EPA-6QQ/4-S5-OU
September 1985
SHORT-TERM METHODS FOR ESTIMATING
THE CHRONIC TOXICITY OF EFFLUENTS AND RECEIVING WATERS
TO FRESHWATER ORGANISMS
Edited
William 8. Homing, II
and
Cornelius I. Weber
Biological Methods Branch
Environmental Monitoring and Support Laboratory- Cincinnati
Cincinnati, Ohio 45268
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY - CINCINNATI
-• OFFICE OF RESEARCH AND DEVELOPMENT
U. 5. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY ^S|rr^ p*
OFFICE OF RESEARCH AND DEVELOPMENT C *
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OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
CINCINNATI. OHIO 4.5268
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*"
DATE: December 4, 1985
SUBJECT: Advance Copy of Chronic Toxicity Test Manual for Use in the New
Water Quality Based Approacn to Permit Writing
J
FROM: Cornelius I. Weber, Ph.D., Chief
Biological Methods Branch
TO: Water Management Division Directors
Environmental Services Division Directors
Permits Branch Chiefs
Regional Water Quality Coordinators
Senior Regional Biologists
Attached is a prepublication copy of the manual, "Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Freshwater Organisms," prepared at the request of the Office of Water
Enforcement and Permits for use in the water quality based approach to
permitting. Publication.of this manual was originally scheduled for
October, 1985, but was delayed because of budget restrictions during the
first two months of FY-86, when the Agency was operating under continuing
resolutions.
Funds are now available for printing, and we anticipate that copies
of the manual will be received from the printer in February. To obtain
additional copies at that time, please contact our Publications
Assistant, Betty Thomas, FTS 684-7301.-
Attachment (1):
As Stated
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NOTICE
This document has been reviewed In accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade
names or commercial products does not constitute endorsement or
recommendation for use.
n
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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. The Environmental Monitoring and Support Laboratory -
Cincinnati (EMSL-Cincinnati) operates an Agency-wide quality assurance
program to assure standardization and quality control of systems for
monitoring water and wastewater, and 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, wastewater, and municipal sludge.
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. _..,.,«
The Federal Water Pollution Control Act Amendments of 1972
(PL 92-500) and the Clean Water Act (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 estimating the chronic toxicity of effluents and
receiving waters to aquatic life for use by the U.S. Environmental
Protection Agency (USEPA) regional and state,programs, and National
Pollutant Discharge Elimination System (NPOES) permittees.
Robert L. Booth
Director
Environmental Monitoring and
Support Laboratory - Cincinnati
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PREFACE
This manual 1s the first Agency methods manual for estimating the
chronic toxicity of effluents and receiving waters. The draft was
reviewed by the Bioassay Subcommittee of the EMSL-Cincinnati Biological
Advisory Committee, USEPA headquarters and regional staff, other Federal
agencies, 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
Bruce Littell, Environmental Services Division, Region 7 .
Leo Mosby, Environmental Services Division, Region 7
Loys Parrish, Environmental Services Division, Region 8
James Lazorchak, Water Management Division, Region 8
Milton Tunzi, Environmental Services Division, Region 9
Joseph Cummins, Environmental Services Division, Region 10
Robert Schneider, National Enforcement Investigations Center, Denver
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, Environmental Research Laboratory - Corvallis
Rick Brandes, National Pollutant Discharge Elimination System
Technical Support Branch, Permits Division, Office of Water
Enforcement and Permits
Edward Bender, Compliance Branch, Enforcement Division, Office of
Water Enforcement and Permits
Thomas Murray, Monitoring Branch, Monitoring and Data Support
Division,"Office of Water Regulations and Standards
Stephen Ells, Environmental Effects Branch, Health and Environmental
Review Division, Office .of Toxic Substances
Cornelius I. Weber, Ph.D.
Chairman, Biological Advisory Committee
Chief, Biological Methods Branch
Environmental Monitoring and Support
Laboratory - Cincinnati
iv
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ABSTRACT
This manual describes short-term (four- to eight-day) methods for
estimating the chronic toxicity of effluents and receiving waters to a
freshwater fish, an invertebrate, and an alga. Also included are
guidelines on'laboratory safety, quality assurance, facilities and
equipment, dilution water, effluent sampling and holding, data analysis,
report preparation, and organism culturing and handling. Listings of
computer programs for Ounnett's Procedure and Probit Analysis are
provided in the Appendix.
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CONTENTS
Foreword . * iii
Preface iv
Abstract v
Figures vii
Tables vii
Acknowledgments viii
1. Introduction 1
2. Short-Term Methods for Estimating Chronic Toxicity ... 3
3. Health and Safety 6
4. Quality Assurance ..... 8
5. Facilities and Equipment ' 13
6. Test Organisms . 15
7. Dilution Water 17
8. Effluent and Receiving Water Sampling and Sample Handling 19
9. Chronic Toxicity Test End Pofnts and Data Analysis ... 22
10. Report Preparation 26
11. Fathead Minnow (Pimephales promelas) Larval Survival
and Growth'Test 28
12. Fathead Minnow (Pimephales promelas) Embryo-larval
Survival and Teratogemcity Test 42
13. Ceriodaphnia Survival.and Reproduction Test 58
14. Algal (Selenastrum capricornutum) Growth Test 76
Selected References 96
Appendix . ^ 105
A. Validating Normality and Homogeneity of Variance
Assumptions . . . 106
8. Arc Sine Square-Root Transformation Ill
C. Dunnett's Procedure 113
0. Steel's Many-one Rank Test 132
E. Fisher's Exact Test 135
F. Probit Analysis 144
VI
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FIGURES
Number " Page
1. Control chart 12
TABLES
Number . page -
1. Preparation of synthetic fresh water. . . . 18
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ACKNOWLEDGMENTS
Materials in this manual were taken in part from the following
sources: USEPA, 1975, Methods for Acute Toxieity Tests with Fish,
Macroinvertebrates, and Amphibians, Environmental Research Laboratory,
U. S. Environmental Protection Agency, Duluth, Minnesota,
EPA-660/3-75-009; USEPA, 1979, Handbook for Analytical Quality Control in
Water and Wastewater Laboratories, Environmental Monitoring and Support
Laboratory - Cincinnati, U. S. Environmental Protection Agency,
Cincinnati, Ohio, EPA-600/4-79/019; USEPA, 1979, Interim NPOES Compliance
Biomonitoring Inspection Manual, Enforcement Division, Office of Water
Enforcement, U. S. Environmental Protection Agency, Washington, D.C.;
Peltier, W. H., and C. I. Weber, 1985, Methods for Measuring the Acute
Toxicity of Effluents to Freshwater and Marine Organisms, Environmental
Monitoring and Support Laboratory - Cincinnati, U. S. Environmental
Protection Agency, Cincinnati, Ohio, EPA-600/4-85/013; Mount, D. I., and
T. J. Norberg, 1984, A Seven-day Life-cycle Cladoceran Test, Environ.
Toxicol. Chem. 3:425-434; Norberg, T., and 0. I. Mount, 1985, A New
Subchronic Fathead Minnow (Pimephales promelas) Toxicity Test, Environ.
Toxicol. Chem. (In press); and Miller, W. £., J. C. Greene, and
T. Shiroyama, 1978r The Selenastrum capricornutum Printz Algal Assay
Bottle Test, Environmental Research Laboratory, U. S. Environmental
Protection Agency, Corvallis, Oregon, EPA-600/9-78-018.
The assistance of the following members of the staff of the Aquatic
Biology Section, Biological Methods Branch, EMSL-Cincinnati, is
gratefully acknowledged: Quentin Pickering prepared the section on the
fathead minnow larval survival and growth test; Timothy Neiheisel
prepared the section on the fathead minnow embryo-larval survival and
teratogenicity test; Philip Lewis prepared the section on the
Ceriodaphnia survival and reproduction test; Ernest Robinson provided the
precision data for Selenastrum capricornutum; James Dryer provided the
listings of the computer programs, and assisted in preparing other
materials on test data analysis; and Cordelia Newell, Diane Schirmann,
Dianne White, and Janice Miller provided valuable secretarial assistance.
Many helpful suggestions for the revision of the manual were provided
by members of the Bioassay Subcommittee in the first and second round
review. William Peltier, Chairman, Bioassay Subcommittee, deserves
special recognition for the valuable assistance provided in the
preparation of the final draft of the manual.
John Menkedick and Florence Kessler, Statisticians, Computer Sciences
Corporation, on-site contractor at the Andrew W. Breidenbach
Environmental Research Center, U. S. Environmental Protection Agency,
Cincinnati, provided materials for inclusion in Section 9, and assisted
in the preparation of materials on data analysis and precision for other
sections and the Appendix.
viii
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The editors also wish to acknowledge the review comments received
from the following persons: Howard Alexander, Dow Chemical U.S.A.,
Midland, Michigan; R. Clifton Bailey, U. S. Environmental Protection
Agency, Washington, DC; James Baker, U. S. Environmental Protection
Agency, Denver, Colorado; Dorothy Berner, Temple University,
Philadelphia, Pennsylvania; F. A. Blanchard, Dow Chemical U.S.A, Midland,
Michigan; K. M. Bodner, Dow Chemical U.S.A, Midland, Michigan; Douglas
Burnhara, Vermont Agency of Environmental Conservation, Montpelier,
Vermont; Oscar Cabra, U. S. Environmental Protection Agency, Dallas,
Texas; Joseph Carra, U. S. Environmental Protection Agency, Washington,
DC;' Larry Claxton, U. S. Environmental Protection Agency, Research
Triangle Park, North Carolina; Gary Collins, U. S. Environmental
Protection Agency, Cincinnati, Ohio; Jody Connor, New Hampshire Water
Pollution Control Commission, Concord, New Hampshire; Nelson Cooley,
U. S. Environmental Protection Agency, Gulf Breeze, Florida; John Cooney,
Battelle Columbus Laboratories, Columbus, Ohio; Frank Covington, U. S.
Environmental Protection Agency, San Francisco, California; U. M.
Cowgill, Dow Chemical U.S.A., Midland, Michigan; Michael OeGraeve,
Battelle Columbus Laboratories, Columbus, Ohio; D. C. Dill, Dow Chemical
U.S.A., Midland, Michigan; Kenneth Dostal, Water Engineering Research
Laboratory, U. S. Environmental Protection Agency, Cincinnati, Ohio;
Thomas Duke, U. S. Environmental Protection Agency, Gulf Breeze, Florida;
Robert Elliott, U. S. Environmental Protection Agency, Seattle,
Washington; Robert Estabrook, New Hampshire Water Pollution Control
Conraission, Concord, New Hampshire; James Fava, EA Engineering, Science,
and Technology, inc., Sparks, Maryland; David Flemer, U. S. Environmental
Protection Agency,, Washington, DC; Peter Gartside, University of
Cincinnati Medical School, Cincinnati, Ohio; F. M. Gersich, Dow Chemical
U.S.A., Midland, Michigan; James Gillett, Cornell University, Ithaca, New
York; Miriam Goldberg, U. S. Environmental Protection Agency, Washington,
DC; Larry Goodman, U. S. Environmental Protection Agency, Gulf Breeze,
Florida; Joseph Gorsuch, Eastman Kodak Company, Rochester, New York;
David Gruber, Biological Monitoring, Inc., Blacksburg, Virginia; Scott
Hall, Johns Hopkins University, Baltimore, Maryland; Jerry Hamelink,
Lilly Research Laboratory, Greenfield, Indiana; David Hansen, U. S.
