversides, Men idia
menidia. In: Persoone, G., P. Sorgeloos, D.A. Roels, and E. Jaspers.
eds., The brine shrimp, Artemia. Vol. 3, Ecology, culturing, use in
aquaculture. Universa Press, Wetteren, Belgium, pp. 249-259.
Bengston, D.A.S., A.D. Beck, S.M. Lussier, D. Migneault, and C.E. Olney.
1984. International study on Artemia. XXXI. Nutritional effects in
toxicity tests: use of different Artemia geographical strains. In: G.
Persoone, E. Jaspers, and C. Claus, eds., Ecotoxicological testing for
the marine environment, Vol. 2. State Univ. Ghent and Inst. Mar. Sci.
Res., Bredene, Belgium. 588 pp.
Johns, D.M., W.J. Berry, and W. Walton. 1981. International study on
Artemia. XVI. Survival, growth and reproductive potential of the
mysid, Mysidopsis bahia Molenock fed various geographical strains of
the brine shrimp, Artemia. J. exp. mar. Biol. Ecol. 53:209-219.
Persoone, G., P. Sorgeloos, 0. Roels, and E. Jaspers, eds. 1980. The
brine shrimp Artemia. Vol. 1. Morphology, genetics, radiobiology,
toxicology. Universa Press, Wetteren, Belgium. 318 pp.
Persoone, G., P. Sorgeloos, 0. Roels, and E. Jaspers, eds. 1980. The
brine shrimp Artemia. Vol. 2. Physiology, biochemistry, molecular
biology. Universa Press, Wetteren, Belgium. 636 pp.
Persoone, G., P. Sorgeloos, 0. Roels, and E. Jaspers, eds. 1980. The
brine shrimp Artemia. Vol. 3. Ecology, culturing, use in aquaculture.
Universa Press, Wetteren, Belgium. 428 pp.
Sorgeloos, P. 1980. Life history of the brine shrimp Artemia. In:
Persoone, G., P. Sorgeloos, D.A. Roels, and E. Jaspers, eds., The brine
shrimp, Artemia. Vol. 1. Morphology, genetics, radiobiology,
toxicology. Universa Press, Wetteren, Belgium, pp. ixx-xxii.
Usher, R.R., and D.A. Bengston. 1981. Survival and growth of sheepshead
minnow larvae and juveniles on diet of Artemia nauplii. Prog.
Fish-Cult. 43:102-105.
140
-------
APPENDIX B
DILUTOR SYSTEMS
Two proportional dilutor systems are illustrated: the solenoid valve
system, and the vacuum siphon system.
1. Solenoid and Vacuum Siphon Dilutor Systems
The designs of the solenoid and vacuum siphon dilutor systems
incorporate features from devices developed by many other Federal and
state programs, and have been shown to be very versatile for on-site
bioassays in mobile laboratories, as well as in fixed (central)
laboratories. The Solenoid Valve system is fully controlled by solenoids
(Figs. 24, 25, and 26), and is preferred over the vacuum siphon system.
The Vacuum Siphon system (Figs. 24, 27, and 28), however, is acceptable.
The dilution water, effluent, and pre-mixing chambers for both systems
are illustrated in Figs. 29, 30, and 31. Both systems employ the same
control panel (Fig. 32).
If in the range-finding test, the LC50 of the effluent falls in the
concentration range, 6.25% to 100%, pre-mixing is Jiot required. The
pre-mixing chamber is bypassed by running a TYGON® tube directly from
the effluent in-flow pipe to chamber E-2 (see Figures 26 and 28), and
Chambers E-l and D-l and the pre-mixing chamber are deactivated.
The dilutor systems described here can also be used to conduct tests
of the toxicity of pure compounds by equipping the control panel with an
auxiliary power receptacle to operate a metering pump to deliver an
aliquot of the stock solution of the pure compound directly to the mixing
chamber during each cycle. In this case, chamber E-l is de-activated and
chamber D-l is calibrated to deliver a volume of 2000 ml, which is used
to dilute the aliquot to the highest concentration used in the toxicity
test.
141
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142
-------
FLOW CONTROL
VALVES
NORMALLY OPEN
SOLENOID VALVES
7 mm (9/32 in.)
DILUTION WATER
INFLOW-*.
EFFLUENT
INFLOW
CYCLE-
COUNTER
DILUTION WATER
CHAMBERS
LAPSE
TIME
CLOCK
MAGNETIC
STIRRER
TEST CHAMBERS 1-20 LITERS CAPACITY
LIQUID LEVEL
SWITCH
DILUTION HATER OVERFLOW
19 mm (3/4 in.) HOLE
12 mm (1/2 in.) WASTE LINE
EFFLUENT
CHAMBER
PRE-MIXING
CHAMBER
NORMALLY OPEN SOLENOID VALVE
7 mm (9/32 in.) ID
EFFLUENT OVERFLOW
19 mm (3/4 in. ) HOLE
12 mm (1/2 in. ) LINE
MIXING CHAMBERS
Figure 25. Solenoid valve dilutor system, general diagram (not to scale)
143
-------
EFFLUENT
CHAMBER'
i i
D-l
0-2
n
i i
10 mm 00
ADJUSTABLE
STANDPIPE DRAIN
PRE-MIXING
CHAMBER
MAGNETIC
STIRRER
DILUTION WATER
CHAMBERS
NORMALLY CLOSED
SOLENOID VALVES
El, 02 - 06 = 7 mm (9/32 In.) ID
Dl =9.5 mm (3/8 in.) ID
6 mm 00 DELIVERY TUBE
NORMALLY OPEN SOLENOID VALVE
7 mm (9/32 in.) ID
6 mm OD DELIVERY TUBE
EFFLUENT CHAMBERS
NORMALLY CLOSED
SOLENOID VALVE
7 mm (9/32 in.) ID
6 mm 00 DELIVERYTUBE
MIXING CHAMBER
1200 ml CAPACITY
10 mm 00 DELIVERY TUBE
TEST CHAMBER
1-20 LITER CAPACITY
NOTE: WHEN 100X EFFLUENT IS USED AS THE HIGHEST EFFLUENT CONCENTRATION,
E-l, D-l, AND THE PRE-MIXING CHAMBER ARE BYPASSED BY CONNECTING A
TYGON TUBE TO THE EFFLUENT INFLOW, AND RUNNING IT DIRECTLY TO E-2.
IN THIS CASE, SOLENOIDS FOR E-l AND D-l, AND THE PRE-MIXING CHAMBER
ARE DISCONNECTED. D-2 + E-3 = SOX EFFLUENT; D-3 + E-4 = 25X EFFFLUENT,
ETC.
Figure 26. Solenoid valve dilutor system, detailed diagram (not to scale),
144
-------
SOLENOID SYSTEM EQUIPMENT LIST
1. Diluter Glass.
2. Stainless Steel Solenoid Valves
a. 3, normally open, two-way, 55 psi, water, 1/4" pipe size, 9/32"
orifice size, ASCO 8262152, for incoming effluent and dilution
water pipes and mixing chamber pipe.
b. 1, normally closed, two-way, 15 psi, water, 3/8" pipe size, 3/8"
orifice size, ASCO 8030B65, for D-l chamber evacuation pipe.
c. 12, normally closed, two-way, 36 psi, water, 1/4" pipe size,
9/32" orifice size. ASCO 8262C38, for remaining dilution
chambers (D2 - D6) and effluent chamber (E1-E6) evacuation pipes.
3. Stainless steel tubing, seamless, austenetic, 304 grade for freshwater
and 316 grade for saline water.
a. 10 ft of 3/8" OD, 0.035" wall thickness, for dilution water and
effluent pipes.
b. 60 ft of 1/4" OD, 0.035" wall thickness, for dilution water and
effluent pipes.
c. 1 ft of 3/4" OD, 0.035" wall thickness, for standpipe in Dl
chamber.
4. Swagelok tube connectors, stainless steel.
a. 4, male tube connectors, male pipe size 1/4", tube OD 3/8."
b. 2, male tube connectors, male pipe size 1/2", tube OD 3/8."
c. 26, male tube connectors, male pipe size 1/4, tube OD 1/4."
d. 2, male tube connectors, male pipe size 3/8", tube OD 3/8."
e. 2, male adapter, tube to pipe, male size 1/2", tube OD 3/8."
5. 7, 1200 mL stainless steel beakers.
6. Several Ibs each of Neoprene stoppers, sizes 00, 0, and 1; 1 Ib of
size 5.
7. 14 - aquarium (1-20 liters).
8. Magnetic stirrer.
9. 2 - PVC ball valves, 1/2" pipe size.
10. Dilutor control panel - see Fig. 32 and equipment list.
11. Plywood sheeting, exterior grade: one - 4' x 8' x 3/4", one - 4' x
8' x 1/2".
12. Pine or redwood board, 1" x 8", 20 ft.
13. Epoxy paint, 1 gal.
14. Assorted wood screws, nails, etc.
15. 25 ft - 1/4" ID, TEFLON^ tubing, to connect the mixing chambers to
the test chambers.
145
-------
FLOW CONTROL VALVES
DILUTION INFLOW
NORMALLY OPEN
SOLENOID VALVES
n (9/32 in.) ID
L
DILUTION WATER CHAMBERS
^- EFFLUENT CHAMBER
•<- PRE-MIXING CHAMBER
<
N
k
u
Jm
LIQUID LEVEL SWITCH
NORMALLY CLOSED
SOLENOID VALVE
5 rum (3/8 1n.) ID
EFFLUENT CHAMBERS
MIXING CHAMBERS
TEST CHAMBERS 1 20 LITERS CAPACITY
Figure 27. Vacuum siphon dilutor system, general diagram (not to scale)
146
-------
0-1
EFFLUENT
CHAMBER
-n
i
11 —
i —
Jx~|
J
^
^ - —
••*
NORMALLY CLOSED
SOLENOID VALVE
9.5 mm (3/8 in.) ID
_ DILUTION WATER CHAMBERS
VACUUM LINE
tmm 00 CONNECTING TUBE T FORM
10 mm 0 D U SHAPE SYPHON TUBE
6mm 0 D VACUUM LINE TUBE
STAINLESS STEEL HOSE CLAMP
10 Km ID CONNECTING TUBES Y FORM
10 mm 0 D DELIVERY TUBE
120 ml BOTTLE VACUUM BLOCK
10 mm 00 DELIVERY TUBE
10 mm 00 DELIVERY TUBE
10 mm 00 AUTOMATIC SYPHON TDK
EFFLUENT CHAMBERS
0.0 U SHAPE SYPHON TUBE
10 nm I D CONNECTING TUBE Y FORM
10 mm 00 DELIVERY TUBE
MIXING CHAMBER 1200 ml CAPACITY
10 mm 0 D. DELIVERY TUBE
TEST CHAMBERS CAPACITY
1-ZO LITERS
Figure 28. Vacuum siphon dilutor system, detailed diagram (not to scale),
147
-------
VACUUM SIPHON SYSTEM EQUIPMENT LIST
1. Diluter Glass.
2. Stainless steel solenoid valves.
a. 2, normally open, two-way, 55 psi, water, 1/4" pipe size, 9/32"
orifice size, ASCO 8262152, for incoming effluent and dilution
water pipes.
b. 2, normally closed, two-way, 15 psi, water, 3/8" pipe size, 3/8"
orifice size, ASCO 8030B65, for dilution water chamber D-6 and
effluent chamber E-2.
3. Stainless steel tubing, seamless, austenetic, 304 grade for freshwater
and 316 grade for saline water.
a. 60 ft of 3/8" OD, 0.035" wall thickness, for dilution water and
effluent pipes.
b. 20 ft of 5/16" OD, 0.035" wall thickness, for standpipes in
mixing chambers.
c. 1 ft of 3/4" OD, 0.035" wall thickness, for standpipe in Dl
chamber.
4. Swagelok tube connectors, stainless steel
a. 4, male tube connectors, male pipe size 1/4", tube OD 3/8."
b. 2, male tube connectors, male pipe size 3/8", tube OD 3/8."
c. 2, male adapter, tube to pipe, male pipe size 1/2", tube OD 3/8."
d. 2, male tube connectors, male pipe size 1/2", tube OD 3/8."
5. 7, 1,200 ml stainless steel beakers.
6. Several Ibs each of NEOPRENER stoppers, sizes 00, 0 and 1; 1 Ib of
size 5.
7. 14, aquarium (1-20 liters)
8. Magnetic stirrer.
9. 2, PVC Ball valves, 1/2" pipe size.
10. Dilutor control panel equipment - see Fig. 32 and equipment list.
11. 7, 120 ml NALGENER bottles.
12. 3 ft, l-in-2 aluminum bar, for siphon support brackets.
13. Stainless steel set screws, box of 50, for securing SS tubing in
siphon support brackets.
14. Stainless steel hose clamps, box of 10, size #4 or 5,(need 3 boxes).
15. 6, NALGENER T's, 5/16" OD.
16. 12, TYGONR Y connectors, 3/8" I.D.
17. TYGONR tubing, 3/8" OD, 10 ft.
18. Plywood sheeting, exterior grade: one - 4' x 8x x 3/4", one - 4' x
8' x 1/2".
19. Pine or redwood board, 1" x 8", 20 ft.
20. Epoxy paint, 1 gal.
21. Assorted wood screws, nails, etc.
22. 25 ft of 5/16" ID, TEFLONR tubing, to connect the mixing chambers
to the test chambers.
148
-------
EFFLUENT CHAMBER
STANDPIPE
DRAIN
DILUTION WATER CHAMBERS
F2
* A B A
y\ / .^f\xy
OVERFLOW
19 Bin (3/4 1n.) DIAMETER HOLE
12 »«i (1/2 in.) BASTE LINE
19 mm (3/4 in.) HOLE
12 m (1/2 1n.) LINE
(FOR VACUUM SIPHON SYSTEM ONLY)
o
C2
O O O O O
T
107
1
U-33-74-*! l*-38 167 208 — 268 335 -405»j
DRAIN HOLES IN BOTTOM PLATE (Cl AND C2) SHOWN FOR SOLENOID
VALVE DILUTOR SYSTEM. FOR VACUUM SIPHON DILUTOR SYSTEM
DRAIN HOLE IS REQUIRED ONLY FOR CHAMBER £1.
INDIVIDUAL PART SIZE AND NUMBER OF PIECES USING 6 mm
(1/4 In.) PLATE 6LASS. NOTE: 1.6 mm (1/16 1n.) No. 304
6RADE (FOR FRESH WATER) OR No. 316 GRADE (FOR SALINE WATER)
STAINLESS STEEL MAY BE SUBSTITUTED FOR GLASS
WIDTH NO. PIECES (15)
Cl:
C2:
Fl:
F2:
4 (END PLATES)
5 (PARTITIONS)
107 mm X 107 mm - 1 (BOTTOM PLATE FOR El)
1 (BOTTOM PLATE FOR D1-D6)
2 (FRONT AND BACK PANELS FOR El)
447 mm X 231 mm - 2 (FRONT AND BACK PANELS FOR D1-D6)
447 mm X 107 mm
107 mm I 231 TO
INSIDE CELL MEASUREMENTS AND APPROXIMATE VOLUMES
El:
Dl:
D2:
D3:
D4:
DS:
D6:
WIDTH LENGTH HEIGHT VOLUME
95 mm X 95 mm X 231 mm • 2085 mL
125 mm n 95 mm X 200 mm
40 mm X 95 mm X 200 mm
50 mm X 95 mm X 200 mm
60 urn X 95 mm X 200 mm
60 mm X 95 mm X 200 mm
70 mm X 95 «m X 200 mm
2375 mL
760 mL
950 mL
1140 mL
1140 mL
1330 mL
Figure 29. Effluent and dilution water chambers (not to scale)
149
-------
EFFLUENT OVERFLOW
-18 .« (3/4 In.) HOLE
12 •» (1/2 1n.) LINE
TT rr
BOTTOM PLATE (C)
A
92
f
0 O
je/ on
»-203 — »237-
0 0
*• I
*!o°*|
DRAIN HOLES IN BOTTOM PLATE (C) SHOWN FOR SOLENOID VALVE
DILUTOR SYSTEM ONLY. FOR VACUUM SIPHON DILUTOR SYSTEM,
A DRAIN HOLE IS REQUIRED ONLY FOR CHAMBER E2.
INDIVIDUAL PART SIZE AND NUMBER OF PIECES USING 6 mm (1/4 in.)
PLATE GLASS ARE SHOWN BELOW. NOTE: 1/16 in. No. 304 (FOR FRESH WATER)
OR NO. 316 STAINLESS STEEL (FOR SALINE WATER) MAY BE SUBSTITUTED
FOR GLASS.
LENGTH
180 mm X
155 mm X
296 mm
WIDTH
80 mm
80 mm
92 mm
296 mm X 180 mm
NO. PIECES (9)
2 (END PLATES)
4 (PARTITIONS)
1 (BOTTOM PLATE)
2 (FRONT AND BACK PLATES)
INSIDE CHAMBER MEASUREMENTS AND APPROXIMATE VOLUMES:
WIDTH LENGTH HEIGHT VOLUME
E2: 110 mm X 80 mm X 155 mm = 1364 mL
E3: 60 mm X 80 mm X 155 mm = 744 mL
E4: 30 mm X 80 mm X 155 mm * 372 mL
E5: 30 mm X 80 mm X 155 mm = 372 mL
E6: 30 mm X 80 mm X 155 mm = 372 mL
Figure 30. Effluent chambers (not to scale).
150
-------
PRE-MIXING CHAMBER
M-2
M-3
21.
M-1
240 mm
SIDE VIEW
\ 25 mm
END VIEW
M-1
Q*^
J+-
19 mm (3/4 in.) HOLE
12 mm (1/2/in.) LINE
15 mm
1 65 mm
INDIVIDUAL PART SIZE AND NUMBER OF PIECES
USING 6 mm (1/4 in.) PLATE GLASS. APPROXIMATE
CAPACITY 4360 mL.
M-1 125 mm X 153 mm
M-2 125 mm X 153 mm
M-3 240 mm X 165 mm
M-4 240 mm X 125 mm
1 (END PLATE, WITH HOLE]
1 (END PLATE)
1 (BOTTOM PLATE)
2 (SIDE PLATES)
Figure 31. Pre-mixing chamber (not to scale),
151
-------
c
O
O
S-
o
ro
-------
DILUTOR CONTROL PANEL EQUIPMENT LIST*
Designation
Al
CTR-1
ET
Fl
Jl
J2
J3
Ll
L2
L.S.
Pi
Si
$2
SJ2
SJ3
SJ4 -
TDR-1
TDR-2
CKT Description
Encapsulated amplifier
Cycle counter
Elapsed time indicator
Input power fuse
Recepticle
Aux A.C. output jack
Main input power cord
Fill indicator light
Emptying indicator light
Liquid level sensor
(Dual Sensing Probe)
Plug
On-off main power switch (spst)
On-off aux power switch (spst)
Solenoid
Additional Solenoids for
Solenoid Valve System
Time delay relay
Aux time delay relay
Manufacturer
Cutler Hammer 13535H98C
Redington #P2-1006
Conrac #636W-AA H&T
Little fuse 342038
Amphenol 91PC4F
Stand. 3-prong AC Rcpt.
Stand. 3-prong AC male plug
Dialco 95-0408-09-141
Dialco 95-0408-09-141
Cutler Hammer 13653H2
Amphenol 91MC4M
Cutler Hammer 7580 K7
Cutler Hammer 7580 K7
(See Solenoid and Vacuum
System equipment lists)
Dayton 5x829
Dayton 5x829
*Consult local electric supply house.
153
-------
APPENDIX C
BIOASSAY MOBILE LABORATORY PLANS
1. TANDEM-AXLE TRAILER
SPOTLIGHT
AIR CONDITIONER
5
WINDOW
REGION IV
ATHENS GEORGIA
o
EXTERNAL SIDE VIEW
185'
) I
75'
CABINETS
DOOR
S
DILUTERS
WINDOW
><
1 SINK 1 36" STANDING fAplfJETS nni nirjr Tjiniif-
WALL CABINETS OVER DRAWERS
TOP VIEW
Figure 33. Mobile bioassay laboratory, tandem-axle trailer. Above
external side view; below - internal view from above.
154
-------
LIGHTS
SWITCH'S
OUTSIDE SPOTLIGHT
.ACEILING LIGHTS
/7
REAR INSIDE
WEATHERPROOF SPOTLIGHT
O
o
n
LICENSE PLATE REAR OUTSIDE
-AIR CONDITIONER*
. DRAWERS-
n
FRONT INSIDE
FRONT OUTSIDE
Figure 34. Mobile bioassay laboratory, tandem-axle trailer, external
and internal end views.
155
-------
LEFTSIDE
. ELECTRIC OUTLETS,
1
1
\
I
J
1
1
m-
—
DILUTER BOARD [
AIR CONDITIONER^
FRONT [^
STAINLESS STEEL TROUGH STAINLESS STEEL TROUGH
CABINETS WITH
SLIDING DOORS
6" OPENING
WITH
SPRING COVER
ON OUTSIDE
DUAL WHEEL WELL
DRAWERS
24"X 16"
RIGHT SIDE
CABINETS 18" X 12"
SLIDING DOORS
I \
FRONT
ELECTRIC OUTLET!
2 DRAWERS
18"X 18"
SWITCH'S P.UMP UNDER SINK
CABINET LIGHTS
3 DRAWERS
24" X 16"
DUAL WHEEL WELL
36" HIGH CABINETS
SLIDING DOORS
•18'-
Figure 35. Mobile bioassay laboratory, tandem-axle trailer, internal
views of side walls.
156
-------
APPENDIX C
BIOASSAY MOBILE LABORATORY PLANS
2. FIFTH WHEEL TRAILER
DILUTOR SYSTEM
UPPER CABINETS
COUNTER TOP
DRAWERS^
AIR CONDITIONER
DILUTOR SYSTEM
DOUBLE SHELVES
FOR STATIC TESTS
PLAN A
PLANB
SINK
WATER TANK'
32" DOOR
CABINET
UPPER CABINETS
COUNTER TOP
SINK
4t
DRAWERS^ WATER TANK/
DOOR
y
DOUBLE SHELVES
FOR STATIC TEST
UPPER CABINETS
COUNTER TOP
AIR CONDITIONER^
X
X
DILUTER SYSTEMS
-i
•125 FEET •
5 5 FEET
-H
Figure 36. Mobile bioassay trailer, fifth-wheel trailer, internal
view from above.
157
-------
APPENDIX D
CHECK LISTS AND INFORMATION SHEETS^
1. BIOASSAY FIELD EQUIPMENT LIST
Truck
Boards
Cinder blocks
Drums: 500 gal nalgene
55 gal metal - diesel fuel
22 gal
" 5 gal
Gas can
Jacks
Jumper cables
Oil
Pumps: (2)
Home lite
_Hoses & couplings
_Shovels
_Spare tires (trailer, generator)
_Brine shrimp eggs
_Broom
_Brushes (wash)
_Buckets
"Camera
"Chlorine kit (w/chem)
_Cleanser
_Clip board (Ig, sm)
_Cork borer set
_Culture dishes (200 mL, Daphnia)
Daphnia food
_Data sheets: Bioassay (static)
Bioassay (flow-thru)
Oiluter volume delivery
Calibarator delivery sheet
Daily events
Trailer
_Acetone
"Aerators (battery operated)
Air line: Clamps
Aerators (battery operated)
"Air line: Clamps
_Dish pan
_Dish rack
_Dissolved oxygen:
KCL membrane solution
Membranes
"Meter (YSI)
log
_Stones
Tubing
"Valves
_
"Probes
Reagent:
_
_Alkaline azide
H2S04
"0.0375 Na thiousulfate
Alcohol
"Aluminum foil
"Alkalinity analysis (0.02 N H2S04)
Boots: safety
wading
Starch
_ TFfsTilled H20
Emergency road kit
"Enamel pans (Ig.sm)
"Erlenmeyer flasks:
_Batteries
Beakers:
Bottles:
D cell
T50 ml nalgene
"200 mL glass (3 boxes)
".0.
"wash
_S ample
_VOA vials
_500 mL plastic
_Glass organic
Qt. w/teflon liner
500 ml
TOOO ml
"2000 mL
(2)
Extension cords
"Fire extinguishers (2)
"First aid kit
"Fish nets, (Ig.sm)
Tlash light
"Generator: Oil
Filter - fuel
Funnel
Grease gun(wheels)
_Credit card
_Lock/key
Siphon hose
^Prepared by the staff of the Environmental Biology Section,
Ecological Support Branch, Environmental Services Division,
U.S. Environmental Protection Agency, Athens, Georgia 30613
158
-------
BIOASSAY FIELD EQUIPMENT LIST (CONT.)