Environmental Protection Agency, Narr.agansett, Rhode Island; David
Hutton, Haskell Laboratory for Toxicology, I.E. DuPont de Nemours and
Company, Newark, Delaware; Kathleen Keating, Rutgers State University,
Park Ridge, New Jersey; Stephen Klaine, Memphis State University,
Memphis, Tennessee; Armond Lemke, U. S. Environmental Protection Agency,
Dulutn, Minnesota; Michael Lewis, Proctor and Gamble Company, Cincinnati,
Ohio; Elizabeth Loevey, U. S. Environmental Protection Agency,
Washington, DC; Donald Lollock, California Department of Fish and Game,
Sacramento, California; Leif Marking, U. S. Fish and Wildlife Service, La
Crosse, Wisconsin; Michael Martin, California Department of Fish and
Game, Monteray, California; Jack Mattice, Electric Power Research
Institute, Palo Alto, California; Foster Mayer, U. S. Environmental
Protection Agency, Gulf Breeze, Florida; M. A. Mayes, Dow Chemical
U.S.A., Midland, Michigan; Robert Medz, U. S. Environmental Protection
Agency, Washington, DC; Gary Neuderfer, New York Department of
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Environmental Control, Avon, New York; Paul Pan, U. S. Environmental
Protection Agency, Washington, DC; Rod Parri|h, U. S. Environmental
Protection Agency, Gulf Breeze, Florida; Gilbert Potter, U. S.
Environmental Protection Agency, Las Vegas, Nevada; Ronald Raschke, U. S.
Environmental Protection Agency, Athens, Georgia; John Rogers, University
of Wisconsin, Superior, Wisconsin; Landon Ross, Florida Department of
Environmental Regulation, Tallahassee, Florida; Richard Scnoettger, U. S.
Fish and Wildlife Service, Columbia, Missouri; William Selconis,
Hoffman-LaRoche, Belvidere, New Jersey; Judith Shaw, American Petroleum
Institute, Washington, DC; Russell Sherer, South Carolina Department of
Health and Environmental Control, Columbia, South Carolina; Richard
Steele, U. S. Environmental Protection Agency, Narragansett, Rhode
Island; Charles Stephan, U. S. Environmental Protection Agency, Duluth,
Minnesota; James Stiebing, U. S. Environmental Protection Agency,
Seattle, Washington; Daniel Sullivan, U. S. Environmental Protection
Agency, Edison, New Jersey; Jerry Stara, U. S. Environmental Protection
Agency, Cincinnati Ohio; I. T. Takahashi, Dow Chemical U.S.A, Midland,
Michigan; James Swigert, Indiana State Board of Health, Indianapolis,
Indiana; Frieda Taub, University of Washington, Seattle, Washington;
William Telliard, U. S. Environmental Protection Agency, Washington, DC;
Roy Thompson, Imperial Chemical Industries, Devon, England; Glen Thursby,
U. S. Environmental Protection Agency, Narragansett,.Rhode Island;
William Tucker, Illinois Environmental Protection Agency, Springfield,
Illinois; William Waller, University of Texas at Dallas, Richardson,
Texas; Gerald Walsh, U. S. Environmental Protection Agency, Gulf Breeze,
Florida; Barbara Walton, Oak Ridge National Laboratory, Oak Ridge,
Tennessee; Thomas Wallingham, U. S. Environmental Protection Agency,
Denver, Colorado; James Whitaker, Illinois Environmental Protection
Agency, Springfield, Illinois; J. I. Whitfield, Dow Chemical U.S.A.,
Midland, Michigan; John Winter, U. S. Environmental Protection Agency,
Cincinnati, Ohio; and William Wuerthele, U. S. Environmental Protection
Agency, Denver, Colorado.
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SECTION 1
INTRODUCTION
1.1 The Federal Water Pollution Control Act Amendments of 1972 (PL 92-500)
and the Clean Water Act (CWA) of 1977 (PL 95-217) were enacted to restore
and maintain the chemical, physical, and biological integrity of the
Nation's waters (Section 101[a]), and contained specific or implied
requirements for the collection of b i onion i tor ing data in at least 15
sections.
1.2 The Declaration of Goals and Policy, Section 101(a)(3), in these two
laws, states that "it is the national goal that the discharge of toxic
pollutants in toxic amounts be prohibited." To achieve the goals of this
legislation, extensive effluent toxicity screening programs were conducted
during the 1970s by the regions and states. Acute toxicity tests (USEPA,
1975; Peltier, 1978) were used to measure effluent toxicity and to estimate
the safe concentration of toxic effluents in receiving waters. However,
for those effluents that were not sufficiently toxic to cause mortality in
acute (one- to four-day) fests, short-term inexpensive methods were not
available to detect the more subtle, low-level, long-term, adverse effects
of effluents on aquatic organisms, such as reduction in growth and
reproduction, and occurrence of terata. Fortunately, rapid developments in
toxicity test methodology during the past five years have resulted in the
availability of several methods that permit detection of the low-level,
adverse effects (chronic toxicity) of effluents in eight days or less.
*>
1.3 As a result of the increased awareness of the value of'effluent
toxicity test data for toxics control in the National Pollutant Discharge
Elimination System (NPOES) permit program, which emerged from the extensive
effluent toxicity monitoring activities of the regions and states, and the
recent availability of short-term chronic toxicity test methods, the U. S.
Environmental Protection Agency (USEPA) issued a national policy statement
entitled, "Policy for the Development of Water Quality-Based Permit
Limitations for Toxic Pollutants," in the Federal Register Vol. 49, No. 48,
Friday, March 9, 1984. A technical support document on the use of effluent
and receiving water toxicity data also has been prepared by the Office of
Water Enforcement and Permits (OWE?) to provide additional guidance on the
implementation of the biomonitoring policy (USEPA, 1985).
1.4 This new Agency policy proposes the use of toxicity data to assess and
control the discharge of toxic substances to the Nation's waters through
the NPDES permits program. The policy states that "biological testing of
effluents is an important aspect of the water quality-based approach for
controlling toxic pollutants. Effluent toxicity data, in conjunction with
other data, cain be used to establish control priorities, assess compliance
with State water quality standards, and set permit limitations to achieve
those standards." All states have water quality standards which include
narrative statements prohibiting the discharge of toxic materials in toxic
amounts.
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1.5 The four short-term tests described in this manual are for use in the
NPOES Program to estimate one or more of the following: (1) the chronic
toxicity of effluents collected at the end of the discharge pipe and tested
with a standard dilution water (moderately hard synthetic freshwater in Table
1, p. 18); (2) the chronic toxicity of effluents collected at the end of the
discharge pipe and tested with dilution water consisting of non-toxic
receiving water collected upstream from the outfall, or with other
uncontaminated surface water or standard dilution water having approximately
the same hardness as the receiving water; (3) the toxicity of receiving water
downstream from the outfall; and (4) the effects of multiple discharges on
the quality of the receiving water. The tests may also be useful in
developing site-specific water quality criteria.
1.6 These methods were developed to provide the most favorable cost-benefit
relationship possible, and are intended for use in effluent toxicity tests
performed on-site, where time is very costly, and for toxicity tests with
effluent samples shipped to central and distant laboratories, where the
volume of waste shipped.must be kept to a minimum.
The tests include:
1. Seven-day, sub-chronic, fathead minnow (Pimephales promelas),
static renewal, larval survival and growth test.
2. Seven-day, (three-brood), chrom'c, Ceriodaphnia dubia, static
renewal, survival and reproduction test.
3. Eight-day, sub-chronic, fathead minnow (Pimephales promelas),
static renewal, embryo-larval survival and teratogenicity test.
4. Four-day, chronic, Selenastrum capricornutum, static, growth test.
1.7 The first two tests were adapted from methods developed by
Or. Donald Mount and Teresa Norberg, Environmental Research Laboratory,
USEPA, Duluth, Minnesota (Mount and Norberg, 1984; Norberg and Mount,
1985). The third test was adapted from a method developed by Drs. Wesley
Birge and Jeffrey Black, Graduate Center for Toxicology, University of
Kentucky, Lexington, Kentucky (Birge and Black, 1981). The fourth test,
a 96-h, multi-generation test utilizing the freshwater alga, Selenastrum
capricornutum, was adapted from the publications of the Environmental
Research Laboratory - Corvallis (USEPA, 1971; Miller et al., 1978).
1.8 The Environmental Monitoring and Support Laboratory - Cincinnati
(EMSL-Cincinnati) has incorporated the short-term chronic and sub-chronic
tests Into this manual for use by regulatory agencies involved in
biological monitoring of wastewater under the NPOES program. Authority
for promulgating test procedures for the analysis of pollutants is
contained in Section 304(h) of the CWA.
1.9 The manual was prepared in the established EMSL-Cincinnati format
(Kopp, 1983) so that each method can be used independently of the other
methods.
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... .... .. SECTION 2
SHORT-TERM METHODS FOR ESTIMATING CHRONIC TOXICITY
2.1 The objective of aquatic toxicity tests with effluents or pure
compounds is to estimate the "safe" or "no effect" concentration of these
substances, which is defined as the concentration which will permit normal
propagation of fish and other aquatic life in the receiving waters. The
endpoints that have been considered in tests to determine the adverse
effects of toxicants include death and survival, decreased reproduction and
growth, locomotor activity, gill ventilation rate, heart rate, blood
chemistry, histopathology, enzyme activity, olfactory function, and
terata. Since it is not feasible to detect and/or measure all of these
(and other possible) effects of toxic substances on a routine basis,
observations in toxicity tests generally have been limited to .only a few
effects, such as mortality, growth, and reproduction.
2.2 Acute mortality is.an obvious and easily observed effect, which
accounts for its wide use in the early period of evaluation of the toxicity
of pure compounds and complex effluents. The results of these tests were
usually expressed as the concentration lethal to 50% of the test organisms
(LC50) over relatively short exposure periods (two to four days).
2.3 As exposure periods of acute tests were lengthened, the LC50 and
lethal threshold concentration were observed to decline for many
compounds. By lengthening the tests to include one or more complete life
cycles and observing the more subtle effects of the toxicants, such as a
reduction in growth and reproduction, more accurate, direct, estimates of
the threshold or safe concentration of the toxicant could be obtained.
However, laboratory life-cycle tests may not accurately estimate the safe
concentration of toxicants because they are conducted with a limited number
of species under highly controlled, steady-state conditions, and the
results do not include the effects of the stresses to which the organisms
would ordinarily be exposed in the natural environment.
2.4 An early published account of a full life-cycle, fish toxicity test
was that of Mount and Stephan (1967). In this study, fathead minnows
(Pimephales promelas) were exposed to a graded series of pesticide
concentrations throughout their life cycle, and the effects of the toxicant
on survival, growth, and reproduction were measured and evaluated. This
work was soon followed by full life-cycle tests using other toxicants and
fish species.
2.5 McKira (1977) evaluated the data from 56 full life-cycle tests, 32 of
which used the fathead minnow, and concluded that the embryo-larval and
early juvenile life-stages were the most sensitive stages. He proposed the
use of partial life-cyle toxicity tests with the early life-stages (ELS) of
fish to establish water quality criteria.
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2.6 Macek and Sleight (1977) found that exposure of critical life-stages
of fish to toxicants provides estimates of chronically safe concentrations
remarkably similar to those derived from full life-cycle toxicity tests,
and reported that "for a great majority of toxicants, the concentration
which will not be acutely toxic to the most sensitive life stages is the
chronically safe concentration for fish, and that the most sensitive life
stages are-the embryos and fry." Critical life-stage exposure was
considered to be exposure of the embryos during most, preferably all, of
the embryogenic (incubation) period, and exposure of the fry for 30 days
post-hatch for warm water fish with embryogenic periods ranging from one to
14 days, and for 60 days post-hatch for fish with longer embryogenic
periods. They concluded that in the majority of cases, the maximum
acceptable toxicant concentration (MATC) could be estimated from the
results of exposure of the embryos during incubation, and the larvae for 30
days post-hatch.
2.7 Because of the high cost of full life-cycle fish toxicity tests and
the emerging concensus that the ELS test data would be adequate for
estimating chronically safe concentrations, there was a rapid shift by
aquatic toxicologists to 30- to 90-day ELS toxicity tests for estimating
chronically safe concentrations in the late 1970s. In 1980, USEPA adopted
the policy that ELS test data could be used in establishing water quality
criteria if data from full life-cycle tests were not available (USEPA,
1980a).
2.8 Published reports of the results of ELS tests indicate that the
relative sensitivity of growth and survival as endpoints may be species
dependent, toxicant dependent, or both. Ward and Parrish (1980) examined
the literature on ELS tests that used embryos and juveniles of the salt
water species, sheepshead minnow (Cyprinodon variegatus), and found that
growth was not a statistically sensitive indicator of toxicity in 16 of 18
tests. They suggested that the ELS tests be shortened to 14 days posthatch
and that growth be eliminated as an indicator of toxic effects.