_Glass cutter
_Gloves (plastic)
Graduated cylinders:
~ 25 mL, 50mL, lOOnt
250nt, 500ml, lOOOmL, 2000mL
_Ground wire & rod
"Hand soap
Hard hats
""Hardness analysis: Buffer
~~EDTA
indicator
_HC1 (20*
_Heaters: Aquarium
~ Space
Hose: Clamps
Connectors
Ice chests
jars: 750 mL (4 boxes)
3 gal (glass) (1)
5 gal (glass) (1)
Sample jugs (2)
JCimwipes (lg,sm)
"Lab coats (2)
_Level
_Lignt 110 V
Log book
"Magnetic stirrers:
JLighted
Other
_Mop
_Paper towels
"Parachute cord
_Paraf ilm
_Pencils, pens
_pH: Meters, Orion
Meters, corning
~ ~~Buffers.4.7.10
Probes (extras)
_Pipets: Bulbs
_Eyedroppers
Volumetric (1 mL, 5 mL, 10 mL)
_Plastic bags
"Quality assurance - SPCP
Rain gear
"Reconstituted hard water
Refactometer
"Respirators (cartridges)
_Rubber bands
_Ruler
_Safety glasses
_Safety manual
_Sample labels
"Scissors
_Screen bioassay cups
"Sea salts (Instant Ocean)
"Separatory funnels & racks
_Silent giants
_Silicon sealant
_Solenoids (spare)
_stainless steel tubing pieces
"Standard Methods Hand Book
_Stirring bars
"Stoppers (assorted)
"Submersible pumps: Ig, sm.
screens
_Super ice
_Tablets (paper)
_Tape: Cellophane
Color coded
Electrician
Masking
Nylon
_Therometers: Dial
~ Glass
_Tools (lock/key)
_Tygon tubing, 1/8", 1/4", 3/8"
_Volumetric flasks (1000 mL, 2000 mL)
_WD40
_Weigh boats
Wire tags
159
-------
APPENDIX D
CHECK LISTS AND INFORMATION SHEETS
2. INFORMATION CHECK LIST FOR ON-SITE INDUSTRIAL
OR MUNICIPAL WASTE TOXICITY TEST.
1. PRE-TRIP INFORMATION
Facility Name:
Address:
Phone number:
Plant Representative(s):
Names, Titles, Addresses of Company Personnel:
A. To Receive Correspondence:
B. To Receive Carbons:
Date of Notification Letter:
State Making Notification and Arrangements:
Special Plant Safety/Security Requirements for EPA Personnel to Observe:
Local Accomodation Recommendations:
Directions to Plant:
Availability of Power Hookups (three 20-amp, 110-V Circuits)
160
-------
Distance from Power Source to Trailer:
Trailer Location:
(Feet)
Possible Source of Dilution Water:
Major Products:
Raw Materials:
Name of Receiving Water:
Schedule of Plant Operation (continuous, weekdays only, etc.)
Treatment Steps:
Treatment Level (BPT, BAT, etc.):
Wastewater Retention Time by Lagoon or Treatment Step:
Lagoon
Designation
Retention Time
Hours Days
Total Wastewater Retention Time: Hours;_
Retention Time Determination: Calculated;
Calculation method:
_Days
Actual
161
-------
Description of Wastewater Tap Point:
Description of Outfall (surface, submerged diffuser, etc.):
Description of Other Waste Disposal Alternatives in Use (spray irrigation,
deepwell, municipal discharge, etc.):
2. ON-SITE INFORMATION
Wastewater General Characteristics:
Color:
Odor:
Solids:
Other:
Serial Number(s) of Discharge(s) to be Tested:
Description of Receiving Water: Uniflow; Tidal;
Approximate amplitude, feet
Color:
Odor:
Solids:
Salinity: High tide ; Low tide
Other:
7Q10: ; Ave. flow
162
-------
Description of Receiving Water Zone of Dilution:
Location and Description of Water Sampling Point(s):
Fresh:
Salt:
Dilution Waste General Characteristics:
Color:
Odor:
Solids:
Other:
Description of Toxicity Test Anomalies (plant production changes, power
failure, rain events, etc.):
Duration
Time & DateTime & Date Anomaly
Description of Plant maintenance:
163
-------
DIAGRAM OF WASTEWATER TREATMENT FACILITIES:
164
-------
3. FOLLOW-UP INFORMATION
Date of follow-up letter:
Wastewater Flow (data supplied by discharger):
Week Prior to Testing Week of Testing
Date Discharge (MGD) Date Discharge (MGD)
Average Discharge (MGD):
Organisms Tested On-site or In-Lab:
Flow-thuStatic
test test
duration duration Test
Species (h) (h) Location Dates Results
Possible Recommended Action as a Result of These Findings:
165
-------
APPENDIX D
CHECK LISTS AND INFORMATION SHEETS
3. DAILY EVENTS LOG
Date: Page of Pages
Site: Day # of Study
Initials: Day # of Flow-through Test
Time: Notes:
166
-------
APPENDIX D
CHECK LISTS AND INFORMATION SHEETS
4. DILUTOR CALIBRATION FORM
Calibration Site:.
Diluter Number:
Calibrator:
Date:"
Effluent
Concentration (%)
Dilution Water (ml)
Trial 1
2
3
Average
Effluent (ml)
Trial 1
2
3
Average
100.0
0
1000
50.0
500
500
25.0
750
250
12.5
875
125
6.25
938
62
3.12
969
31
1.56
984
16
Mixing Chamber (%):_
Waste water (mL):
Dilution Water (mL):
Vol (mL)
Trial 1
2
3
Average
Dilution
Water
Effluent
Remarks:
167
-------
APPENDIX D
CHECK LISTS AND INFORMATION SHEETS
5. DAILY DILUTOR CALIBRATION CHECK
1
£
n
o
4->
i.
>> "
W 4->
•*-> }c/i
I/) ..
3 O> +>*
_ TJ -4-» Wl
0 C *£
1"
z
>-
0£
UJ
>
!U
d
y
^
3
^
>
o:
g
o
C
Ol
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X QJ
0 ^
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O X
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oi h^ ""7
3 OJ ••-
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OJ O ^ cn c z
•^ 0
OJ C
oj a) o «o
i tu*" 3
•— 3C -C C
O (JO
> (D 'f-
UJ •»-*
O
1
0
o
C
o
4->
ie
> L-
i~ a
o z
1
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> 0)
II
3 t-
4-* 3
n c
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j ui r- o CM LO
168
-------
APPENDIX E
COMPUTER PROGRAMS FOR CALCULATING THE LC50 AND 95% CONFIDENCE INTERVAL
(Prepared by Games Dryer)
Five computer programs are described in this section. Four of the
programs are provided for calculating the LC50 and 95% confidence intervals
for acute toxicity test data.
0 Moving average-angle
0 Probit
0 Trimmed Spearman-Karber
o TOXDAT (Multi-method)
A fifth program (LC50) provides a statistical test to determine if LC50s
from different samples are significantly different. All of the programs are
written to run on a POP 11/70 under IAS version 3.1. The program descriptions
include information on the program language, examples of the use of the
program with toxicity test data, and a complete program listing.
169
-------
APPENDIX E
COMPUTER PROGRAMS FOR CALCULATING THE LC50 AND 95% CONFIDENCE INTERVAL
(Prepared by James Dryer)
1. MOVING AVERAGE-ANGLE PROGRAM
This program was written in BASIC PLUS 2, version 1.61, by James Dryer,
EMSL-Cincinnati, and calculates the LC50 and 95% confidence interval for
toxicity tests with fish and invertebrates. The program was based on a method
published by Harris, "Confidence Limits for the LD50 using the Moving
Average-Angle Method," Biometrics Vol. 3, Sept. 1959. The program is
interactive, and prompts the user at each stage of input.
NOTE: a. All concentrations are assumed to have the same number of fish at the
start of the test.
b. For effluent toxicity tests, concentrations must be expressed as
percent effluent.
c. Mortality is expressed as a percent, and not the actual number of
animals that died.
d. Control information is not used in calculating the LC50.
e. Data must be entered from highest to lowest concentration
f. Use of a decimal is mandatory for concentration and mortality values,
but is not used for the "number of concentrations."
EXAMPLE #1
A. INPUT
COMPUTER PROMPT:
RESPONSE:
COMPUTER PROMPT:
RESPONSE:
COMPUTER PROMPT:
RESPONSE:
COMPUTER PROMPT:
RESPONSE:
COMPUTER PROMPT:
RESPONSE:
Enter the number of fish at each concentration at the
beginning of test
20
Number of Concentrations (excluding the control)?
6
Concentration Number 1?
100.0
Mortality in number 1 concentration?
100.0
Concentration Number 2?
50.0
170
-------
COMPUTER PROMPT: Mortality in number 2 concentration?
RESPONSE: 80.0
The remaining prompts and responses are listed in abbreviated form below.
Concentration Number 3? = 25.0
Mortality in number 3 concentration? = 55.0
Concentration Number 4? = 12.5
Mortality in number 4 concentration? = 35.0
Concentration Number 5? = 6.25
Mortality in number 5 concentration? = 10.0
Concentration Number 6? = 3.12
Mortality in number 6 concentration? = 0.
B. OUTPUT
The printout for this set of data is as follows:
Confidence Limits (95%) for the LC50 using the
Lower Limit
LC50
Upper Limit
G = 0.156a
Effl.
Cone.
I*)
100.
50.
25.
12.5
6.25
3.12
15.08
20.6165
29.8379
Logic
Cone.
2.0000
1.69897
1.39794
1.09691
.79588
.494155
1.1784
1.31421
1.47477
Response
(*)
100
80
55
35
10
0
Moving Average-Angle Method
Angle
83.5804
63.3525
47.8692
36.2712
18.4349
6.41962
Average
Angle
64.9340
49.1643
34.1918
20.3753
-
alf G is greater than 0.2, caution should be exercised in using the
LC50 and confidence intervals. As G approaches 1.0, the LC50 and
confidence limits become meaningless.)
171
-------
EXAMPLE #2
A. INPUT:
The following concentrations and mortality data are input as
described in Example #1, above.
Effluent
Concentration
Mortality
100.0
50.0
25.0
12.5
6.25
3.125
100.0
90.0
40.0
0.
0.
0.
B. OUTPUT:
The printout for this set of data is as follows:
Confidence Limits (95%) for
Lower Limit
LC50
Upper Limit
Effl.
Cone.
f a/ \
\ *^ /
100.
50.
25.
12.5
6.25
3.125
24.1671
29.4784
35.2333
the LC50 using the Moving Average-Angle Method
Logic
Cone.
2.0000
1.69897
1.39794
1.09691
.79588
.49485
1.38322
1.4965
1.54695
Response
100
90
40
0
0
0
Angle
83.5804
71.0055
39.2315
6.41962
6.41962
6.41962
Average
Angle
64.6058
38.8855
17.3569
6.41962
G = 0.053 (For the significance of "G," see p. 171)
172
-------
COMPARISON OF LC50s FROM TWO TESTS
The moving average-angle program will also test for statistical
significance between two LC50s, such as obtained from the data input in
Examples #1 and #2, above. The comparison can be called up by inputting
the value, "0.1," for "Number of Concentrations" when prompted by the
program after completion of calculations for the second set of data.
A. INPUT
Enter the number of fish at each concentration at
the beginning of test - 20
Number of Concentrations (excluding the control) - "0.1"
B. OUTPUT
The following output is produced by the program:
Code #
1
vs Code #
2
LC50(1)
29.4784
LC50(2)
20.6165
RL
-.228217
The two LC50s will be judged significantly different if:
(Log10 A - Logic B) + RL = 0
Where:
A = the concentration just below the LC50 value in one set of data
(25% in Example #2), and
B = the like value for the other set (12.5% in Example #1).
In this case:
(Logic A - LogiO B) + RL = (1.39794 - 1.09691) - 0.2282
= 0.07283
Since the sum of these values is greater than zero, the two LC50s are
considered to be significantly different.
For a detailed explanation of the calculation of RL, see Harris,
(1959).
173
-------
LISTING OF COMPUTER PROGRAM FOR MOVING AVERAGE-ANGLE METHOD
10 DIM DK25),D2(25),R1<25),P1(25),AO(25),X<150), J
XH150),Y<150),Y1(150),KO(150)rD7(150)
20 COn Z2,N,M,G,D4,Y2,D3,Y3fL4,L6
21 COM L5fD8,D9,R,R2,KrC1,M1fXO,Y4FD1,D2,R1,P1fAO,K5,X8
JO C4=.4342944819
40 05=2.302585093
60 Z»1.96
61 INPUT" ENTER THE NUMBER OF FISH IN EACH TANK AT BEGINNING OF TEST",N
72 PRINT
80 M=0
90 Z2=Z**2
100 C1=6305.60224
110 FOR I = 1 TO 20
120 X(I)=0
130 AO(I)=0
140 NEXT I
150 E1=N
160 E=E1
170 A1=SQR(0.25/E1>
175 F*ATN
-------
LISTING OF COMPUTER PROGRAM FOR MOVING AVERAGE-ANGLE METHOD (CONT.)
308 PRINT "ftORTALITY (IN PERCENT) IN NUMBER "^"CONCENTRATION = "
310 INPUT RHI)
311 RKI)=RKI>/100
330 NEXT I
340 K9=2
350 60 TO 480
360 FOR I = 1 TO M1
370 PRINT "CONCENTRATION NUMBER ";l;" = "
371 INPUT DKI)
372 IF DKI)=0 THEN 370
380 PRINT "MORTALITY (IN PERCENT) IN NUhBER "Jl^CONCENTRATION = "
381 INPUT RKI)
382 R1(I)=R1(I)/100
383 IF R1(I)=0 THEN 380
385 IF RKI) = .001 THEN 387
386 60 TO 390
387 R1(I)=0
390 NEXT I
410 K9=3
420 GO TO 480
430 FOR I = 1 TO H1
440 PRINT "CONCENTRATION NUMBER ";l;" = "
441 INPUT DKI)
442 IF D1(I)=0 THEN 440
450 PRINT "MORTALITY (IK PERCENT) IN NUMBER ", I;"CONCENTRATION = "
451 INPUT RKI)
452 RKI)=RKI)/100
453 IF RKI)=0 THEN 450
454 IF RKI)*.001 THEN 456
455 GO TO 460
456 RKI)=0
460 NEXT I
470 K9=1
480 FOR I = 1 TO M1
490 IF (RKD)OO THEN 520
500 PKI)=PO
510 GO TO 580
520 IF (RKD-1XO THEN 560
530 IF (R1(I)-1)>0 THEN 2580
540 PKI)=P9
550 60 TO 580
560 A1=SQR(RKI)>
570 F31=A1+.1666666667*A1**3+.075*A1**5+.0446428571*A1**7
571 F32=.0303819444*A1**?+.022372159*A1**11+.0173527644*A1**13
572 F34=.00976160952*A1**19+.0083903358*A1**21+.00731252587*A1**23
573 F33=.0139648437*A1**15+.0115518008*A1**17
576 F3=F31+F32+F33+F34
577 F4=F3*180/PI
175
-------
LISTING OF COMPUTER PROGRAM FOR MOVING AVERAGE-ANGLE METHOD (CONT.)
579 PKI)=F4
580 D2(I)=LOG(DHI»*C4
590 RKI)=100*R1U)
600 NEXT I
610 PRINT "CONFIDENCE LIMITS FOR THE LC(50) USING THE"
611 PRINT " MOVING AVERAGE - ANGLE METHOD"
621 PRINT "CONC LOG CONC MORTALITY ANGLE AVERAGE'
639 M2=M1-1
640 C=3
650 K6=2
660 FOR I = K6 TO M2
670 AO(I)*(P1(I-mP1tP1(I+l)
680 NEXT I
690 IF <0 THEN 850
700 IF (P1(M1)-45)>0 THEN 2560
710 C8=2
720 Y1(M)=AO(«2)
730 X1(M)=D2(H2)
740 J=M1
750 FOR I = 1 TO Ml
760 IF (PKJ)-45»=0 THEN 820
770 J=J-1
780 NEXT I
790 X(M)=D2(1)
800 Y(M)=P1(1)
810 GO TO 1090
820 X(H)=D2(J+1)
830 Y(M)=P1(J+1)
840 GO TO 1090
850 IF 0 THEN 990
860 IF (PHD-45KO THEN 2560
870 C8=1
880 Y(M)=AO(K6)
890 X(«)=D2-45»=0 THEN 960
930 J=J-1
940 NEXT I
950 60 TO 2560
960 XKM)=D2(J)
970 YI(M)sPKJ)
980 GO TO 1090
990 J=M2
1000 FOR I « K6 TO M2
1010 IF
-------
LISTING OF COMPUTER PROGRAM FOR MOVING AVERAGE-ANGLE METHOD (CONT.)
1040
1050
1060
1070
1080
1090
1100
1110
1120
1130
1140
1150
1170
1180
1190
1200
1210
1220
1230
1240
1242
1250
1251
1252
1253
1259
1260
1261
1271
1280
1300
1310
1320
1330
1340
1350
1360
1370
1380
1390
1420
1430
1440
1450
1460
1470
1480
1490
C8=3
XKM)=D2(J)
X-Y(I)
Y3=Y1(I*1)-Y(I+1)
XO=X(I+1)-X(I>
Y4=Y(I)-Y(I+1)
X7=D7(I+1)
X2=D7
-------
LISTING OF COMPUTER PROGRAM FOR MOVING AVERAGE-ANGLE METHOD (CONT.)
1500 X2=D7(I+1)
1530 GOSUB 3350
1540 X3=X7*C5
1550 X4=X2*C5
1560 X5=£XP(X3)
1570 X6=£XPU4)
1580 PRINT KO(I),KO(I+1),X5lX6yR2
1590 IF
U20 Y2=Y1(I)-Y(I)
1630 Y3=YKI+2>-Y(I+2>
1640 XO=X(I+2)-X
1650 Y4=Y(I)-Y(I+2)
1660 X7=D7(I+2)
1670 X2=D7(I)
1700 60 TO 1810
1710 D4=X1(I+2)-Xa+2>
1720 D3=X1(I)-X(I)
1730 Y2=YKI+2)-Y(I+2)
1740 Y3=Y1U)-Y(I)
1750 XO=X(I)-X(I+2)
1760 Y4=Y
-------
LISTING OF COMPUTER PROGRAM FOR MOVING AVERAGE-ANGLE METHOD (CONT.)
2040 Y4=YU+2)-YU + 1)
2050 X7=D7U-M)
2060 X2=D7(I+2)
2090 60SUB 3350
2100 X3=X7*C5
2110 X4=X2*C5
2120 X5=EXP(X3>
2130 X6=EXP(X4)
2140 PRINT KO,X5,X6,R2
2150 NEXT I
2160 M3=«-6
2170 L1=1
2180 L2=3
2190 FOR J= L1 TO L2
2200 J1=J+3
2210 FOR I = J1 TO rt STEP 3
2220 IF -XU>
2240 D3=X1(I)-X(I)
2250 Y2«YKJ)-Y(J)
2260 Y3=Y1
-------
LISTING OF COMPUTER PROGRAM FOR MOVING AVERAGE-ANGLE METHOD (CONT.)
2560 PRINT "AVERAGE AND TRANSFORMED ANGLES DO NOT BRACKET 45 DEGREES'
2570 GO TO 230
2580 PRINT Rim,"HAS EXCEEDED 100 PERCENT"
2590 GO TO 230
2740 PRINT D1(1)fD2(1),R1(1)fP1(1)
2750 J=H1-1
2760 FOR I = 2 TO J
2770 PRINT D1(I),D2(I),R1,P1,P1(H1)
2800 RETURN
2870 05=2.302585093
2880 G=C1/(E*C*Y2**2)
2890 G1=1-G
2?00 U=(SQR(G)/G1)*(SBR(A**2+G1/2))
2910 A5=A/G1
2920 D8=X8+D4*
2?40 X9=D8*C5
2950 L4=EXP(X9)
2960 X9=D9*C5
2970 LA=EXP(X9)
2980 RETURN
3030 C5=2.302585093
3040 6=C1/(E*C*Y2**2>
3050 G1=1-G
3060 A5=(A-6)/G1
3070 U=(SQR(G)/G1)*SQR((A-1)**2+G1/2)
3080 D8=(A5-U)*04tX8
3090 D9=
-------
LISTING OF COMPUTER PROGRAM FOR MOVING AVERAGE-ANGLE METHOD (CONT.)
3360 V2=1641.4//
-------
APPENDIX E
COMPUTER PROGRAMS FOR CALCULATING THE LC50 AND 95% CONFIDENCE INTERVAL
2. PROBIT PROGRAM
This program is written in FORTRAN PLUS IV v.3, and calculates the
LC50 and 95% confidence limits using the Probit transformation. It will
also conduct a test to determine if two sets of toxicity test data are
significantly different.
The program was written by You Yen Yang, a statistician formerly with
the USEPA at the Robert A. Taft Sanitary Engineering Center, Cincinnati,
Ohio, and was modified by James Dryer.
NOTE: a. Control information is not used in calculating the LC50.
b. Data must be input from the HIGHEST to the LOWEST
concentration.
c. Input the NUMBER of animals that died, NOT THE PERCENT.
d. Decimals must be used for data on the concentration and
mortality, but not for the "Number of Concentrations."
EXAMPLE #1
A. INPUT
Number of Concentrations (excluding the control) = 6
Concentration = 100. (%)
No. of animals in this concentration at the start of the test = 20.
No. of animals that died at this concentation = 20.
Concentration = 50.
No. of Animals in this concentration at the start of the test = 20.
No. of Animals that died at this concentation = 16.
Concentration = 25.
No. of animals in this concentration at the start of the test = 20.
No. of Animals that died at this concentation =11.
Concentration = 12.5
No. of Animals in this concentration at the start of the test = 20.
No. of Animals that died at this concentation = 7.
Concentration = 6.25
No. of Animals in this concentration at the start of the test = 20.
No. of Animals that died at this concentation = 2.
Concentration = 3.125
No. of Animals in this concentration at the start of the test = 20.
No. of Animals that died at this concentation = 0.
182
-------
B. OUTPUT:
The printout for this set of data is as follows:
PROBIT ANALYSIS
CONC
L06<10) NUMBER OF NUMBER OF
CONC OBSERVATIONS RESPONSES
RATIO
1
2
3
4
5
6
100.00
50.00
25.00
12.50
6.25
3.12
2.00000
1.69897
1.39794
1.09691
0.79588
0.49485
20
20
20
20
20
20
INTERCEPT
SLOPE
T-VALUE FOR B
WEIGHTED MEAN LOGdO) CONC
UEIGHTED MEAN PROBIT T
CHI SQUARE FOR LINEARITY
P = 0.731
20 1.0000
16 0.8000
11 0.5500
7 0.3500
2 0.1000
0 0.0000
1.49849
2.67833
6.76726 UITH 4 DF
1.29800
4.97497
2.02629 UITH 4 DF
LC50
UPPER LIMIT OF LC50
LOUER LIMIT OF LC50
20.29309
26.53896 (AT .95)
15.57846
UEIGHTED SUM OF SQ OF X
UEIGHTED N
G
6.38408
43.71228
0.08389
For information on the significance of "G" see comments p. 171,
183
-------
EXAMPLE #2
A. INPUT:
The second set of test data listed below, is input as described for
the previous example.
Effluent
Concentration
100.0
50.0
25.0
12.5
6.25
3.125
Number
Responding
20
18
8
0
0
0
B. OUTPUT:
The printout for this set of data is listed below:
PKOBIT ANALYSIS
COHC
LOS(10) NUMIER OF RUMIER OF RATIO
CONC OBSERVATIONS RESPONSES
100.00
50.00
25.00
12.50
4.25
3.12
.00000
.69897
.39794
.09691
0.79588
0.49485
20
20
20
20
20
20
INTERCEPT
SLOPE
T-VALUE FOR 1
WEIGHTED HEAN 106(10) CONC
UEI6HTEB MEAN PROBIT T
CHI SQUARE FOR LINEARITY
P * 0.958
20 1.0000
18 0.9000
e 0.4000
0 0.0000
0 0.0000
0 0.0000
-1.71367
« 3.96015
« 4.96001 IITH 4 DF
1.44267
5.0047J
0.64423 UITH 4 DF
LC50
UPPER LIHIT OF LC50
LOUER LIMIT OF LCSO
21.94440
34.79466
24.09461
(AT .95)
KEI6HTED »UM OF SO OF X
UEIBHTED N
6
0.49243
20.12352
0.15415
For information on the significance of "G" see comments p. 171,
184
-------
LISTING OF COMPUTER PROGRAM FOR PROBIT METHOD
IMPLICIT REAL*8(A-H,0-Z)
DIMENSION X(25),RN(25>,UPRO<25),Y(25),SXX<2)fSXY(2),A<3),B(3),
1SYY(2),SXl2),SY<2>,SWN(2),XBAR(2>fYBAR(2),EP(25),UN<25),
2RP(25>,U<25),R<25),P<25),SN<2>,Tn30),TITLE<20),PRnO),KJ<10)
INTEGER ANSUER,COhP
DATA TT/12.706,4.303,3.182,2.776,2.571,2.444,2.365,2.306,2.262,2.2
128,2.201,2.17?,2.16,2.145,2.131,2.12,2.11,2.101,2.093,2.086,2.08,
22.074,2.069,2.064,2.06,2.056,2.052,2.048,2.045,2.0427
DATA PR/3.36,3.72,3.96,4.16,4.33,5.67,5.84,6.04,6.28,6.64/
DATA KJ/5,10,15,20,25,75,80,85,90,95/
C
i.;
I. PROBIT ANALYSIS
C
t:
TYPE*,' PROBIT - ANALYSIS'
TYPE*,' '
1 VARYS= 0.
STXX = 0.