2.9 In a review of the literature on 173 fish full life-cycle and ELS
tests performed to determine the chronically safe concentrations of a wide
variety of toxicants, such as metals, pesticides, organics, inorganics,
detergents, and complex effluents, Woltering (1984) found that at the
lowest effect'concentration, significant reductions were observed in fry
survival in 57%, fry growth in 36%, and egg hatchability in.19% of the
tests. He also found that fry survival and growth were very often equally
sensitive, and concluded that the growth response could be deleted from
routine application of the ELS tests. The net result would be a
significant reduction in the duration and cost of screening tests with no
appreciable impact on estimating MATCs for chemical hazard assessments.
Benoit, et al., (1982), however, in early life-stage tests with four
organic chemicals, found larval growth to be the most significant measure
of effect, and survival to be equally or less sensitive than growth.
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2.10 Efforts to further reduce the length of partial life-cycle toxicity
tests for fish without compromising their predictive value have resulted in
the devtlopratnt of an eight-day, embryo-larval survival and teratogenicity
test for fish and and other aquatic vertebrates (Birge and Black, 1981;
Birge et al., 1985), and a seven-day larval survival and growth test (Mount
et al., 1984; Norberg and Mount, 1985).
2.11 The similarity of estimates of chronically safe concentrations of
toxicants derived from short-term, embryo-larval survival and
teratogenicity test to those derived from full life-cycle tests has been
demonstrated by Birge et al. (1981), Birge and Cassidy (1983), and Birge
et al. (1985).
2.12 Use of a seven-day, fathead minnow larval survival and growth test
was first proposed by Norberg and Mount at the 1983 annual meeting of the
Society for Environmental Toxicology and Chemistry (Norberg and Mount,
1983). This test was subquently used by Mount and associates in field.
demonstrations at Lima,.Ohio (Mount, et al., 1984), and-at many other
locations. Growth was frequently found to be more sensitive than survival
in determining the effect of complex effluents.
2.13 Norberg and Mount (1985) performed three single toxicant fathead
minnow larval growth tests with zinc, copper, and DURSBANR, using
dilution water from Lake Superior. The results were comparable to, and had
confidence intervals that overlapped with, chronic values reported in the
literature for both ELS and full life-cycle tests.
2.14 "Efforts to develop a short-term cladoceran chronic toxicity test as a
substitute for the 21- to 28-day Daphnia chronic toxicity test have
resulted in a seven-day (three-brood) survival and reproduction test using
Ceriodaphm'a (Mount and Norberg, 1984). This test has been shown to
provide data comparable to the previous, longer-term Daphnia chronic tests,
and has also been used extensively by Mount and associates in field
demonstrations.
2.15 The algal toxicity test described in this manual is a shortened
version of the Agency's algal growth potential test (Miller et al., 1978).
The 96-h length of the toxicity test period spans several generation times
and, therefore, more than meets the requirements for a full life-cycle
chronic test. "
2.16 The use, of short-term,-subchronic and chronic toxicity tests in the
NPDES Prograa is especially attractive because they provide a more direct
estimate of the safe concentration of effluents in receiving waters than
was provided by acute toxicity tests, at an only slightly increased level
of effort, compared to that required by the fish full life-cyle chronic and
(30-day) ELS tests and the 21- to 28-day cladoceran tests.
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SECTION 3
:. <:.- :,,«•• -•¥
HEALTH AND SAFETY1
3.1 GENERAL PRECAUTIONS
3.1.1 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 take all safety
precautions necessary for the prevention of bodily injury and illness which
might result from ingestion or invasion of infectious agents, inhalation or
absorption of corrosive or toxic substances through skin contact, and
asphyxiation due to lack of oxygen or presence of noxious gases.
3.1.2 Prior to sample collection and laboratory work, personnel should
determine that all necessary safety equipment and materials have been
obtained and are in good condition.
3.2 SAFETY EQUIPMENT
3.2.1 Personal Safety Gear
Personnel should use safety equipment, as required, such as rubber
aprons, laboratory coats, respirators-, gloves,, safety glasses, hard hats,
and safety shoes.
3.2'.2 Laboratory Safety Equipment
Each laboratory (including mobile laboratories) should be provided with
safety equipment such as first-aid kits, fire extinguishers, fire blankets,
emergency showers, and eye fountains.
3.3 GENERAL LABORATORY AND FIELD OPERATIONS
3.3.1. Work with effluents should be performed in compliance with accepted
rules pertaining to the handling of hazardous materials (see safety
manuals, Paragraph 3.5). It is recommended that personnel collecting
samples and performing toxicity tests should not work alone.
3.3.2. 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.
3.3.3. It is advisable to cleanse exposed parts of the body immediately
after collecting effluent samples.
3.3.4. All containers should be adequately labeled to indicate their
contents.
Adapted from: Peltier and Weber (1985).
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3.3.5. Good housekeeping contributes to safety and reliable results.
3.3.6. Electrical equipment or extension cords not bearing the approval of
Underwriter Laboratories must not be used. Ground-fault interrupters must
be installed in all "wet" laboratories where electrical equipment is used.
3.3.7. Mobile laboratories should be properly grounded to protect against
electrical shock."
3.4 DISEASE PREVENTION
3.4.1 Personnel handling samples which are known or suspected to contain
human wastes should be immunized against tetanus, typhoid fever, and polio.
3.5 SAFETY MANUALS
3.5.1 For further guidance on safe practices when collecting effluent
samples and conducting toxicity tests, check with the permittee and consult
general industrial safety manuals, including USEPA (1977), and Walters and
Jameson (1984).
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SECTION 4
QUALITY ASSURANCE1
4.1 INTRODUCTION
4.1.1 Quality Assurance (QA) practices for effluent toxicity tests consist
of all aspects of the test that affect data quality, 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) replication; (7) use of reference toxicants; (8) record
keeping; and (9) data evaluation. For general guidance on good laboratory
practices related to toxicity'testing, see: FDA, 1978; USEPA, 1979d,
1980b, and 1980c; and DeWoskin, 1984.
4.2 EFFLUENT AND RECEIVING WATER SAMPLING AND HANDLING
4.2.1 Effluent samples.collected for on-site and off-site testing must be
preserved as described in Section 8, Effluent and Receiving Water Sampling
and Sample Handling.
4.3 TEST ORGANISMS
4.3.1 The test organisms used in the procedures described in this manual
are the fathead minnow, Pimephales promelas, the cladoceran, Ceriodaphnia
dubla, and the green alga, SelenaTtrum capricornutunu The organjsms used
should be disease-free, and snould be positively identified to species.
4.4 FACILITIES, EQUIPMENT, AND TEST CHAMBERS
4.4.1 Laboratory and bioassay temperature control equipment must be
adequate to maintain recommended test water temperatures. Recommended
materials must be used in the fabrication of the test equipment which comes
in contact with the effluent (see Section 5, Facilities and Equipment).
4.5 ANALYTICAL METHODS
4.5.1 Routine chemical and physical analyses must include established
quality assurance practices outlined in Agency methods manuals (USEPA,
1979a,b).
4.6 CALIBRATION AND STANDARDIZATION
4.6.1 Instruments used for routine measurements of chemical and physical
parameters such as pH, DO, temperature, conductivity, alkalinity, and
hardness, must be calibrated and standardized according to instrument
manufacturers procedures as indicated in the general section on quality
assurance (see EPA Methods 150.1, 360.1, 170.1, and 120.1, USEPA, 1979b).
Adapted from: Peltier (1978), Peltier and Weber (1985),
and USEPA (1979a).
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4.6.2 Wet chemical methods used to measure hardness and alkalinity must be
standardized according to the procedures for those specific EPA methods (see
EPA Methods 130.2 and 310.1, USEPA 1979b). '
4.7 DILUTION WATER
4.7.1 The-dilution water used in the toxicity tests may be synthetic water,
receiving water, or ground water, appropriate to the objectives of the study
and logistical constraints, as discussed in Section 7.
4.8 TEST CONDITIONS
4.3.1 Water temperature must be maintained within the limits specified for
each test. Dissolved oxygen (DO) concentrations and pH should be checked at
the beginning of the test and daily throughout the test period.
4.9 TEST ACCEPTABILITY
4.9.1 The results of the fathead minnow or Ceriodaphnia 24-h reference
toxicant tests are unacceptable if the survival in the controls is less than
90%. For effluent toxicity tests to be acceptable, control survival must be
at least 80%. The results of the algal toxicity test are unacceptable if
the cell density in the controls after 96 h is less than 106 cells/mL.
4.9.2 An individual test may be conditionally acceptable if temperature,
DO, and other specified conditions fall outside specifications, depending on
the degree of the departure and the objectives of the tests (see test
condition summaries). The acceptability of the test would depend on the
best professional judgment and experience of the investigator. The
deviation from test specifications must be noted when reporting data from
the test.
4.10 PRECISION
4.10.1 The ability of the laboratory personnel to obtain consistent, precise
results must be demonstrated with reference toxicants before they attempt to
measure effluent toxicity. The single laboratory precision of each type of
test to be used in a laboratory should be determined by performing five or
more tests with a reference toxicant. In cases where the test data are used
in the Probit Analysis (see Section 9), precision can be described by the
mean, standard deviation, and relative standard deviation (percent
coefficient of variation, or CV) of the calculated end points from the
replicated tests. However, in cases where the results are reported in terms
of the No-Observed-Effect Concentration (NOEC) and Lowest-Qbserved-Effect
Concentration (LOEC) (see Section 9), precision can only be described by
listing the NOEC-LOEC interval for each test. In this case, it is not
possible to express precision in terms of a commonly used statistic. For
instance, when all tests of the same toxicant yield the same NOEC-LOEC
interval, maximum precision has been attained. However, the "true" no
effect concentration could fall anywhere within the interval, NOEC +
(NOEC-LOEC).
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4.10.2 It should be noted here that the dilution factor selected for a
test deteraines the width of the NOEC-LOEC Interval and the Inherent
maxima® precision of the test. As the absolute value of the dilution
factor decreases, the width of the NOEC-LOEC interval increases, and the
Inherent maximum precision of the test decreases. When a dilution factor
of 0.3 is used, the NOEC could be considered to have a relative variability
as high as- + 300%. With a dilution factor of 0.5, the NOEC could be
considered to have a relative variability of + 100%. Other factors which
can affect test precision include test organism age, condition, and
sensitivity, temperature control, and feeding.
4.11 REPLICATION AND TEST SENSITIVITY
4.11.1 The sensitivity of the tests will depend in part on the number of
replicates, the probability level selected, and the type of statistical
analysis. The minimum recommended number of replicates varies with the
test and the statistical method used, .and is discussed in Section 9 and in
each method. The sensitivity of the test will increase as the number of
replicates 1s increased.
4.12 QUALITY OF TEST ORGANISMS
4.12.1 If the laboratory does not have an ongoing test organism culturing
program and obtains the test organisms from an outside source, the
sensitivity of each batch of test organisms must be evaluated with a
reference toxicant in a toxicity test performed concurrently with the
effluent and/or receiving water toxicity tests. If the laboratory
maintains breeding cultures, the sensitivity of the offspring should be
determined in a toxicity test performed with a reference toxicant at least
once each month. If preferred, this reference toxicant test may be
performed concurrently with an effluent toxicity test. A 24-h acute
toxicity test is used to determine the sensitivity of fathead minnows and
Ceriodaphnia. For the acute toxicity test design, see Peltier and Weber
(1985). A 96-h toxicity test is used to determine the sensitivity of
Selenastrum.
4.12.2 The sensitivity of fathead minnow larvae is determined with newly
hatched larvae as used in the growth and survival or embryo-larval test.
The sensitivity of Ceriodaphnia is determined using animals less than 24 h
old, and which were released within the same 4-h period.