STXY = 0.
STYY = 0.
SUttX s 0.
SUflY = 0.
STUN = 0.
SUMN = 0.
CHI = 0.
ZZZZ = 0.
1=0
30 1=1+1
IF(I.EQ.3) 60 TO 13
TYPE 45
45 FORHATC NUHBER OF CONCENTRATIONS = ',$)
ACCEPT 44,M
44 FORHATU2)
DO 5 J=1,H
TYPE 880
880 FORMAT(' CONCENTRATION = ',*)
ACCEPT 881,U(J)
881 FORMAT(F5.2)
TYPE 882
882 FORMATC NO. OF ANIMALS AT START OF TEST IN THIS CONCEN. = ',«)
ACCEPT 883,RN(J)
883 FORMAT(F2.0)
TYPE 884
884 FORMAT(' NO. OF ANIHALS RESPONDING IN THIS CONCENTRATION » ',*)
ACCEPT 885,R(J)
885 FORrtAT(F2.0)
TYPE*/ '
185
-------
LISTING OF COMPUTER PROGRAM FOR PROBIT METHOD (CONT.)
TYPE*/ '
X(J)=DLQG10(U(J»
RP(J)=R(J)/RN(J)
IF(RP(J))3,3,4
I EP(J)=0.
GO TO 5
\ T=DSQRT(DLOG(1./RP(J)*»2»
EPU)=T-(2.515517+.802853+H.010328*T*T)/(1.+1.432788*1+.18926V*T+
1T+.001308*T**3)
EP(J)=5.-EP(J)
5 CONTINUE
SSX = 0.
SSY = 0.
XY = 0.
SMX = 0.
SflYX= 0.
SMY = 0.
00 6 J=1,H
SSX=SSX+X(J)**2
XY=XY+X(J)*EP(J)
SMX=ShX+X(J)
I S«Y=S«Y+EPU)
F«=«
B(I)=(XY-S«X*ShY/FH)/(SSX-SMX**2/F«)
Aa) = (S«Y-B(I)*SMX)/FH
' SXX(I) = 0.
SYY(I) = 0.
SXYU) = 0.
SXU) = 0.
SYU) = 0.
SN(I) = 0.
SWN(I) = 0.
DO 10 J=1,«
Y(J)=A(I)+B(I)*X(J)
YY=Y(J)
CALL NID
P(J)=PP
IF(Y(J)-5.)9,8,8
} P(J)=1.-P(J)
1 Q=1.-P(J)
U=Z*Z/(P(J)*Q)
UN(J)=RN(J)*U
UPRO
-------
LISTING OF COMPUTER PROGRAM FOR PROSIT METHOD (CONT.)
SUN(I)=SUN(I)+UN(J)
10 CONTINUE
B/SUN-SXU>**2/SUNU»
A(I)=(SY(I)-B(I)*SX(I))/SUN(I)
DO 11 J=1,M
D=DABS(A(1)+B
500 FORMAT <•' PROBIT ANALYSIS ')
URITE(5,501)
501 FORflATC ')
URITE<5,504)
URITE(5,502)
502 FORMATC L06(10) NUMBER OF NUMBER OF RATIO')
URITE(5,503)
503 FORMAT (' CONC CONC OBSERVATIONS RESPONSES')
URITE(5,504)
504 FORMATC ')
TYPE*,1' •'
XBAR(I)=SX(I)/SUN(I)
YBAR(I)=SY(I)/SUN(I)
UUUU=SXY(I)-SX(I)*SY(I)/SWN(I)
XXXX=SXX ( I ) -SX ( I )**2/SUN ( I )
YYYY=SYY
GO TO 19
187
-------
LISTING OF COMPUTER PROGRAM FOR PROBIT METHOD (CONT.)
18 T=2.0
19 S=YYYY/DF
20 G=S*T*T/
UED=AM+G*(At1-XBAR(I))/(1.-6)+CON*T/(Bm*(1.-6))
UED=10.**UED
FED=10.**FED
GO TO 25
24 UED=0.
FED=0.
25 DO 28 J=1,rt
N=RN(J)
K=R(J)
CON=(1.-6)/SUN(I)+(X(J)-XBAR(I))**2/XXXX
IF(CON)27,26,26
26 CON=DSQRT(CON)
PU=X(J)+G*(X(J)-XBAR(I))/(1.-6)+CON*T/(B(I)*(1.-G»
PL=X(J)+G*(X(J)-XBAR(I))/(1.-G)-CON*T/(B(I)*(1.-G))
PU=A(I)+PU*B(I)
PL=A(I)+PL*B(I)
CALL NID(PU,Z,Q)
IF(PU.GT.5.)Q=1-Q
CALL NID(PL,Z,QQ)
IF(PL.GT.5.)QQ=1.-Q8
GO TO 28
27 0=0.
Q0=0.
28 URITE(5,29)J,U(J),X(J),N,KfRP(J)
29 FOR«AT
URITE(5y213)YBAR(I)
URITE(5f214)YYYYfrthrtM,CHIP
URITE(5,215)ED
URITE(5,2U)UED
URITE(5,217)FED
URITE(5,200)XXXX
URI7E(5f218)SUN(I)
URITE(5,219)G
188
-------
LISTING OF COMPUTER PROGRAM FOR PROBIT METHOD (CONT.)
IF(COttP.EQ."YES-')GO TO 13
TYPE*,"' IF YOU MOULD LIKE TO COMPARE ANOTHER SET OF DATA TO THIS'
TYPE*/ SET, TYPE IN "YES" FOR "COMPARE?". OTHERWISE, TYPE "NO" '
TYPE 651
651 FORHATC COMPARE ?',$.)
ACCEPT 652,COMP
652 FQRHAT(20A2>
IFtCONP.EQ.'NO')BO TO 556
GO TO 30
13 TYPE*,'****WHEN THE CONFIDENCE LIMITS TO P AND LC50 ARE 0,"
TYPE*,'THEY ARE NOT REALLY 0, BUT THEY ARE NOT ESTIMATABLE.'
TYPE*,'6 IS (BY FINNEY S NOTATION) TOO BIG.****'
DF=SUrtN-4.
ND=SUMN-4.
CHIP=CHIPR(DF,CHI)
IF(CHIP-.05)32,32,35
32 S=CHI/DF
IF(DF-30.)33,33,34
33 IDF=DF
T=TT(IDF)
GO TO 36
34 T=2.
60 TO 36
35 S=1.
T=1.96
36 CHI1=ZZZZ-STXY**2/STXX
PP=CHIPR(1.,CHI1)
37 B<3)=STXY/STXX
VARB=S/STXX
VARYS=VARYS*S
POTE=XBAR(1)-XBAR(2)-(YBARU)-YBAR(2))/B(3)
POTEN=10.**POTE
6=S*T*T/(B(3)**2*STXX)
D=POTE-XBAR(1)+XBAR<2)
IF(G-1.)39,38,39
38 G=.9?9?99
39 E=<1.-G)*VARYS+D*D/STXX
IF(E)41,40,40
40 E=T*DSQRT(E)/B(3)
FLOU=XBAR<1)-XBAR(2)+(D-E)/<1.-G)
UPPER=XBAR(1)-XBAR(2)+(D+E)/(1.-6)
UPPER=10.**UPPER
FLOU=10.**FLOU
60 TO 42
41 UPPER=0.
FLOU=0.
42 URITE(5,43)CHI,ND,CHIP
43 FORHAT(10X,'CHI SQ. FOR LINEARITY',F12.4,-'UITH',14,'DF,P='FF8.4)
URITE(5,201)CHI1,PP
201 FORMAT(10X,'CHI SQ FOR PARALLEL ISM',F12.4,' UITH 1 DF,P =',F8.4)
189
-------
LISTING OF COMPUTER PROGRAM FOR PROBIT METHOD (CONT.)
URITE(5,202)B(3)
202 FORMATdOX,'POOLED SLOPE',12X,F1 2.4)
URIT£(5,203)VARB
203 FORMAT(1OX,"VARIANCE FOR B',10X,F12.4)
URITE(5,204)VARYS
204 FORnAT(10X,'VAR(Y8AR(S)-YBAR
WRITE(5,206)UPPER
206 FORMATd OX,'UPPER LIMIT OF R (AT .95>',F11.4)
URITE(S,20?)FLOU
207 FORMAT(10X,'LOUER LIMIT OF R (AT .95)',m»*>
47 FORMAT (/,14X,'INTERCEPT-', 18X,' = ',F14.5)
210 FORMAT (14X,'SLOPE', 22X,'=-',F14.5)
211 FORf1AT(14X,'T-VALUE FOR B',14X,'=',F14.5,' UITH',12," DF')
212 FORrtAT(14X,'UEIGHTED MEAN LOGdO) CONC =',F14.5)
213 FORHAT(14X,-'UEIGHTED MEAN PROBIT Y-',5X,' = ',F14.5)
214 FOR«AT(14X,'CHI SQUARE FOR LINEARITY =',F14.5,' WITH',12,' DF'
1/,14X,' P = ',F5.3)
215 FORMAT(//,14X,aC50',23X,'=',F14.5)
216 FORHATd4X,'UPPER LIHIT OF LC50',8X,'=' ,F14.5,' (AT .95)')
217 FORHAT(14X,'LOUER LIMIT OF LC50',8X,'=',F14.5)
200 FQR«AT(//,14X,'UEIGHTED SUtt OF SQ OF X',4X,'=',F14.5)
218 FORMAT(14X,'UEI6HTED N',17X,'=',F14.5)
21? FORrtATd4X,'G',26X,' = ',F14.5,//////)
IFtPP.GT..05)60 TO 1
DO 51 J=1,10
DO 48 1=1,2
48 EP(I)=XBAR(I)-KPR(J)-YBAR(I))/B(I)
POT£=EP(1)-EP<2)
P(J)=10.**POTE
D=POTE-XBAR(1)+XBAR(2)
E=d .-G)*VARYS+D*D/STXX
IF(E)49,50,50
49 X(J)=0.
Y(J)=0.
GO TO 51
50 E=T*DSQRT(E)/B(3)
X(J)=XBARd)-XBAR(2) + (D-E)/(1.-G)
Y(J)=XBARd )-XBAR(2)*(D+E)/d .-G)
X(J)=10.**X(J)
Y(J)=10.**Y(J)
51 CONTINUE
URITE<5,52)
52 FOR«AT(1H1 ,14X/THE RELATIVE POTENCY AND ITS .95 LIMITS AT'
1/1H ,14X,'DIFFERENT PERCENTILES UHEN PARALLELISM WAS REJECTED',//
21H f31X,'R(L>VXf'R',6Xt'R(U)'f/>
DO 53 J«1,10
IF (J.EQ.6)URITE(5f54)
53 URITE(5,55)KJ(J),X(J),P(J),Y(J)
190
-------
LISTING OF COMPUTER PROGRAM FOR PROBIT METHOD (CONT.)
54 FORHAK/1H )
55 FORMAT(20X,I6/Z'r3F9.4>
IF
ACCEPT 654,ANSUER
654 FORHAH20A2)
IF (ANSUER.EQ.'YES') 60 TO 1
56 STOP
END
SUBROUTINE NID(Y,Z,P>
IftPLICIT REAL*8(A-H,0-Z)
E=DABS (Y-5.)
T=1./(1.+.2316419*E)
Z=0.3989422804*DEXP(-0.5*E*E)
P=Z*<.31938153*T-.356563782*T*T+1.781477937*T**3-1.821255978*T*
1*4+1.33027442?*T**5)
RETURN
END
FUNCTION CHIPR(DF,CHSQ)
IMPLICIT REAL*8(A-H,0-Z)
A=.5*DF
X=.5*CHSQ
IF(X)101f101,100
101 CHIPR=1.
RETURN
100 TERM=1.
SUH = 0.
COFN=A
IF(13.-X)110,110,120
110 IF(A-X)140,140,120
120 CON=1.
FACT=-A
130 T£MP=SUn
SUh=SUMtTERM
COFN=COFN+1.
TER«=TERM*X/COFN
IF(SU«-TEMP)160,160,130
MO CON=0.
FACT=X
150 TE«P=SU«
SUM=SUrt+TERrt
COFN=COFN-1.
RATIO=COFN/X
TERrt=TERM*RATIO
IF
-------
APPENDIX E
COMPUTER PROGRAMS FOR CALCULATING THE LC50 AND 95% CONFIDENCE INTERVAL
3. TRIMMED SPEARMAN-KARBER PROGRAM
This program was written in FORTRAN PLUS IV v.3, at the Fisheries
Bioassay Laboratory, Montana State University, Bozeman, Montana, by M.A.
Hamilton and S.M. Hinkins, and is discussed in the publication:
Hamilton, M.A., R.C. Russo, and R.V. Thurston, 1977, Trimmed
Spearman-Karber method for estimating median lethal concentrations in
toxicity bioassays, Environ. Sci. Technol. 11(7) :714-719. The program is
interactive and prompts the user for data input.
Note: Decimals must be used with data on concentration values and
numbers of animals.
EXAMPLE
A. INPUT:
Enter the number of concentrations: 6
Do you want automatic trim calculations (yes or no): yes
Enter each concentration in ascending order: 3.125
6.25
12.5
25.0
50.0
100.0
Are the number of animals equal in all concentrations? Yes
Enter the number animals per concentration: 20.
Enter the number of animals that died in each concentration: 0.
0.
0.
8.
18.
20.
B. OUTPUT:
The printout includes the following information:
1. Raw data:
Concentrations: 3.125, 6.25, 12.5, 25.0, 50.0, 100.0
Number of organisms in each concentration: 20., 20., 20., 20.,
20., 20.
Mortality in each concentrations: 0., 0., 0., 8., 18., 20.
2. Spearman-Karber Trim (Calculated) 0%
3. Spearman-Karber Estimates:
LC50 = 28.72
Lower 95% confidence limit = 24.03
Upper 95% confidence limit = 34.31
192
-------
LISTING OF COMPUTER PROGRAM FOR THE TRIMMED SPEARMAN-KARBER METHOD
(J*
C* THIS LISTING DESCRIBES THE FORTRAN SUBPROGRAMS FOR CALCULATING * 2.
C* THE TRIMMED SPEARMAN-KARBER ESTIMATE OF THE LC50 UHICH IS IN * 3.
C* USE AT THE FISHERIES BIOASSAY LABORATORY, MONTANA STATE UNIV., * 4.
C* BOZEMAN AS OF JUNE, 1978. THE PROGRAMS UERE NOT ORIGINALLY * 5.
C* WRITTEN FOR CIRCULATION AND HAVE NOT BEEN DOCUMENTED. THE * 6.
C* USER UILL NEED TO URITE A MAIN PROGRAM UITH APPROPRIATE INPUT- * 7.
C* OUTPUT ROUTINES. THE FISHERIES BIOASSAY LABORATORY ASSUMES NO * 8.
C* RESPONSIBILITY FOR THE TECHNICAL ACCURACY OF THESE PROGRAMS. * 9.
C* PROGRAMMERS: M.A. HAMILTON AND S.M. HINKINS. * 10.
C* * 11.
C* MODIFIED ON JUNE 3,1980 BY BRAD GREENUOQD FOR THE EPA AT DULUTH *
C* MINNESOTA TO ALLOW FOR AUTOMATIC TRIM OPTION. *
C* *
C* *
C* FOR REFERENCE, CITE: » 12.
C* HAMILTON, M. A., R.C. RUSSO, AND R.M. THURSTON. 1977. * 13.
C* TRIMMED SPEARMAN-KARBER METHOD FOR ESTIMATING MEDIAN * 14.
C* LETHAL CONCENTRATIONS IN TOXICITY BIOASSAYS. ENVIRON. SCI. * 15.
C* TECHNOL. 11(7): 714-719. CORRECTION 12(4): 417 (1978) * 16.
C* — * 17.
C...CHKSK 19.
IMPLICIT REAL*8 (A-H,0-Z) 20.
INTEGER ANS
DIMENSION X(10),KR(10),N(10>,P(10>,PT(10> 21.
DIMENSION XI (10) i ERLD MODIFICATION
C—
C—
C—
LOGICAL FLAGX,FLAGN,FLAGP,FLAGTtFLAGC,FLAGQ,FLAGLP
LOGICAL*! FLAG, YES, NO
JATA YES,NO/'YVN'/
C—
C—
C—
C MAIN PROGRAM TO PERFORM 100A TRIMMED SPEARHAN-KARBER CALCULATIONS 22.
C CALLS MNTN AND SKA 23.
C ANALYSIS OF LOG(CONCENTRATION) 24.
C-
C—
*"""*
FLAGX'.TRUE.
FLAGN=.TRUE.
FLAGP=.TRUE.
FLAGQB.TRUE.
FLAGT=.TRUE.
FLA6C-.TRUE.
193
-------
LISTING OF COMPUTER PROGRAM FOR THE TRIMMED SPEARMAN-KARBER METHOD (CONT.)
FLA6LP=. FALSE.
C--
C—
C—
£****:¥********:t**********************+t*****:t:*Xt*t:t.***«** 25.
C* * 26.
C* INSERT HERE THE CODE TO READ IN AND/OR * 27.
C* CALCULATE K, ( X(I),N(I), AND P(I) FOR * 28.
C* 1 = 1, K), AND AA * 2V.
C* * 30.
C—
TYPE 10
IF(FLAGLP) PRINT 10
10 FORMAT*' TRIMMED SPEARMAN-KARBER METHOD. MONTANA STATE UNIV',/)
TYPE 20
20 FORMATC ENTER THE NUMBER OF DOSE CONCENTRATIONS: ',$>
ACCEPT*, K
IF( K .GT. 10 ) STOP ' ERROR. TOO MANY DOSES (NAX=10) — ABORTED'
11 CONTINUE
IF(FLAGLP) PRINT 10
IF( FLAGC ) TYPE 31
31 FORMATC DO YOU WANT AUTOMATIC TRIM CALCULATION (YES OR N0)s',$>
IF( FLAGC ) ACCEPT 210, FLAG
1F( (FLAG. EQ. YES). AND. FLA6C) FLAGT=. FALSE.
IF( (FLAG. EQ. YES). AND. FLAGC) AA'O.ODO
IF( FLAGT ) TYPE 30
30 FORMATC ENTER THE % TRIM REQUESTED: ',$)
IF( FLAGT ) ACCEPT*, AA
IF(FLAGC. OR. FLAGT) AA=AA*.01 ! CONVERT TO Z
IF( FLAGX ) TYPE 40, K
40 FORHATC ENTER THE', 13,' DOSE CONCENTRATIONS (IN INCREASING ORDER)
1 /10X,'? ',«)
IF( FLAGX ) ACCEPT*, (XKI), 1 = 1, K)
IF(FLAGQ) FLAG^NO
IF( FLAGQ ) TYPE 51
SI FORMAT (' ARE THE NUMBER OF ANIMALS IN EACH TANK EQUAL?', $)
IF( FLAGQ ) ACCEPT 210, FLAG
IF( (FLAG. EQ. YES) .AND. FLAGQ) FLAGN*. FALSE.
IF((FLAG.EO. YES). AND. FLAGQ) TYPE 52
52 FORMATC ENTER THE NUMBER FISH PER TANK:',*)
IFtFLAGQ. AND. (FLAG. EQ. YES)) ACCEPT*, KT
IF(FLA6Q) GO TO 53
60 TO 55
53 CONTINUE
DO 54 1=1, K
N(I)=KT
194
-------
LISTING OF COMPUTER PROGRAM FOR THE TRIMMED SPEARMAN-KARBER METHOD (CONT.)
54 CONTINUE
55 CONTINUE
IF< FLAGN ) TYPE 50
50 FORMAT! ' ENTER THE TOTAL NUMBER OF ANIMALS IN EACH DOSE EXPOSURE.',
1 /10X,'? ',$>
IF( FLAGN ) ACCEPT*, (N(I), 1=1, K)
IF< FLAGP ) TYPE 60
60 FORflATC ENTER THE TOTAL NUMBER OF ANIHALS ',
1 'THAT EXPIRED IN EACH DOSE.',/10X/? ',*)
IF( FLAGP ) ACCEPT*, (P
-------
LISTING OF COMPUTER PROGRAM FOR THE TRIMMED SPEARMAN-KARBER METHOD (CONT.)
C* * 5?.
C~
C—
.C—
TYPE 90
IF(FLAGLP) PRINT 90
90 FORflAT
91 FORMATC DOSE:',T20, 10F7.2)
TYPE 92,(N(I),I=1,K)
IF(FLAGLP) PRINT 92,
92 FORMATC NUMBER :',T20, 1017)
TYPE 93,(P(I),I*1,K)
IF(FLAGLP) PRINT 93,
ACCEPT 210, ANS
IF(ANS.NE.'N'.AND.ANS.NE.'Y') GO TO 99
IF(ANS.EQ.-'N-') GO TO 101
FLAGX=. FALSE.
FLA6C=. FALSE.
FLAGN=. FALSE.
FLAGP*. FALSE.
FLAGQ=. FALSE.
FLAGLP=.TRUE.
FLAGT=. FALSE.
60 TO 11
101 CONTINUE
TYPE 200
200 FORBATC WOULD YOU LIKE TO MODIFY SOME OF YOUR DATA? ',$)
ACCEPT 210, FLAG
196
-------
LISTING OF COMPUTER PROGRAM FOR THE TRIMMED SPEARMAN-KARBER METHOD (CONT.)
210 FORMAT (AD
IF( FLAG .EQ. NO ) STOP ' RUM COfiPLETED '
C
C
FLA6X=.FALSE.
FLAGC=.FALSE.
FLAGN=.FALSE.
FLAGP=.FALSE.
FLA60=.FALSE.
FLAGLP=.FALSE.
FLAGT=.FALSE.
TYPE 220
220 FORflAK-' DO YOU UANT TO flODIFY ANY OF THE DOSES (YES,NO)? '•,*>
ACCEPT 210,FLAG
IF( FLAG .EQ. YES ) FLAGX=.TRUE.
TYPE 230
230 FORMATC DO YOU UANT TO MODIFY THE NUMBER OF ANIMALS? ',»
ACCEPT 210,FLAG
IF( FLAG .EB. YES ) FLAGN'.TRUE.
TYPE 240
240 FORMATC DO YOU UANT TO MODIFY THE MORTALITIES? ',*>
ACCEPT 210,FLAG
IF( FLAG .EQ. YES ) FLAGP=.TRUE.
TYPE 250
250 FORMATC DO YOU UANT TO CHANGE THE TRIM :»:»:«*t#«***t«:**#*:»::»»****:******************:********** 60.
555 URITE ( 5,500) AA 62.
CALL EXIT 63.
500 FORMAT (F8.4,' SK NOT CALCULABLE') 64.
STOP 65.
END 66.
C* HNTN 67.
SUBROUTINE HNTN(K,N, PT) 68.
C* M. HAMILTON * DEPT. OF MATH. * MONTANA STATE UNIVERSITY*BOZEHAN * 6?.
C SUBROUTINE TO ADJUST PROPORTIONS TO BE MONOTONE INCREASING 70.
C***K=NO. OF DOSE LEVELS 71.
C***N(I)-NO. OF SUBJECTS AT DOSE I 72.
C***PT(I)=PROPORTION OF RESPONSE AT DOSE I; ENTERS AS OBSERVED 73.
C*** PROPORTION AND RETURNS AS ADJUSTED PROPORTION 74.
C********** 75.
IMPLICIT REAL*8
-------
LISTING OF COMPUTER PROGRAM FOR THE TRIMMED SPEARMAN-KARBER METHOD (CONT.)
NS=0 79.
1=0 80.
16 CONTINUE 81.
1=1+1 82.
DD=PT(I + 1)-PTU> 83.
IF(DD .GT. -1.0D-8) 60 TO 8 84.
KA = I 85.
IF (I .EQ. 1) 60 TO 10 86.
J-I+1 87.
20 CONTINUE 88.
J=J-1 89.
IF 100.
IF(DD .LT. 1.0D-8) GO TO 11 101.
KB = J 102.
GO TO 12 103.
11 CONTINUE 104.
12 CONTINUE 105.
NCUM=0 106.
CUH=O.ODO 107.
DO 13 J=KA,KB 108.
CUM = CUH + PTU)*N(J) 109.