4.12.3 Three reference toxicants are available from EMSL-Cincinnati to
establish the precision and validity of toxicity data generated by
biomonitoring laboratories: sodium dodecylsulfate (SDS), sodium
pentachlorophenate (NaPCP), and cadmium chloride (Cdd2). The reference
toxicants may be obtained by contacting the Quality Assurance Branch,
Environmental Monitoring and Support Laboratory, U. S. Environmental
Protection Agency, Cincinnati, Ohio, 4526S; FTS 684-7325, comm'l 513-569-7325.
Instructions for the use and the expected toxicity values for the reference
toxicants are provided with the samples. To assure comparability of QA
10
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data on a national scale, all laboratories must use the same source of
reference toxicant (EMSL-C1ncinnati) and the same formulation of dilution
water — moderately hard synthetic water,.described in Table 1, p. 18* for
fathead minnows and Ceriodaphhia. and algal growth medium described in
Tables 1 and 2, Section 14, for Selenastrum.
4.13 FOOD QUALITY
4.13.1 The quality of the food for fish and invertebrates is an important
factor in toxicity tests. Suitable trout chow, Artemia. and other foods
must be obtained as described in the manual. Limited quantities of
reference Artemia cysts, information on commerical sources of good quality
Artemia cysts, and procedures for determining cyst suitability as food are
available from the Quality Assurance Branch, Environmental Monitoring and
Support Laboratory, U. S. Environmental Protection Agency, Cincinnati,
Ohio, 45268. The suitability of each new supply of food must be determined
in a side-by-side test in which the response of test organisms fed with the
new food is compared with the response of organisms fed a reference food or
a previously used, satisfactory food.
4.14 CONTROL CHARTS
4.14.1 A control chart should be prepared for each reference-toxicant-
organism combination, and successive toxicity values should be plotted and
examined to determine if the results-are within prescribed limits
(Fig. 1). In this technique, a running plot is maintained for the toxicity
values (Xi) from successive tests with a given reference toxicant. The
type of control chart illustrated (USEPA, 1979a) is used to evaluate the
cumulative trend of the statistics from a series of samples. The mean (1)
and upper and lower control limits (± 2S) are re-calculated 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. At the
P0.05 probability level, one in 20 tests would be expected to fall
outside of the control limits by chance alone.
4.14.2 If the toxicity value from a given test with the reference toxicant
does not fall in the expected range for the test organisms when using the
standard dilution water, 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 should be repeated with a
different batch of test organisms.
4.15 RECORD KEEPING
4.15.1 Proper record keeping is required. Bound notebooks should be used
to maintain detailed records of the test organisms such as species, source,
age, date of receipt, and other pertinent information-relating to their
history and health, and information on the calibration of equipment and
instruments, test conditions employed, and test results. Annotations
should be made on a real-time basis to prevent the loss of information.
11
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to
«J
UPPER CONTROL LIMIT(X+2S)
CENTRALTENOENCY
LOWER CONTROL LI MIT (X - 2S)
L I I I I I I I I I I I I I I I I I
• L
OS 10 15 20
TOXIC1TY TEST WITH REFERENCE TOXICANTS
*»
• Figure 1. Control chart.
n- I
Where:
X-f » Successive LCSO's from toxicity tests.
n 3 Nunier of tests.
' X » Mean LC50.
S » Standard deviation.
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SECTION 5
FACILITIES AND EQUIPMENT^
5.1 GENERAL REQUIREMENTS
5.1.1 Effluent toxicity tests may be performed in a fixed or mobile
laboratory. Facilities should include equipment for rearing, holding, and
accl-imating 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.
Dechlorination can be accomplished by aeration (allowing the water to stand
in an open vessel for 24 h), carbon filtration, or the use of sodium
thiosulfate. Use of 1.0 mg (anhydrous) sodium thiosulfate/L will reduce
1.5 mg chlorine/L. After dechlorination, total residual chlorine should be
non-detectable. Air used for aeration must be free of oil and fumes. Test
facilities must be well ventilated and free of fumes. During rearing,
holding, acclimating, and testing, test organisms should be shielded from
external disturbances.
5.1.2 Materials used for exposure chambers, tubing, etc., which come in
•contact with the effluent should be carefully chosen. Tempered glass and
perfluorocarbon plastics (TEFLONR) 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, TY60NR, etc., may be used as test
chambers or to store effluents, but caution should be exercised in their
use because they could introduce toxicants when new, or carry over
toxicants from one test to another if reused. The use of glass carboys is
discouraged for safety reasons.
5.1.3 New plastic products of a type not previously used should be tested
for toxicity before initial use by exposing the test organisms in the test
system where the material is used. Equipment (pumps, valves, etc.) which
cannot 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 material should be flushed or rinsed thoroughly with the test
media 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.
1Adapted from: Peltier and Weber (1985).
13
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5.1.4 Silicone adhesive used to construct glass test chambers absorbs some
organochlorint 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.
5.2 TEST CHAMBERS
5.2.1 Test chamber size and shape are varied according to size of the test
organism. Requirements are specified in each test.
5.3 CLEANING
5.3.1 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 below).
5.3.2 It is recommended that all sample containers, test vessels, pumps,
tanks, and other equipment that has come in contact with effluent be washed
after use in the manner described below to remove surface contaminants.
Special cleaning requirements for glassware used in algal toxicity tests
are described in Section 14.
1. Soak 15 minutes, and scrub with detergent in tap water, or clean in
an automatic dishwasher.
2. Rinse twice with tap water.
3. Carefully rinse once with -fresh dilute (20% V:V) nitric acid or
hydrochloric acid to remove scale, metals and bases. To prepare a
20X solution of acid, add 20 ml of concentrated acid to 80 mL of
distilled water.
4. Rinse twice with tap water.
5. Rinse once with full-strength acetone to remove organic compounds.
6. Rinse well with tap water.
7. Rince twice with dilution water.
5.3.3 All test chambers and equipment must be thoroughly rinsed with the
dilution water immediately prior to use in each test.
14
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SECTION 6
TEST ORGANISMS
6.1 SPECIES
6.1.1 The organisms used in the chronic tests described in this manual are
the fathead minnow, (Pimephales promelas), the cladoceran, Ceriodaphnia
dubia, and the green alga, SeTehastrum capricornutum.
6.2 SOURCE
6.2.1 The test organisms are easily cultured in the laboratory.
Culturing, care, and handling procedures for Ceriodaphnia and Selenastrum
are described in the respective test methods sections. A fathead minnow
culturing procedure using laboratory water is described in Peltier and
Weber (1985). .
6.2.2 Starter cultures of Selenastrum capricornutum are available from the
following sources:
1. Aquatic Biology Section, Biological Methods Branch, Environmental
Monitoring and Support Laboratory, U.S. Environmental Protection
Agency, Cincinnati, Ohio 45268.
2. Environmental Research "Laboratory, U.S. Environmental Protection
Agency, 200 SW 35th Street, Con/all is, Oregon 97330.
3* American Type Culture Collection (Culture No. ATCC 22662), 12301
Parklawn Drive, Rockville, Maryland 10852.
4. Culture Collection of Algae, Botany Department, University of Texas,
Austin, Texas 78712.
6.2.3 Starter cultures of the fathead minnow (Pimephales promelas) and
Ceriodaphnia dubia (Berner, 1985) can be obtained from the Aquatic Biology
Section, Biological Methods Branch, EMSL-Cincinnati Newtown Facility,
Environmental Monitoring and Support Laboratory, U. S. Environmental
Protection Agency, Newtown, Ohio 45244 (Phone: FTS 778-8350;
Commercial 513-527-8350).
6.2.4 If because of their source there is any uncertainty concerning the
identity of the organisms, it is advisable to have them examined by a
second party to confirm their identification.
15
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6.3 SHIPMENT ;
6.3.1 Many states have strict regulations regarding the importation of
non-native fishes. Required clearances should be obtained from state
fisheries agencies before arrangements are made for the interstate shipment
of fathead minnows. ;
6.4 DISPOSAL
6.4.1 Test organisms must be destroyed after use.
16
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SECTION 7 "
DILUTION WATER
7.1 The source of dilution water used in the tests will depend largely on
the objectives of the study as described in Section 1: (1) If the
objective of the test is to estimate the inherent chronic toxicity of the
effluent, a standard dilution water (moderately hard water, Table 1) is
used; (2) If the objective of the test is to estimate the chronic toxicity
of the effluent in uncontaminated receiving water, the test may be
conducted using dilution water consisting of a single grab sample of
non-toxic receiving water collected upstream from the outfall, or with
other uncontaminated surface water or standard dilution water having
approximately the same hardness (+; 1056) as the receiving water; (3) If the
objective of the test is to determine the additive effects of the discharge
on already contaminated receiving water, the test is performed using
dilution water consisting of receiving water collected daily upstream from
the outfal1. -
7.2 When the dilution water is to be taken from the receiving water
"upstream" from the outfall, 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 sample should be collected immediately
prior to the test, but never more than 96 h before the test begins. Except
where it is used within 24 h, the sample should be chilled to 4°C during
or immediately following collection, and maintained at that temperature
until used.
7.3 Where toxicity-free dilution water is required in a test, the water is
considered acceptable if test organisms show adequate survival, growth, and
reproduction in the controls during the test.
7.4 Dechlorinated water should be used as dilution water only as a last
resort, because it is usually difficult to completely remove all the
residual chlorine or chlorinated organics, which may be very toxic to the
test organisms. Sodium thiosulfate is recommended for dech1orination
(1.0 mg anhydrous sodium thiosulfate/L will reduce 1.5 mg chlorine/L ).
After dech1 orination, total residual chlorine must be non-detectable.
7.5 If it is necessary to pass the dilution water through a deionizer to
remove unacceptably high concentrations of copper, lead, zinc, fluoride, or
other toxic substances before use, it must be reconstituted to restore the
calcium and magnesium removed by the deiom'zation process.
7.6 To prepare a synthetic fresh water, use the reagents listed in
Table 1. For example, to prepare 20 L of moderately hard synthetic water:
1. Place 19 L of distilled or deionized water in a properly cleaned
plastic carboy.
2. Add sufficient MgS04, NaHCOs and KC1 to the carboy, and stir
well.
17
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3. Add sufficient CaS04.2H20 to 1 L of distilled or deionized
water in a.separate flask, place on a magnetic stirrer until the
calcium sulfate has dissolved and add to the carboy and stir well.
4. Aerate vigorously for 24 h (with air filtered through cotton to
remove oil) to dissolve the added chemicals and stabilize the
medium.
7.7 The measured pH, hardness and alkalinity of the aerated water will be
approximately as indicated under "Final Water Quality" in Table 1.
TABLE 1. PREPARATION OF SYNTHETIC FRESH WATER4
Reagent Added (mg/L)b
Final Water Quality
Water
Type
Very soft
Soft
Moderately
Hard
Very hard
«aHC03 (
12.0
48.0
Hard 96.0
192.0
384.0
AND A • 2Ho
7.5
30.0
60.0
120.0
240.0
7.5
30.0
60.0
120.0-
240.0
KCL
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-
Hardnessd linityd
10-13
40-48
80-100
160-180
280-320
10-13
30-35
60-70
110-120
225-245
aTaken iri part from Marking and Dawson (1973).
bAdd reagent grade chemicals to distilled or deionized water.
cApproximate equilibrium pH after 24 h of aeration.
dExpressed as mg CaC03/L .
18
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SECTION 8
EFFLUENT AND RECEIVING WATER SAMPLING AND SAMPLE HANDLING
8.1 EFFLUENT SAMPLING
8.1.1 The effluent sampling point usually should be the same as that
specified in the NPOES discharge permit (USEPA, I979c). Conditions for
exception would be: (1) better access to a sampling point between the
final treatment and the discharge outfall; (2) if the processed waste is
chlorinated prior to discharge to the receiving waters, it may also be
desirable to take samples prior to contact with the chlorine to determine
toxicity of the unchlorinated effluent; or (3) in the event there is a
desire to evaluate the toxicity of the influent to municipal waste
treatment plants or separate wastewater streams in industrial facilities
prior to their being combined with other wastewater streams or non-contact
cooling water, additional sampling points may be chosen.
3.1.2 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 effluent
quality varies considerably with timet which can occur where holding times
are short, grab samples may seem preferable because of the ease of
collection and the potential of observing peaks (spikes) in toxicity.