13 NCUM = Ncun + N
-------
LISTING OF COMPUTER PROGRAM FOR THE TRIMMED SPEARMAN-KARBER METHOD (CONT.)
C***K=NO. OF DOSE LEVELS 127.
C***Z(I)=DOSE LEVEL I, ENTERED IN INCREASING ORDER 128.
C***N(I)=KlinBER OF SUBJECTS AT DOSE Z(I> 129.
i;***PTa)=PROPORTION OF RESPONSE AT DOSE Z(I); HONDECREASIN6 IN I 130.
C***PT=P-TILDA OF E,S, i T (1977) PAPER 131.
C********CALLS SUBROUTINE SK( )********* 132.
O*****«******************************************************** 133.
IhPLICIT REAL*8
-------
LISTING OF COMPUTER PROGRAM FOR THE TRIMMED SPEARMAN-KARBER METHOD (CONT.)
IF(DABS(A-0.5) .GT. 1.00-8) GO TO 2 16?.
SKAEST=Z(JLT)+(Z(JLT+1>-Z 199.
IF (KLT .EO. (KUT-D) GO TO 2 200.
IF (KLT .EQ. (KUT-2)) GO TO 3 201.
C...CALCULATIONS FOR (KUT-KLT) .GE. 3 202.
Y1=(Z(KLT+1)-Z(KLT))*(A-PT(KLT))*(PT(KLT+1)-A) 203.
Y1=Y1/(2.DO*(PT(KLT+1)-PT(KLT))) 204.
Y2=(Z(KLT)+Z(KLT+1))*(PT(KLT*1)-A)/2.DO 205.
Y4=(Z(KUT-1)+Z(KUT))*(1.DO-A-PT(KUT-1»/2.DO 206.
Y5=(Z(KUT)-Z(KUT-1))*(1.DO-A-PT(KUT-1))*
-------
LISTING OF COMPUTER PROGRAM FOR THE TRIMMED SPEARMAN-KARBER METHOD (CONT.)
V1=(Z*(())**2) 215.
V1 = W**2)*PT 216.
V2=(Z(KLT+1)-Z*PT(KLT+1)*(1.DO-PT-Z(KUT-1»*«U.DO-A-PT*n.DO-PT(KUT))/(N(KUT)*4.DO> 224.
V3=O.DO 225.
IF (KUT .EQ. (KLT+3)) 60 TO 8 226.
KLT2=KLT+2 227.
KUT2=KUT-2 228.
DO 9 I=KLT2,KUT2 229.
9 V3=V3+«Z(I + 1)-ZU-1»**2)*PTU)*<1.DO-PT**2> 231.
60 TO 10 232.
C...CALCULATIONS FOR (KUT-KLT) .EQ. 1 233.
2 CONTINUE 234.
SKEST=Z(KLm<0.5DO-PT-PT 237.
V3=((Z(KUT)-Z(KLT))/(2.DO*(PT(KUT)-PT(KLT))**2))**2 238.
VARSK=V3*(V1+V2) 239.
C *** WARNING *** 240.
C IF PT(KLT)=0.0 J/OR PT(KUT)=1.0 THEN 241.
C VARSK IhrtEDIATELY ABOVE IS INAPPROPRIATE 242.
GO TO 10 243.
C...CALCULATIONS FOR (KUT-KLT) .EQ. 2 244.
3 Y1=(A-PT(KLT))*(PT(KLT+1)-A)*(Z(KLT*1)-2(KLT)) 245.
Y1=Y1/(2.DO*(PT(KLT+1)-PT*(((PT(KLT*1)-A)/(PT(KLT+1)-PT(KLT)))**2) 252.
V1=(V1**2)*PT(KLT)*(1.DO-PT(KLT))/(4.DO*N(KLT» 253.
V2=(Z(KLT)-Z(KUT))/2.DO 254.
VV=(Z(KUT)-Z(KLT+1))/2.DO 255.
V2=V2+VV*<«PT(KLT+2)-1.DO+A)/(PT(KLT*2)-PT(KLT+1)))**2) 256.
VV=(Z(KLT+1)-Z(KLT))/2.DO 257.
V2=V2-VV*(«A-PT(KLT))/(PT
-------
APPENDIX E
COMPUTER PROGRAM FOR CALCULATING THE LC50 AND 95% CONFIDENCE INTERVAL
4. PROGRAM FOR STATISTICAL COMPARISON OF LC50s
This program is written in BASIC PLUS 2, version 1.61, by James Dryer
and determines whether two LC50 values are significantly different by
comparing the two LC50 values and their upper confidence limits using the
standard error of mean differences. The program is based on the report
by Sprague and Fogels (1977), "Watch the Y in Bioassay," published in the
Proceedings of the 3rd Aquatic Toxicity Workshop, Halifax, Nova Scotia,
November 2-3, 1976, Environmental Protection Service Technical Report
No. EPS-5-AR-77-1, Halifax, Nova Scotia, Canada, pp. 107-118.
The program uses the two LC50s and their upper confidence limits
(UL), and is based on the following relationships:
Log10
UL(1)
LC50(1)
2
+
Log10
UL(2)
LC50(2)
2
Where:
H =
7
L ~
Larger LC50
Larg
Smal
Smaller LC50
If Z is larger than H, the LC50s are considered to be significantly
different.
If Z is smaller than H, the difference between the LC50 values is not
considered significant.
202
-------
EXAMPLE
A. INPUT
LC50 of first test? 20.62
Upper Confidence Limit of First Test? 29.84
LC50 of second test? 29.48
Upper Confidence Limit of First Test? 35.23
The following calculations are carried out by the program:
G =
Log10
29.84
2
Loa 35.23
910 29.48
2
H = 10G = 10 0.1619 = 1.51
Z =
29.48
20.62
1.43
Results:
Z is smaller than H. Therefore, the LC50s are not considered to be
significantly different.
B. OUTPUT
The following output is produced by the program:
"THE LC50 VALUES ARE SUFFICIENTLY CLOSE AND MAY BE
FROM THE SAME POPULATION"
H = 1.51
Z = 1.43
203
-------
LISTING OF COMPUTER PROGRAM FOR STATISTICAL COMPARISON OF LC50S
10 INPUT "LC50 OF FIRST TEST",A
20 INPUT "UPPER CONFIDENCE LIMIT OF FIRST TEST",C
30 INPUT "LC50 OF SECOND TEST",B
40. INPUT "UPPER CONFIDENCE LIMIT OF SECOND TEST",D
50 E=(L0610(C/A))**2
60 F=(LOG10(D/B)>**2
70 G=SQR(E+F)
80 H=10**(G)
90 IF A>B THEN 120
100 Z=B/A
110 GO TO 130
120 Z=A/B
130 IF H>Z THEN 160
140 PRINT "THE LC50 VALUES ARE SUFFICIENTLY REHOVED AND ARE PROBABLY"
141 PRINT "FROH DIFFERENT POPULATIONS."
142 PRINT "A = ",H
143 PRINT "B = ",Z
150 GO TO 170
160 PRINT'THE LC50 VALUES ARE SUFFICIENTLY CLOSE AND MAY BE FROH THE '
161 PRINT "SAME POPULATION"
162 PRINT "A = ",H
163 PRINT "B = %Z
170 END
204
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APPENDIX E
COMPUTER PROGRAMS FOR CALCULATING THE LC50 AND 95% CONFIDENCE INTERVAL
5. TOXDAT MULTI-METHOD PROGRAM (BIONOMIAL, MOVING AVERAGE,
AND PROBIT METHODS)
This program is written in BASIC PLUS 2, Version 1.61. It was
originally obtained from Charles Stephen, Environmental Research
Laboratory, USEPA, Duluth, Minnesota, and calculates the LC50 and 95%
confidence limits by each of the following three methods:
1. Binomial Distribution
2. Moving Average
3. Probit
The program was obtained from the Environmental Research Laboratory,
U. S. Environmental Protection Agency, Duluth, Minnesota.
NOTE: a. Data are input from highest to lowest concentration.
b. Decimals must be used for concentration values and number of
organisms that died, but not for "Number of Concentrations."
EXAMPLE #1
A. INPUT:
Enter the number of concentrations - it should be greater than 1 ? 6
Enter in descending order the concentrations used in the test:
? 100.
? 50.
? 25.
? 12.5
? 6.25
? 3.125
Enter the number of animals exposed at each concentration:
? 20.
? 20.
? 20.
? 20.
? 20.
? 20.
205
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Enter the number of organisms that died at each concentration:
? 20.
? 16.
? 11.
? 7.
? 2.
? 0.
B. OUTPUT:
Where do you want your output to go? On your terminal (TT) or line
printer (LP)?
Enter TT or LP? TT
A printout of the results is as follows:
CQNC.
100
50
25
12.5
6.25
3.125
NUMBER
EXPOSED
20
20
20
20
20
20
NUMBER
DEAD
20
16
11
7
2
0
PERCENT
DEAD
100
80
55
35
10
0
BINOMIAL
PROB.(Z)
.0000953674
.590897
41.1901
13.1588
.0201225
.0000953674
THE BINOMIAL TEST SHOUS THAT 6.25 AND 50 CAN BE
USED AS STATISCALLY SOUND CONSERVATIVE 95 PERCENT
CONFIDENCE LIMITS SINCE THE ACTUAL CONFIDENCE LEVEL
ASSOCIATED UITH THES€ LIMITS IS 99.389 PERCENT.
AN APPROXIMATE LC50 FOR THIS SET OF DATA IS 21.0569
»»»»RESULTS CALCULATED USING THE HOVIN6 AVERAGE METHOD
SPAN G LC50 95 PERCENT CONFIDENCE LIMITS
5 .05135 19.865 15.6704 25.5024
>»»»RESULTS CALCULATED USING THE PROSIT METHOD
ITERATIONS
6
G H
.0838854 1
GOODNESS OF FIT PROBABILITY
.730927
SLOPE = 2.67833
95 PERCENT CONFIDENCE LIMITS = 1.90261
AND
3.45405
LC50 * 20.2931
95 PERCENT CONFIDENCE LIMITS
LCI » 2.74538
95 PERCENT CONFIDENCE LIHITS
15.5785 AND 26.539
1.16044 AND 4.52059
206
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EXAMPLE #2
A. INPUT:
The following data are entered as described in Example #1:
Effluent
Concentration
100.
50.
25.
12.5
6.25
3.125
Number of
Organisms
Exposed
20.
20.
20.
20.
20.
20.
Number of
Organisms
That died
20.
18.
8.
0.
0.
0.
B. OUTPUT:
The printout of the results is as follows:
CONC.
100
50
25
12.5
4.25
3.125
NUMBER
EXPOSED
20
20
20
20
20
20
NUMBER
DEAD
20
18
8
0
0
0
BINOMIAL
.0000953674
.0201225
25.1722
.0000953674
.0000953674
.000095,5674
PERCENT
DEAD
100
90
40
0
0
0
THE BINOMIAL TEST SHOUS THAT 12.5 AND 50 CAN BE
USED AS STATISCALLY SOUND CONSERVATIVE 95 PERCENT
CONFIDENCE LIHITS SINCE THE ACTUAL CONFIDENCE LEVEL
ASSOCIATED UITH THESE LIMITS IS 99.9798 PERCENT.
AH APPROXIMATE LC50 FOR THIS SET OF DATS IS 28.3375
»»»/>RESULTS CALCULATED USING THE MOVING AVERAGE METHOD
SPAN G LC50 95 PERCENT CONFIDENCE LIMITS
4 .0513499 29.7135 24.135 37.2718
>»»»RESULTS CALCULATED USING THE PROSIT METHOD
ITERATIONS
8
H
.156151
GOODNESS OF FIT PROBABILITY
.958014
SLOPE = 5.96063
95 PERCENT CONFIDENCE LIKITS = 3.60523
AND
8.31602
LC50 = 28.9644
95 PERCENT CONFIDENCE LIMITS = 24.0946 AND 34.7947
LCI = 11.7897
95 PERCENT CONFIDENCE LIMITS = 6.29974 AND 15.8096
207
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SUMMARY OF RESULTS:
A. Example #1
(1) Binomial Test
LC50 =21.1
Upper 95% confidence limits = 50.0
Lower 95% confidence limits = 6.25
(2) Moving Average
LC50 = 19.9
Upper 95% confidence limits = 25.5
Lower 95% confidence limits = 15.7
(3) Probit
LC50 = 20.3
Upper 95% confidence limits = 24.4
Lower 95% confidence limits = 16.9
A. Example #2
(1) Binomial Test
LC50 = 28.3
Upper 95% confidence limits = 50.0
Lower 95% confidence limits = 12.5
(2) Moving Average
LC50 = 29.7
Upper 95% confidence limits = 37.3
Lower 95% confidence limits = 24.1
(3) Probit
LC50 = 29.0
Upper 95% confidence limits = 34.8
Lower 95% confidence limits = 24.1
208
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LISTING OF TOXDAT MULTI-METHOD PROGRAM
10 DIM A(10),B<10)fC(10),D
30 INPUT "NUMBER OF CONCENTRATIONS =";n
40 FOR J=1 TO n
50 PRINT "CONCENTRATION NUMBER";J,"'= "
51 INPUT C3
52 C.5 THEN 290
270 N2=D(J)
280 GO TO 300
290 N2=EU)-DU)
300 FOR N=0 TO N2
310 B2,B3=1
320 FOR IMN1-N+1) TO N1
330 B2=B2*I
340 NEXT I
350 FOR I = 2 TO N
360 B3=B3*I
370 NEXT I
380 B1=B1+(B2/B3)
390 NEXT N
400 B(J)=100*B1*(0.5*N1)
410 PRINT C(J>,E(J>,D(J),100*P
450 A1=SQR(D(J)/(E(J) + 1»
460 A2=ATN(A1/(SQR(1-A1*A1)»
470 IF D(J)=E(J) THEN 510
480 A3=SQR((D(J) + 1)/(E(J)tD)
490 A4-ATN(A3/(SQR(1-A3*A3)»
500 GO TO 520
510 A4=1.57079633
209
-------
LISTING OF TOXDAT MULTI-METHOD PROGRAM (CONT.)
520 A(J>=180*(A2+A4)/(2*PI)
530 NEXT J
540 FOR J=1 TO n
550 IF P(J)>0 THEN 610
560 IF J>1 THEN 5?0
570 Y(J)=-2.67*(1-PU+1>/2)
580 GO TO 810
590 Y(J)=-2.A7*(1-PU-1)/2>
600 GO TO 810
610 IF PUK1 THEN 665
620 IF J0 THEN 790
770 X3=C(J)
780 GO TO 800
7*0 IF X3=C(J) THEN 810
800 KO*KO+1
810 NEXT J
1000 84,85,66,67=0
1010 FOR J=1 TO fl
1020 IF BUK1776 THEN 1040
1030 GO TO 1230
1040 IF PUK.5 THEN 1090
1050 IF BU»2.5 THEN 1090
1060 B4=C(J>
1070 B5=B(J)
1080 NEXT J
1090 FOR J=H TO 1 STEP -1
1100 IF P(J».5 THEN 1150
1110 IF B(J)>2.5 THEN 1150
1120 B6=C(J)
1130 B7=B(J)
1140 NEXT J
1150 B8=100-B5-B7
210
-------
LISTING OF TOXDAT MULTI-METHOD PROGRAM (CONT.)
1160 IF B4>0 THEN 1190
1170 PRINT "THE BINOMIAL TEST SHOUS THAT ";B6;"AND + INFINITY CAN BE"
1180 60 TO 1200
1190 PRINT "THE BINOMIAL TEST SHOUS THAT ";B6;MAND ";B4J" CAN BE"
1200 PRINT "USED AS STATISCALLY SOUND CONSERVATIVE 95 PERCENT"
1210 PRINT "CONFIDENCE LIMITS SINCE THE ACTUAL CONFIDENCE LEVEL"
1220 PRINT "ASSOCIATED UITH THESE LIMITS IS "JB8;" PERCENT."
1230 FOR J=1 TO M
1240 IF PUK0.5 THEN 1270
1250 61=J
1260 NEXT J
1270 FOR J=M TO 1 STEP -1
1280 IF P(J»0.5 THEN 1310
1290 G2=J
1300 NEXT J
1310 IF P(G2KP(G1> THEN 1340
1320 N1 = ((C(G1))*r.5
1330 60 TO 1370
1340 G3M45-A(G2))/
1350 G4=L(G2)+(L(G1)-L(62))*G3
1360 M1=10~G4
1370 PRINT "AN APPROXIMATE LC50 FOR THIS SET OF DATA IS "JM1
1380 PRINT
1390 IF K0>1 THEN 1440
1400 PRINT 'WHEN THERE ARE LESS THAN TWO CONCENTRATIONS AT UHICH THE PERCENT1
1410 PRINT "DEAD IS BETUEEN 0 AND 100, NEITHER THE MOVING AVERAGE NOR THE"
1420 PRINT "PROBIT METHOD CAN GIVE ANY STATISTICALLY SOUND RESULTS."
1430 GO TO 3850
1440 14=0
1450 FOR S=H-1 TO 1 STEP -1
1460 FOR N=1 TO M-S+1
1470 IF S<3 THEN 1580
1480 FOR J=1 TO S-2
1490 J2 THEN 1640
1620
211
-------
LISTING OF TOXDAT MULTI-METHOD PROGRAM (CONT.)
1630 60 TO 1650
1640 M(J)=U2-T(K1)*W1
1650 G(JfN)=MU)*(0.5+E(J»
1660 V(N)=V(N)+G(J,N)
1670 K1=K1+1
1680 NEXT J
16?0 K(N>=0
1700 F(N)=0
1710 FOR J=N TO N+S-1
1/720 K(N)=K(N)+G(J,N)*L(J)/V(N)
1730 F(N)=F(N)+G(J,N)*A(J)/V(N)
1740 NEXT J
1750 F(N) = (INT((1E6)*F(N>+0.5»/(1E6)
1760 NEXT N
1770 K2=1
1780 FOR J=1 TO M-S
1790 X(K2)=0
1800 K4=0
1810 FOR N=J TO J+S
1820 IF P(N)=0 THEN 1850
1830 IF P(N)=1 THEN 1850
1840 K4=K4+1
1850 NEXT N
1860 IF K4<2 THEN 1920
1870 IF FU)=FU-H> THEN 1920
1880 IF FUK45 THEN 1920
1890 IF FU+1)>45 THEN 1920
1?00 X(K2)=J
1910 K2=K2+1
1920 NEXT J
1930 IF X(1>=0 THEN 2360
1?40 IF 14=1 THEN 1980
1?50 PRINT "»»»»RESULTS CALCULATED USING THE flOVING AVERAGE METHOD"
1?60 PRINT "SPAN","G",MLC50"f"95 PERCENT CONFIDENCE LIMITS"
1970 14=1
1980 FOR N=1 TO K2-1
1990 P=X(N)
2000 Q=X(N)+1
2010 Y=F(Q)-F(P)
2020 A=(45-F(P))/Y
2030 M2=K(P)+(K(Q)-K(P))*A
2040 H3=10"«2
2050 V1,V2,V3=0
2060 FOR J-1 TO S
2070 V1=V1 + <(3282.81*
2080 V2=V2+((3282.81*(G(Q+J-1,Q)"'2))/((4*E(0+J-1)+2)*(V(Q))'V2))
2090 IF J*1 THEN 2110
2100 V3*V3+((3282.81*G(P+J-1,P)*G<8+J-2,Q»/<(4*E
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LISTING OF TOXDAT MULTI-METHOD PROGRAM (CONT.)
2110 NEXT J
2120 V4=VUV2-2*V3
2130 V5=V1-V3
2140 2=1.96
2150 G=Z*Z*V4/U"2)
2160 IF G=1 THEN 2330
2170 R=V1-2*A*V5-KA~2)*V4-G*
2200 V7=(A-G*V5/V4)/(1-G)
2210 L2=K(P)MK(Q)-K(P))*1 THEN 2470
2430 PRINT "WHEN THERE ARE LESS THAN TUO CONCENTRATIONS AT UHICH THE PERCENT"
2440 PRINT "DEAD IS BETUEEN 0 AND 100, THE PROBIT METHOD CANNOT GIVE ANY"
2450 PRINT"STATISTICALLY SOUND RESULTS."
2460 GO TO 3850
2470 S1,S2,S3,S4,K3FI3=0
2480 FOR J-1 TO h
2485 SI-SUL(J)
2490 S2=S2+Y(J)
2500 S3=S3+L(J)*L(J)
2510 S4=S4+L(J)*Y(J)
2520 NEXT J
2530 B=(S4-S1*S2/M)/(S3-S1*S1/H)
2540 A=(S2-B*S1)/M
2550 F1,F2,F3,F4,F5,F6=0
2560 FOR J=1 TO M
2570 Z(J)=A*B*L(J)
213
-------
LISTING OF TOXDAT MULTI-METHOD PROGRAM (CONT.)
2580 Z1=ABS(ZU»
2590 IF Z1>8 THEN 2700
2600 Z2=0.39894228*EXP(-.5*Z1*Z1)
2410 Z3=1/(1+0.2316419*Z1)
2620 Z4=((1.330274*23-1.821256)*Z3+1.781478>*Z3
2630 P=Z2*Z3*«Z4-.356563782)*Z3+.31938153)
2640 IF ZUKO THEN 2680
2650 Q=P
2660 P=1-P
2670 GO TO 2730
2680 Q=1-P
2690 GO TO 2730
2700 P=1E-16
2710 Z2=1E-15
2720 GO TO 2640
2730 U3=E(J)*(Z2/P)*(Z2/Q)
2740 U4=Z(J)+(P(J)-P)/Z2
2750 F1=F1+L(J)*U3
2760 F2=F2+U3*U4
2770 F3=F3+L(J)*L20 THEN 3220
3020 J=1
3030 FOR I*V TO 2 STEP -2
3040 J=J*I
3050 NEXT I
214
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LISTING OF TOXDAT MULTI-METHOD PROGRAM (CONT.)
3060 C2=Cr=0.001 THEN 3200
3190 C6=0
3200 V=M-2
3210 60 TO 3250
3220 C6=0
3230 60 TO 3250
3240 C6=1492
3250 IF C6<=0.05 THEN 3290
3260 T5=1.96
3270 H=1
3280 60 TO 3370
3290 IF V=1 THEN 3330
3300 IF V=2 THEN 3350
3310 T5=1.95996+1/(.413*V-.423)
3320 60 TO 3360
3330 T5=12.706
3340 GO TO 3360
3350 T5=4.303
3360 H=C1/V
3370 G=H*T5~2/B"2/F8
3380 E1=SQR(H/F8)
3390 E2=B-T5*E1
3400 £3=B+T5*E1
3410 PRINT "»»>»R£SULTS CALCULATED USING THE PROBIT METHOD"
3415 PRINT
3420 PRINT-ITERATIONSVGVHVGOODNESS OF FIT PROBABILITY"
3430 IF C6=1492 THEN 3460
3440 PRINT K3,G,H,C6
3450 60 TO 3470
3460 PRINT K3,GfH," (CANNOT BE CALCULATED)"
3470 PRINT
3480 IF C6>0 THEN 3510
3490 PRINT "A PROBABILITY OF 0 HEANS THAT IT IS LESS THAN 0.001"
3SOO PRINT
3510 IF C6>0.05 THEN 3550
3520 PRINT "SINCE THE PROBABILITY IS LESS THAN 0.05, RESULTS CALCULATED'
215
-------
LISTING OF TOXDAT MULTI-METHOD PROGRAM (CONT.)
3530 PRINT "USING THE PROFIT METHOD PROBABLY SHOULD NOT BE USED."
3540 PRINT
3550 PRINT-SLOPE =",B
3560 PRINT "95 PERCENT CONFIDENCE LIMITS ="JE2; " AND ";E3
3570 PRINT
3580 «4=-A/B
35?0 H5=10"<«4)
3600 PRINT "LC50 = %M5
3610 IF 6=1 THEN 3770
3620 H1=H*«1-G)/F6+(M4-F9r2/F8)
3630 IF HKO THEN 3770
3640 H2=SQR(H1)
3650 L4=M4+G*(M4-F9)/(1~G)-H2*T5/((ABSF9 THEN 3730
3710 PRINT "95 PERCENT CONFIDENCE LIMITS = 0 AND " ;U5
3720 GO TO 3790
3730 PRINT "95 PERCENT CONFIDENCE LIMITS = "JL5; " AND + INFINITY'
3740 GO TO 3790
3750 PRINT "95 PERCENT CONFIDENCE LIMITS = " ;L5j " AND " ;U5
3760 GO TO 3790
3770 PRINT "95 PERCENT CONFIDENCE LIMITS =0 AND + INFINITY"
3790 IF 13=1 THEN 3850
3800 M=(-2.32679-A)/B
3810 n5=10~M4
3820 PRINT "LCI = ",rt5
3830 13=1
3840 GO TO 3610
3850 GO TO 10
3860 END
216
U. S. GOVERNMENT PRINTING OFFICE: 1986/646-116/20722
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