However, the sampling duration of a grab sample is so short that full
characterization of an effluent over a 24-h period would require a
prohibitive number of separate samples and tests. Collection of a 24-h
composite sample, however, may dilute toxicity spikes, and averages the
quality of the effluent over the sampling period. A lengthy discussion of
the advantages and disadvantages of using grab or composite samples is
found in Peltier and Weber (1985).
8.1.3 Aeration during collection and transfer of effluents should be
minimized to reduce the loss of volatile chemicals.
8.1.4 Definitive tests performed for NPOES permit purposes require daily
effluent sample collection and daily renewal of test solutions.
8.2 RECEIVING WATER SAMPLING
8.2.1 It is common practice to collect grab samples for receiving water
toxicity studies.
8.2.2 When non-toxic receiving water is required for a test, it may be
collected upstream from the outfall or from other uncontaminated surface
water having approximately the same hardness (^ 10%) as the receiving
water. If the objective of the test is to determine the additive effects
of the discharge on receiving water which may already be contaminated, the
test is performed using dilution water consisting of receiving water
collected daily upstream from the outfall.
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8.2.3 Dilution water to be taken from the receiving water "upstream" from
the outfall, 1s collected at a point as close as possible to the outfall,
but upstrean from or outside of the zone influenced by the effluent.
8.2.4 To determine the extent of the zone of toxicity in the receiving
water downstream from the outfall, receiving water samples are collected
at several, distances downstream from the discharge. The time required for
the effluent-receiving-water mixture to travel to sampling points
downstream from the outfall may be difficult to ascertain, and it may not
be possible to correlate downstream toxicity with effluent toxicity at the
discharge point unless a dye study is performed. The toxicity of
receiving water samples from five stations downstream from the discharge
point can be evaluated using the same number of test vessels and test
organisms as used in one effluent toxicity test with five effluent
dilutions.
8.3 SAMPLE HANDLING AND PRESERVATION _
8.3.1 .If the data from the samples are to be acceptable for use in the
NPDES Program, the lapsed time from collection of a grab or composite
sample and the initiation of the test must not exceed 72 h. Composite
samples should be chilled during collection, where possible. Except when
used within 24 h of collection, samples must be chilled after collection
and maintained at 4°C until used.
8.3.2 Samples Used in On-Site Tests
8.3.2.1 Samples collected for on-site tests should be used within 24 h.
8.3.3 Samples Shipped to Off-Site Facilities
8.3.3.1 Samples collected for off-site toxicity testing are to be chilled
to 4°C when collected, shipped iced to the central laboratory, and there
transferred to a refrigerator (4QC) until used. Every effort must be
made to initiate the-test with an effluent sample on the day of arrival in
the laboratory.
8.3.3.2 Samples may be shipped in 4-L (1-gal) glass jugs, CUBITAIN£RSR,
or new plastic "milk" jugs. All sample containers should be rinsed with
source water before being filled with sample. Glass jugs can be cleaned
and reused (see p. 12), whereas CUBITAINERSR and plastic jugs are not
reused. Plastic containers used for effluents or toxic surface water
samples should be punctured after use to prevent reuse.
8.4 SAMPLE PREPARATION
8.4.1 With the Ceriodaphnia and fathead minnow tests, effluents and
surface waters must be filtered through a (30 urn) plankton net to remove
indigenous organims that may attack,or be confused with the test organisms
(see Ceriodaphnia test method for details). Surface waters used in algal
20
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toxicity tests must be filtered through a 0.45 urn pore diameter filter
before use. It may be necessary to first coarse-filter the dilution
and/or waste water through a nylon sieve having 2- to 4-nro holes to remove
debris and/or break up large floating or suspended solids.
8.4.2 The DO concentration in the dilution water should be near
saturation prior to use. Aeration will bring the 00 and other gases into
equilibrium with air, minimize oxygen demand, and stabilize the pH.
8.4.3 If the dilution water ana effluent must be warmed to bring them to
the prescribed test temperature, supersaturation of the dissolved gases
may become a problem. To prevent this problem, the effluent and dilution
water are heated to 25°C and checked for dissolved oxygen (DO) with a
probe. If the 00 exceeds 8.5 mg/L (100% saturation), the solutions are
aerated virorously with an air stone (usually 1-2 min) until the DO is
lowered to 100% saturation.
21
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SECTION 9 '
CHRONIC TOXICITY TEST END POINTS AND DATA ANALYSIS
9.1 END POINTS
9.1.1 Numerous terms are used to define the end points employed in
chronic toxicity tests, which have their origin in the earlier, full
life-cycle tests. As shorter "chronic" tests were developed, it became
common practice to apply the same terminology to the end points. The
primary terms in current use are listed below:
9.1.1.1 Safe Concentration - the highest concentration which will permit
normal propagation of fish and other aquatic life in receiving waters.
9.1.1.2 No Observed Effect Concentration (NOEC) - The highest
concentration of toxicant to which organisms are exposed in a full
life-cycle or partial life-cycle test, which causes no statistically
significant adverse effect on the observed parameters (usually
hatchability, survival, growth, and reproduction).
9.1.1.2. Lowest Observed Effect Concentration (LOEC) - The lowest
concentration of toxicant to which organisms are exposed in a life-cycle
or partial life-cycle test, which causes a statistically significant
adverse effect on the observed parameters (usually hatchability, survival,
growth, and reproduction).
*
9.1.1.3. Maximum Acceptable Toxicant Concentration (MATC) - An
undetermined concentration within the interval bounded by the NOEC and
LOEC.
9.1.1.4. Chronic Value (ChV) - A value lying between the NOEC and LOEC;
derived by calculating the geometric mean of the NOEC and LOEC. The term
is sometimes used interchangably with MATC.
9.1.1.5. LC (or EC) - Lethal concentration (LC) or effective
concentration (EC). A point estimate of the toxicant concentration that
would adversely affect a given percent of the test organisms, calculated
by regression (such as Probit Analysis). The LCI (or EC!) is the
estimated concentration of toxicant that adversely affects 1% of the test
population^ and is defined here as the threshold concentration, or lowest
concentration that would cause an adverse effect on the observed
parameters, and falls in the range of the NOEC and LOEC (Birge, et. al.
1981).
9.2 DATA ANALYSIS
9.2.1 Role of the Statistician
9.2.1.1 The choice of a statistical method to analyze toxicity test data
and the interpretation of the results of the analysis of the data from, any
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of the toxicity tests described in this manual can become problematic
because of the Inherent variability and sometimes unavoidable anomalies in
biological data. Analysts who are not proficient in statistics are
strongly advised to seek the assistance of a statistician before selecting
the method of analysis and using any of the results.
9.2.2 Plotting the Data
9.2.2.1 It is recommended that the data always be plotted as a
preliminary step, to help spot problems and detect unsuspected trends or
patterns in the responses.
9.2.3 Data Transformations
9.2.3.1 Transformations of the data, such as arc sine and logs, can be
used if they help the data meet the assumptions of the proposed analyses.
9.2.4 Analysis of Growth and Reproduction Data
9.2.4.1 Growth data from the fathead minnow larval survival and growth
test, and reproduction data from the Ceriodaphnia survival and
.reproduction test, are analyzed using Dunnett's Procedure (Dunnett, 1955)
if the assumptions of normality and homogeneity of variance are met (see
Appendix for details). If the assumptions are not met, the data are
analyzed using Steel's Many-One Rank Test (Steel, 1959; Miller, 1981).
9.2.4.2 The growth response data from the algal toxicity test may be
converted to a proportion of the growth of the controls, which may then be
analyzed by Probit Analysis (Finney, 1971) or, the growth response data,
after an appropriate transformation if necessary to meet the assumptions
of normality and homogeneity of variance, may be analyzed by Dunnett's
Procedure or Steel's Many-One Rank Test.
9.2.5 Analysis of.Mortality Data
9.2.5.1 Mortality data from the fathead minnow larval survival and growth
test and the fathead minnow embryo-larval survival and teratogenicity test
are used in .a Probit Analysis to determine the LCI, if Probit Analysis is
appropriate (see discussion below).
9.2.5.2 Fisher's Exact Test is used to analyze the mortality data from
the Ceriodaphnia survival and reproduction test, prior to the analysis of
the reproduction data.
9.2.5.3 Mortality data from the fathead minnow larval survival and growth
test, and the fathead minnow embryo-larval survival and teratogenicity
test, can be analyzed by Ounnett's Procedure or Steel's Many-One Rank Test
after transforming the square root of the proportion of dead organisms to
an arc sine value (see Appendix). This transformation is performed by the
computer program for Dunnett's Procedure provided in the Appendix.
23
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9.2.6 Ounnett's Procedure
9.2.6.1 Dunnett's Procedure consists of an analysis of variance (ANOVA)
to determine the error term, which is then used in a multiple comparison
method for comparing each of the treatment means with the control mean, in
a series of paired tests. Use of Dunnett's Procedure requires at least
two replicates per treatment.
9.2.6.2 The asumptions upon which the use of Ounnett's Procedure are
contingent are that the observations are independent and normally
distributed, with homogeneity of variance. Before analyzing the data, the
assumptions are checked using the procedures provided in the Appendix.
9.2.6.3 Some indication of the sensitivity of the analysis should be
provided by calculating: (1) the minimum difference between means that can
be detected as statistically significant, and (2) the percent change from
the control mean that this minimum difference represents for a given
test. Calculation of beta levels (Type II error, which results when the
null hypothesis is not rejected when it should be) as an indication of the
power of the test would be another alternative.
9.2.6.4 The safe concentration derived from this test is reported in
terms of the NOEC. A step-by-step example of Dunnett's Procedure is
provided in the Appendix.
9.2.6.5 If, after suitable transformations have been carried out, the
normality assumptions have not been met, the Steel Many-One Rank Test
should be used. • .
9.2.7 Steel's Many-One Rank Test
9.2.7.1 Steel's Many-One Rank Test is a multiple comparison method for
comparing several treatments with a control which is similar to Ounnett's
Procedure, except that it is not necessary to meet the assumption for
normality. The data are ranked, and the analysis is performed on the
ranks rather than on the data themselves. If the data are normally or
nearly normally distributed, Dunnett's Procedure would be more sensitive
(would detect smaller differences between the treatments and control).
For data that are not normally distributed, Steel's Many-One Rank Test can
be much more efficient (Hodges and Lehmann, 1956). It is necessary to
have at least four replicates to use Steel's test. The sensitivity of
this test cannot be stated in terms of the minimum difference between
treatment means and the control mean.
9.2,7.2 The safe concentration is reported as the NOEC. A step-by-step
example of Steel's Many-One Rank Test is provided in the Appendix.
9.2.8 Probit Analysis
9.2.8.1 Probit Analysis is used to analyze percentage data from
concentration-response tests. The analysis can provide an estimate of the
24
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concentration «f toxicant lethal to a giverr percent of the test organisms
and provide a. confidence interval for the estimate. Probit Analysis also
assumes normal distribution of log tolerances and independence of the
individual, responses. To use Probit Analysis, at least two partial
mortalities must be obtained.
9.2.9 Fisher's Exact Test
9.2.9.1 Fisher's Exact Test (Finney, 1948; Pearson and Hartley, 1962) is
a statistical method based on-the hypergeometric probability distribution
that can be used to test that the probability of a response is the same in
two binomial populations. When used with the Ceriodaphnia data, it
provides a conservative test of the equality of any two survival
proportions assuming only the independence of responses from a binomial
population.
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SECTION 10
REPORT PREPARATION!
The following general format and content are recommended for the
report:
10.1 INTRODUCTION
1. Permit number
2. Toxicity testing requirements of permit
3. Plant location
4. Name of receiving water body
5. Contractor (if contracted)
a. Name of firm
b. Phone number
c. Address
10.2 PLANT OPERATIONS
1. Product(s)
2. Raw materials
3. Operating schedule
4. Description of waste treatment
5. Schematic of waste treatment
6. Retention time (if applicable)
7. Volume of waste flow (MGD, CFS, GPM)
10.3 SOURCE OF EFFLUENT (AMBIENT) AND DILUTION WATER
1. Effluent Samples
a. Sampling point
b. Collection dates and times
c. Sample collection method
d. Physical and chemical data
2. Surface Mater Samples
a. Sampling point
b. Collection dates and times
c. Sample collection method
d. Physical and chemical data
e. Streamflow (at 7Q10 and at time of sampling)
1Adapted from: Peltier and Weber (1985). Prepared by Lee Tebo and
William Peltier, Environmental Services Division, U. S. Environmental
Protection Agency, Athens, Georgia.
26
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3. Dilution Water Samples
a. Source
b. Collection date and time
c. Pretreatment
d. Physical and chemical characteristics
10.4 TEST METHODS
1. Toxicity test method used
2. End point(s) of test
3. Deviations from reference method, if any, and the reason(s)
4. Date and time test started
5. Date and time test terminated
6. Type of test chambers
7. Volume of solution used/chamber
8. Number of organisms/test chamber
9. Number of replicate test chambers/treatment
10. Acclimation of test organisms (mean and range)
11. Test temperature, (mean and range)
10.5 TEST ORGANISMS
1. Scientific name
2. Age
3. Life stage
4. Mean length and weight (where applicable)
5. Source
6. Diseases and treatment (where applicable)
10.6 QUALITY ASSURANCE.
1. Standard toxicant used and source
2. Date and time of most recent test
3. Dilution water used in test
4. Results (LC50 or, where applicable, NOEC and/or EC!)
5. Physical and chemical methods used
10.7 RESULTS
1. Provide raw biological data in tabular form, including daily
records of affected organisms in each concentration (including
controls)
2. Provide table of LCSO's, NOECs, etc
3. Indicate statistical methods to calculate end points
4. Provide summary table of physical and chemical data
5. Tabulate QA data
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SECTION 11
TEST METHOD1.2
FATHEAD MINNOW (PIMEPHALES PROMELAS) LARVAL SURVIVAL AND GROWTH TEST
METHOD 1000.0
1. SCOPE AND APPLICATION
1.1 This method estimates the chronic toxicity of whole effluents and
receiving water to the fathead minnow (Pimephales promelas), using newly
hatched larvae in a seven-day, static renewal test".The effects include
the synergistic, antagonistic, and additive effects of all the chemical,
physical, and biological components which adversely affect the
physiological and biochemical functions of the test organisms.
1.2 Detection limits of the toxicity of an effluent or pure substance
are organism dependent.
1.3 Single or multiple excursions in acute toxicity may not be detected
using 24-h composite samples. Also, because of the long sample
collection period involved in composite sampling, and because the test
chambers are not sealed, highly volatile and highly degradeable toxicants
in the source may not be detected in'the test.
1.4 This method should be restricted to use by or under the supervision
of professionals experienced in aquatic toxicity testing.
2. SUMMARY OF METHOD
2.1 Larvae (preferrably less than 24-h old) are exposed in a static
renewal system for seven days to different concentrations of effluent or
to receiving water. Test results are based on the survival and growth
(increase in weight) of the larvae.
3. DEFINITIONS
(Reserved for addition of terms at a later date.)
4. INTERFERENCES
4.1 Toxic substances may be introduced by contaminants in dilution
water, glassware, sample hardware, and testing equipment (see Section 5,
Facilities and Equipment).
format used for this method was taken from Kbpp, 1983.
2This method was adapted from Norberg and Mount, 1985.
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4.2 Adverse effects of low dissolved oxygen (DO) concentrations, high
concentrations of suspended and/or dissolved solids, and extremes of pH,
may mask the. presence of toxic substances.
4.3 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling and Sample
Handling). -
4.4 Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.
4.5 Food added during the test may sequester metals and other toxic
substances and confound test results.
5. SAFETY
5.1 See Section 3, Health and Safety. .
6. APPARATUS AND EQUIPMENT
6.1 Fathead minnow and brine shrimp culture units — see Peltier and
Weber (1985). This test requires 150-300 newly hatched larvae. It is
preferable to obtain this fish from an inhouse fathead minnow culture
unit. If it is not feasible to culture fish inhouse, newly hatched larvae
can be shipped in well oxygenated water in insulated containers.
6.2 Samplers — automatic sampler, preferrably with sample cooling
capability, that can collect a 24-h composite sample of 4 L.
6.3 Sample containers — for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling and Sample Handling).
6.4 Environmental chamber or equivalent facility with temperature control
(25+ 2°C).
6.5 Water purification system — Millipore Super-Q or equivalent.
6.6 Balance — analytical, capable of accurately weighing larvae to
0.0001 g.
6.7 Reference weights, Class S ~ for checking performance of balance.
Weights should bracket the expected weights of the weighing pans and the
expected weights of the pans plus fish.
6.8 Test chambers — borosilicate glass or non-toxic disposable plastic
labware. A minimum of two 1-L beakers are required for each concentration
and control. To avoid potential contamination from the air, the chambers
should be covered during the test.
6.9 Volumetric flasks and graduated cylinders — Class A, borosilicate
glass or non-toxic plastic labware, 10-1000 ml for making test solutions.
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6.10 Volumetric pipets-- Class A, 1-100 ml.
6.11 Serological pipets— 1-10 ml, graduated.
6.12 Pipet bulbs and fillers — PropipetR, or equivalent.
6.13 Droppers, and glass tubing with fire polished edges, 4mm ID — for
transferring larvae.
6.14 Wash bottles — for washing embryos from substrates and containers
and for rinsing small glassware and instrument electrodes and probes.
6.15 Glass or electronic thermometers — for measuring water temperatures.
6.16 Bulb-thermograph or electronic-chart type thermometers — for
continuously recording temperature.
6.17 National Bureau of Standards Certified thermometer (see USEPA Method
170.1, USEPA 1979b).
6.18 pH, DO, and specific conductivity meters — for routine physical and
chemical measurements. Unless the test is being conducted to specifically
measure the effect of one of the above parameters, a portable, field-grade
instrument is acceptable.
6.19 Miscellaneous apparatus and equipment — transfer containers, pumps,
and automatic dilution devices should be constructed of materials as
indicated in Section 5, Facilities and Equipment.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Reagent water — defined as activated-carbon-filtered distilled or
deionized water that does not contain substances which are toxic to the
test organisms. A water purification system may be used to generate
reagent water (see paragraph 6.5 above).
7.2 Effluent, surface water, and dilution water — see Section 7,
Dilution Water, and Section 8, Effluent and Surface Water Sampling and
Sample Handling.
7.3 Reagents for hardness and alkalinity tests (see USEPA Methods 130.2
and 310.1, USEPA 1979b).
7.4 pH buffers 4, 7, and 10 (or as per instructions of instrument
manufacturer) for standards and calibration check (see USEPA Method 150.1,
USEPA 1979b).
7.5 Membranes and filling solutions for dissolved oxygen probe (see USEPA
Method 360.1, USEPA 1979b), or reagents for modified Winkler analysis.
7.6 Laboratory quality assurance samples and standards for the above
methods.
30 .
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7.7 •Specific conductivity standards (see USEPA Method 120.T, USEPA 19795).
7.8 Reference toxicant solutions (see Section 4, Quality Assurance).
7.9 Formalin (4%) for use as a preservative for the fish larvae.
7.10 Brine Shrimp (Artemia) Cysts — see Peltier and Weber (1985).
7.10.1 Although there are many commercial sources of brine shrimp eggs,
the Brazilian or Colombian strains are preferred because the supplies
examined have had low concentrations of chemical residues. (One source is
Aquarium Products, 180 L Penrod Ct., Slen Burnie, MD, 21061). Each new
batch of Artemia cysts should be evaluated for nutritional suitability
against known suitable reference cysts by performing a larval growth
test.. It is recommended that a sample of newly-hatched Artemia nauplii
from each new batch of cysts be chemically analyzed to determine that the
concentration of total organic chlorine does not exceed 0.15 ug/g wet
weight or the total concentration of organochlorine pesticides plus PCBs
does not exceed 0.3 ug/g wet weight (USEPA, 1982). If those values are
exceeded, the Artemia should not be used.
7.10.2 Limited quantities of reference Artemia cysts, information on
commerical sources of good quality Artemia cysts, and procedures for
determining cyst suitability are available from the Quality Assurance
Branch, Environmental Monitoring and Support Laboratory, U. S.
Environmental Protection Agency, Cincinnati, Ohio, 45268.
7.11 Test organisms — Newly-hatched fathead minnow larvae (see Peltier
and Weber, 1985).
8. SAMPLE COLLECTION, PRESERVATION AND STORAGE
8.1 See Section 8, Effluent and Receiving Water Sampling and Sample
Handling..
9. CALIBRATION AND STANDARDIZATION
9.1. See Section 4, Quality Assurance.
10. QUALITY CONTROL
10.1 See Section 4, Quality Assurance.
11. PROCEDURES
11.1 TEST SOLUTIONS
11.1.1 Surface Waters
11.1.1.1 Surface water toxicity is determined with samples used directly
as collected.
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11.1.2 Effluents
11.1.2.1 The selection of the effluent tesl concentrations should be
based on the objectives of the study. One of two dilution factors,
approximately 0.3 or 0.5, is commonly used. A dilution factor of
approximately 0.3 allows testing between 100% and IX effluent using only
five effluent concentrations (100%, 30%, 10%, 3%, and 1%). This series of
dilutions minimizes the level of effort, but because of the wide interval
between test concentrations provides poor test precision (+ 300%).
A dilution factor of 0.5 provides greater precision (+• 1005?), but requires
several additional dilutions to span the same range oT* effluent
concentrations. Improvements in precision decline rapidly as the dilution
factor is increased beyond 0.5
11.1.2.2 If the effluent is known or suspected to be highly toxic, a
lower range of effluent concentrations should be used, beginning at 10%.
If a high rate of mortality is observed during the first 1 to 2 h of the
test, additional dilutions at.the lower range of effluent concentrations
can be added.
11.1.2.3 The volume of effluent required for daily renewal of two
replicates per concentration, each containing 500 ml of test solution, is
approximately 2 L. Prepare enough test solution (approximately 1400 mi.)
at each effluent concentration to provide 400 mL additional volume for
chemical analyses.
11.2 START OF THE TEST
11.2.1 Tests should begin as soon as possible, preferably within 24 h of
sample collection. If the persistence of the sample toxicity is not
known, the maximum holding time should not exceed 36 h. In no case should
the test be started more than 72 h after sample collection. Just prior to
testing, the temperature of the sample should be adjusted to (25 +_ 2°C)
and maintained at that temperature until portions are added to the
dilution water.
11.2.2 The test is initiated by placing larvae one or two at a time, into
each test.chamber in sequential order, until each chamber contains 10
larvae, for a- total of at least 20 larvae for each concentration. The
test organisms should come from a pool of larvae consisting of at least
three separate spawnings. The amount of water added to the chambers when
transferring the larvae to the compartments should be kept to a minimum to
avoid unnecessary dilution of the test concentrations.
11.2.3 Randomize the position, of test chambers at the beginning of the
test.
11.3 LIGHT, PHOTOPERIOD AND TEMPERATURE
11.3;1 The light quality and intensity should be at ambient laboratory
levels, which is approximately 10-20 u£/m2/s, or 50 to 100 foot
32
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candles (ft-c), with a photoperiod of 16 h of light and 8 h of darkness.
The water temperature in the test chambers should be maintained at
25 + 2°C.
11.4 DISSOLVED OXYGEN (DO)
11.4.1 Aeration may affect the toxicity of effluents and should be used
only as a last resort to maintain satisfactory 00 concentrations. The 00
concentrations should not fall below 40% saturation. If it is necessary
to aerate, all concentrations and the control should be aerated. The
aeration rate should not exceed 100 bubbles/min, using a pipet with an
orifice of approximately 1.5 mm, such as a 1-mL, Kimax serological pipet,
No. 37033, or equivalent. Care should be taken to ensure that turbulence
resulting from aeration does not cause undue physical stress to the fish.
11.5 FEEDING
11.5.1' The fish in each test chamber are fed 0.1 ml (approximately 700 to
1000) of a concentrated suspension of newly hatched (less than 24-h old)
brine shrimp nauplii three times daily at 4-h intervals (at the beginning,
midway, and at the end of the work day). The nauplii should be rinsed
with freshwater before use.
11.6 DAILY CLEANING OF TEST CHAMBERS .
11.6.1 Before the .daily renewal of test solutions, uneaten and dead brine
.shrimp and other debris are removed from the bottom of the test chambers
with a siphon hose. Alternately, a large pipet (50 ml) fitted with a
rubber bulb can be used. Because of their small size during the first few
days of the tests, larvae are easily drawn into the siphon tube when
cleaning the test chambers. By placing the test chambers on a light box,
inadvertent removal of larvae can be greatly reduced because they can be
more easily seen. If the water siphoned from the test chambers is
collected in a white plastic tray, the larvae caught up in the siphon can
be retrieved and returned to the chambers.
11.7 TEST, SOLUTION RENEWAL
11.7.1 The test solutions are renewed daily using freshly collected
samples, immediately after cleaning the test chambers. The vater level in
each chamber is lowered to a depth of 7 to 10 mm, which leaves 15 to 20%
of the test solution. New test solution (500 mL) should be added slowly
by pouring down the side of the test chamber to avoid excessive turbulence
for the larvae.
11.8 ROUTINE CHEMICAL AND PHYSICAL ANALYSIS
11.8.1 At a minimum, the following measurements are made:
11.8.1.1 DO is measured at the beginning and end of each 24-h exposure
period at all test concentrations and in the control.
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11.8.1.2 Temperature, pH, and conductivity,are measured at the beginning
of each 24-h exposure period at aFI test cohcerftrations and in the control.
11.8.1.3 Alkalinity and hardness are measured at the beginning of each
24-h exposure period in 100% effluent and in the control.
11.9 OBSERVATIONS DURING THE TEST
11.9.1 The number of live and dead larvae in each test chamber are
recorded daily, and the dead larvae are discarded.
11.9.2 Protect the larvae from unnecessary disturbance during the test by
carrying out the daily test observations, solution renewals, and removal
of dead larvae, carefully. Make sure the larvae remain immersed during
the performance of the above operations.
11.10- TERMINATION OF THE TEST ' •
11.10.1 The test is terminated after seven days of exposure. At
termination, the larvae in each test chamber are counted and preserved as
a group, in 4% formalin, and are dried and weighed at a later date.
Immediately prior to the dry weight analysis, the preserved larvae are
rinsed in distilled water. The group of rinsed larvae from each test
chamber are transferred to a tared weighing boat and dried at 100°C for
a minimum of 2 h. Immediately upon removal from the drying oven, the
weighing boats are placed in a dessicator to prevent the absorption of
moisture from the air, until weighed. The weights should be measured to
the nearest 0.1 mg.
11.11 ACCEPTABILITY OF TEST RESULTS
11.11.1 For the test results to be acceptable, survival in the controls
must be at least 80%, except where survival in any test concentration is
80% or better.
11.12 SUMMARY OF TEST CONDITIONS
11.12.1 A summary of test conditions is listed in Table 1.
12. CALCULATIONS.
12.1 The endpoints of toxicity tests using the fathead minnow larvae are
based on the adverse effects on survival and growth. Probit Analysis
(Firiney, 1971), Ounnett's Procedure (Dunnett, 1955), and Steel's Many-One
Rank Test (Steel, 1959; Miller, 1981), are used to evaluate the data. See
the Appendix for examples of the manual computations, and the program
listings and examples of data input and program output.
12.2 The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent. Tests for
normality and homogeneity of variance are included in the Appendix. The
assistance of a statistician is recommended for analysts who are not
proficient in 'statistics.
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12.3 Table 2 shows the .survival and growth response after seven days of
exposure to the reference toxicant NaPCP. The data for an effluent would
be similar except'that the test concentrations would be expressed as a
percent.
12.4 Analysis of Survival Data - Probit Analysis
12.4.1 Probit Analysis (Finney, 1971) is used to determine the
concentration causing }% mortality (LCI). In this analysis, the total
survival data from all test replicates at a given concentration are
combined (see Table 2, Mean Proportion Surviving). A listing of the
computer program and an example of data input and program output for the
Probit Analysis are provided in the Appendix. Note that for the data in
Table 2, the number of animals exposed in each concentration would be 40,
and number that died at each concentration could be calculated from the
proportions in the table. The program transforms the concentration values
to logig and the percent mortality to probits, and then performs a
regression analysis.
12.4.2 Report the LCI and its 95/6 confidence 1-imits. The LCI is an
estimate of the threshold (chronically toxic) concentration. For the
sample data in Table 2, the LCI is 128 ug NaPCP/L, with upper and lower
95% confidence intervals of 30.5 .ug/L and 198 ug/L, respectively.
12.4.3 If the data do not meet the assumptions necessary for the Probit
analysis, analyze the data using Dunnett's Test, as described below.
12.5 Analysis of Survival Data - Dunnett's Procedure
12.5.1 The survival data meet the normality assumptions (see Appendix),
which indicates that analysis b'y Dunnett's Procedure is appropriate.
If the data had not met the assumptions, Steel's Many-One Rank Test would
have been used (see Section 9). Dunnett's Procedure (Dunnett, 1955; Steel
and Torrie, 1960) includes an analysis of variance (ANOVA), followed by a
comparison of each toxicant concentration mean with the control mean. The
error value calculated in the ANOVA is used in the comparison of the
control and treatment means.
12.5.2 It is necessary to have at least duplicate test chambers at each
treatment concentration to perform this test.
12.5.3 The computer program listed in the Appendix generates output which
includes an ANOVA table, a statement about each treatment mean that can be
used.to identity the NOEC and LOEC, and the minimum difference between
treatment and control means that can be detected as statistically
significant.
12.5.4 The computer program makes the necessary transformation of the
survival data by converting the square root of the proportion of surviving
organisms to arc sine during the analysis, and includes a special
modification of the arc sine transformation which is required where the
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proportion of surviving organisms is 0 or 1 (Bartlett, 1937). For a more
detailed information on the arc sine transformation, see the Appendix.
12.5.5 The results of the analysis of variance of the data from Table 2
are shown in Table 3, and indicate a statistically significant difference
in survival among NaPCP concentrations.
12.5.6 The results of the comparison of the control with the treatment
effects, using 18 degrees of freedom and with a Dunnett's "t" value of
2.41 (P » 0.05), indicate that the NOEC is Concentration 5 (256 ug/L) and
the LOEC is Concentration 6 (512 ug/L). The computer printout of these
results is as follows:
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 2
(32 US NAPCP/L) AND CONTROL.
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 3
(64 UG NAPCP/L) AND;CONTROL.
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 4
(128 UG NAPCP/L) AND CONTROL.
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 5
(256 UG NAPCP/L) AND CONTROL.
THERE IS A SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 6
(512 UG NAPCP/L) AND CONTROL.
12-.5.7 For this set of data, the minimum difference that can be detected
as statistically significant is 0.177. This represents a 19% reduction in
the mean response (survival) from the control.
12.6 The chronic value (ChV) is the geometric mean of the NOEC and LOEC
and is calculated as follows:
Logic NOEC = Logic 256 = 2.4082
Logic LOEC = Logic 512 * 2.7093
ChV = Antilog (2.4082 + 2.7093)/2 = Antilog 2.5588
ChV = 362 ug/L NaPCP
12.7 Analysis of"Growth Data - Dunnett's Procedure
12.7.1 The use of Ounnett's Procedure to analyze the growth data,
expressed in terms of average dry weight (Table 2), is similar to that for
the survival data, except that the weight data are not transformed.
12.7.2. The average dry weight of the larvae from each replicate test
chamber (Table 2) is entered into the program.
12.7.3. The results from the analysis of variance of the data in Table 2
are found in Table 4. The analysis indicates a statistically significant
difference in the effects on larval growth at the various concentrations
of NaPCP.
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12.7.4. The results of the comparison of the average weights for each
treatment with the average weights for the control, using 18 degrees of
freedom and with, a Dunnett's "t" value of 2.41 (P-= 0.05), indicate that
the NOEC is Concentration 4 (128 ug NaPCP/L) and the LOEC is
Concentration 5 (256 ug NaPCP/L). The computer output from the Ounnett
program is as follows (the control is Concentration 1):
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 2
(32 UG NAPCP/L) AND CONTROL.
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 3
(64 UG NAPCP/L) AND CONTROL.
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATIONS
(128 UG NAPCP/L) AND CONTROL.
THERE IS A SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 5
(256 UG NAPCP/L) AND CONTROL.
THERE IS A SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 6
(512 UG NAPCP/L) AND CONTROL.
For this set of data, the minimum difference that can be detected as
statistically significant is 0.095. This represents a 13.3% reduction in
the mean response from the control.
12.6.5 The chronic value (ChV) is the geometric mean of the NOEC and LOEC
and is calculated as follows:
Logic NOEC = Logio 128 = 2.1072
Log-jo LOEC * Logic 256 * 2.4082
ChV = Antilog (2.1072 + 2.40S2)/2 = Antilog 2.2577
ChV « 181 ug/L NaPCP
12.6.6 The results of the test indicate that growth was a more sensitive
index of the effects of NaPCP than was survival.
13. PRECISION AND ACCURACY
13.1 PRECISION
13.1.1 Information on the single laboratory precision of the fathead
minnow larval survival and growth test is presented in Table 5.
13.2 ACCURACY
1312.1 The accuracy of toxicity tests can not be determined.
37
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TABLE 1. SUMMARY OF RECOMMENDED TEST CONDITIONS FOR FATHEAD MINNOW
(PIMEPHALES PROMELAS) LARVAL SURVIVAL AND GROWTH TEST
1. Test type:
2. Temperature (°C):
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test chamber size:
7. Test solution volume:
8. Renewal of test
concentrations:
9. Age of test organisms:
10. Larvae/test chamber
and control:
Replicate
chambers/concentration:
11.
12. Feeding regime:
13.
14.
Cleaning:
Aeration:
15. Dilution water:
16. Effluent concentrations:
17. Dilution factor:
18. Test duration:
19. Effects measured:
Static renewal
25 + 2°C
Ambient laboratory illumination
10-20 u£/m2/s (50-100 ft-c)(ambient lab
levels)
16 h light, 8 h darkness
1-L containers
500 mL/replicate
Daily
Newly hatched larvaed
10 larvae/chamber;
Minimum of 20 larvae/test concentration
Minimum of 2
Feed 0.1 mL newly hatched brine shrimp nauplii
three times daily, 4 h between feedings (at the
begining, midway, and the end of the work day)
Siphon daily, immediately before test solution
renewal
None, unless DO concentration falls below 4055
saturation. Rate should be less than 100
bubbles/min.
Moderately hard standard water, receiving
water, other surface water, ground water, or
synthetic water similar to receiving water
At least 5 and a control
Approximately 0.3 or 0.5
7 days
Survival and growth (increase in weight)
38
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TABLE 3. ANALYSIS OF VARIANCE OF SURVIVAL DATA IN TABLE 2.
Source
Among
Within
Total
OF
5
18
23
Sum of Squares
1.575
0.426
2.001
Calculated Tabular
Mean Square F ^0.05
0.315 13.3* 2.77
0.024
*
Significant at P s 0.01.
TABLE 4. RESULTS FROM ANALYSIS OF VARIANCE OF
DRY WEIGHT DATA IN TABLE 2.
Source
Among
Within
Total
OF
5
18
23
Sum of Squares
0.155
0.060
0.215
Calculated Tabular
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0.031 9.31* 2.77
0.003
Significant at P = 0.05
40
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TABLE 5. PRECISION OF THE FATHEAD MINNOW LARVAL SURVIVAL
AND GROWTH TEST, USING NAPCP AS A REFERENCE TOXICANT*
NOEC
Test (ug/L)
1 256
2 128
3 256
4 128
5 . 128
6 256
LOEC
(ug/L)
512
256
512
256
256
512
Chronic
Value
(ug/L)
362
181
362
181
181
362
aFor a discussion of the precision of data from chronic toxicity
tests see Section 4, Quality"Assurance.
41
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, . SECTION 12
TEST METHOD1.2
FATHEAD MINNOW (PIMEPHALES PROMELAS)
EMBRYO-LARVAL SURVIVAL AND TERATQGENICITY TEST
METHOD 1001.0
1. SCOPE AND APPLICATION
1.1 This method estimates the chronic toxicity of whole effluents and
receiving water to the fathead minnow (Pimeohales promelas). using embryos
and larvae in an eight-day, static renewal test. The effects include the
synergistic, antagonistic, and additive effects of all the chemical,
physical, and biological components which adversely affect the
physiological and biochemical functions of the test organisms. The test
is useful in screening for teratogens because organisms are exposed during
embryonic development.
1.2 Detection limits of the toxicity of an effluent or pure substance are
organism dependent.
1.3 Single or multiple excursions in acute toxicity may not be detected
using 24-h composite samples. Also,- because of the long sample collection
period involved in composite sampling, and because the test chambers are
not sealed, highly volatile and highly degradeable toxicants in the source
may not be detected in the-test.
1.4 This method should be restricted to use by or under the supervision
of professionals experienced in aquatic toxicity testing.
2. SUMMARY OF METHOD
2.1 Fathead minnow embryos and larvae are exposed in a static renewal
system, from shortly after fertilization of the eggs through four days
posthatching (total of eight days), to different concentrations of
effluent or to receiving water. Test results are based on the total
frequency of both mortality and gross morphological deformities (terata).
3. DEFINITIONS
(Reserved for addition of terms at a later date.)
4. INTERFERENCES
4.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see -Section 5,
Facilities and Equipment).
format used for this method was taken from Kopp, 1983.
2This method was adapted from Birge and Black, 1981.
42
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4.2 Adverse effects of low dissolved oxygen (DO), high concentrations of
suspended and/or dissolved solids, and extremes of pH may mask the
presence of toxic substances.
4.3 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling and Sample
Handling).-
4.4 Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.
5. SAFETY
5.1 See Section 3, Health and Safety.
6. APPARATUS AND EQUIPMENT
6.1 Laboratory fatheadl minnow and brine shrimp culture units — See
Peltier and Weber, 1985. To test effluent toxicity on-site or in the
laboratory, sufficient numbers of newly fertilized eggs must be available,
preferably from a laboratory fathead minnow culture unit. If necessary,
embryos can be shipped in well oxygenated water in insulated containers.
6.2 Samplers — automatic sampler, preferrably with sample cooling
capability, that can collect a 24-h composite sample of 2 L.
6.3 Sample containers — for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling and Sample Handling).
6.4 Environmental chamber or equivalent facility with temperature control
(25± 2°C).
6.5 Water purification system — Millipore Super-Q or equivalent.
6.6 Balance — analytical, capable of accurately weighing 0.0001 g.
6.7 Reference weights, Class S — for checking performance of balance.
6.8 Test chambers — borosilicate glass or non-toxic plastic labware; a
minimum of two 500 mL beakers or deep petri dishes with covers per test
concentration. The chambers should be covered during the test to avoid
potential contamination from the air. Care must be taken to avoid
inadvertently removing.embryos or larvae when test solutions are decanted
from the'Chambers. The covers are removed only for observation and
removal of dead organisms.
6.9 Dissecting microscope, or long focal length magnifying lens, hand or
stand supported — for examining embryos and larvae in the test chambers.
6.10 Light box, microscope lamp, or flashlight — for illuminating
embryos and chambers during examination and observation of embryos and
larvae.
43
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6.11 Volumetric flasks and graduated cylinders — Class A, borosillcate
glass or non-toxic plastic labware, 10-1000 ml, for making test solutions.
6.12 Volumetric pi pets— Class A, 1-100 ml.
6.13 Serological pi pets— 1-10 ml, graduated.
6.14 Pipet bulbs and fillers — PropipetR, or equivalent.
6.15 Droppers, and glass tubing with fire polished edges, 2 -mm ID — for
transferring embryos, and 4-mm ID — for transferring larvae.
6.16 Wash bottles ~ for washing embryos from substrates and containers
and for rinsing small glassware and instrument electrodes and probes.
6.17 Glass or electronic thermometers — for measuring water temperatures.
6.18 Bulb-thermograph or electronic-chart type thermometers — for
continuously recording temperature.
6.19 National Bureau of Standards Certified thermometer (see EPA Method
170.1, USEPA 1979b).
6.20 pH, 00, and specific conductivity meters — for routine physical and
chemical measurements. Unless the tes't is being conducted to specifically
measure the effect of one of the above parameters, a portable, field-grade
instrument is -acceptable.
lS' JS1*iseetl«!wbos "apparatus and equipment — transfer containers, pumps,
and automatic dilution devices should be constructed of materials as
indicated in Section 5, Facilities and Equipment.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Reagent water — defined as carbon-filtered distilled or deionized
water which does not contain substances which are toxic to the test
organisms. A water purification system may be used to generate reagent
water (see paragraph 6.5 above).
7.2 Effluent, surface water, and dilution water — see Section 7,
Dilution Water, and Section 8, Effluent and Surface Water Sampling and
Sample Handling*
7.3 Reagents; for; hardness and alkalinity tests (see EPA Methods 130.2 and
310.1, USEPA T979b|.
7.4 pH buffers 4, 7, and 10 (or as per instructions of instrument
manufacturer) for standards and calibration check (see USEPA Method 150.1,
USEPA 1979b).
7.5 Membranes and filling solutions for dissolved oxygen probe (see USEPA
Method 360.1, USEPA 1979b), or reagents for modified Winkler analysis.
44
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7.6 Laboratory quality assurance samples and standards for the above
methods.
7.7 Specific conductivity standards (see EPA Method 120.1, USEPA 1979b).
7.8 Reference toxicant solutions (see Section 4, Quality Assurance).
7.9 Test Organisms — fathead minnow embryos, .2- to 24-h old (preferably
less than 12-h old), and spawned over less than an 8-h period, are used
for the test (for fathead minnow culturing methods (Peltier and Weber,
1985). Spawning substrates with the newly-spawned, fertilized embryos are
removed from the spawning tanks or ponds, and the embryos are separated
from the spawning substrate by using the index finger and rolling the
embryos gently with a circular movement of the finger (See Gast and
Brungs, 1973). The embryos are then washed from the spawning substrate
into a petri dish, using the spray from a wash bottle filled with fresh
culture water, and are examined using a dissecting microscope or other
suitable magnifying device. Damaged and infertile eggs are discarded. It
is strongly recommended that the embryos be obtained from fish cultured
inhouse, rather than from outside sources, to eliminate the uncertainty of
damage caused by shipping and handling that may not be observable, but
which might affect the results of the test. ..
7.9.1 The embryos from several substrates are then pooled in a single
container to provide a sufficient number to conduct the required number of
tests. These embryos may be used immediately to start a test or may be
placed in a suitable container and transported for use at a remote
locationt When overnight transportation is required, embryos should be
obtained when they are 6- to 8-h old. This permits the tests at the
remote site to be started with less than 24-h old embryos. Embryos should
be transported or. shipped in clean, opaque, insulated containers, in well
aerated or oxygenated fresh culture or dilution water, and should be
protected from extremes of temperature and any other stressful conditions
during transport. Instantaneous changes of water temperature when embryos
are transferred from culture unit water to test dilution water, or from
transport container water to on-site test dilution water, should be less
than 2°C. Instantaneous changes of pH, dissolved ions, osmotic
strength, and DO should also be kept to a minimum.
7.9.2 The number of embryos needed to start the test will depend on the
number of tests to be conducted and the objectives. The test is usually
conducted with at least duplicate test chambers at each toxicant
concentration and in the control. At least 40 embryos are exposed per
test concentration (20 embryos in each of duplicate test chambers),
although a larger number, such as 100 (50 embryos in each of duplicate
test chambers), will provide better precision. If fewer than 40 embryos
are used per concentration, test precision will be significantly reduced.
Using more than 100 embryos per test concentration will not provide
sufficient advantage to offset the increased time and effort required for
observation and counting and removal of dead embryos during the test. The
use of 50 embryos per replicate and TOO embryos per test concentration
(a total of 600 embryos for five test concentrations and a control) is
considered optimal.
45
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8. SAMPLE COLLECTION, PRESERVATION AND HANDLING
8.1 See Section 8, Effluent and Receiving Water Sampling and Sample
Handling.
9. CALIBRATION AND STANDARDIZATION
9.1 See Section 4, Quality Assurance.
10. QUALITY CONTROL
10.1 See Section 4, Quality Assurance.
11. PROCEDURES
11.1 TEST SOLUTIONS
11.1.1 Surface Waters ; . -
11.1.1.1 Surface water toxicity is determined with samples used directly
as collected. -
11.1.2 Effluents . '
11.1.2.1 The selection of the effluent test concentrations should be
based on the objectives of the study. One of two dilution factors,
approximately 0.3 or 0.5, is commonly used. A dilution factor of
approximately 0.3 allows testing between 100% and 1% effluent using only
five effluent concentrations (100%, 30%, 10%, 3%, and 1%). This series of
dilutions minimizes the level of effort, but because of the wide interval
between test concentrations provides poor test precision (+ 300%).
A dilution factor of 0.5 provides greater precision (+ 100%), but requires
several additional dilutions to span the same range oT effluent
concentrations. Improvements in precision decline rapidly as the dilution
factor is increased beyond 0.5
11.1.2.2 If the effluent is known or suspected to be highly toxic, a
lower range of effluent concentrations should be used (such as 10%, 3%,
1%, 0.3%, and 0.1%). If a high rate of mortality is observed during the
first 1 to 2 h of the test, additional dilutions at the lower range of
effluent concentrations can be added.
11.1.2.3 The volume of effluent required for daily renewal of two
replicates per concentration, each containing 400 mL of test solution, is
approximately 2 L. Prepare enough test solution (approximately 1200 mL)
at each effluent concentration to provide 400 mL additional volume for
chemical analyses.
11.1.2.4 The hardness of the test solutions must exceed 25 mg/L
to insure hatching success.
46
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11.2 START OF THE TEST
11.2.1 Tests should begin as soon as possible, preferably within 24 h of
sample collection. If the persistence of the sample toxicity is not
known, the maximum holding time should not exceed 36 h. In no case should
the test be started more than 72 h after sample collection. Oust prior to
testing, the temperature of the sample should be adjusted to 25 + 2°C
and maintained at that temperature until portions are added to tFe
dilution water.
11.2.2 Gently agitate and mix the embryos to be used in the test in a
large container so that eggs from different spawns are evenly dispersed.
11.2.3 Randomly add the embryos to each test chamber. Accomplish this by
adding a small number of embryos (5 to 15) to test chambers selected in a
random fashion using a small bore (2mm) glass tube calibrated to contain
approximately the desired number of embryos. Repeat the process until the
appropriate number of embryos, have been added to each chamber.
Alternately, the total number of embryos are added at the same time to
randomly selected test chambers.
11.2.4 After the embryos have been distributed to each test chamber,
examine and count them. Remove and discard damaged or infertile eggs and
replace with new undamaged embryos. It may be more convenient and
efficient to transfer embryos to intermediate containers of dilution water
for examination and counting. After the embryos have been examined and
counted in the intermediate container, assign them to the appropriate test
chamber and transfer them with a minimum of dilution water. If ntore than
one test is being performed, the exposure is started for all of the tests
at approximately the same time.
11.2.5 Randomize the position of the test chambers at the beginning of
the test.
11.3 LIGHT, PHOTOPERIOD AND TEMPERATURE
11.3.1 The light quality and intensity should be at ambient laboratory
levels, approximately 10-20 uE/m2/s, or 50 to 100 foot candles (ft-c),
with a photoperiod of 16 h of light and 8 h of darkness. The test water
temperature should be maintained at 25 + 2°C.
11.4 DISSOLVED OXYGEN (DO)
11.4.1 Aeration may affect the toxicity of effluents and should be used
only as a last resort to maintain satisfactory DO concentrations. The DO
concentrations should not fall below 40% saturation. If it is necessary
to aerate, all concentrations and the control should be aerated. The rate
should not exceed 100 bubbles/min, using a pipet with a 1-2 mm orifice,
such as a 1-mL Kimax Serological Pipet No. 37033, or equivalent. Care
should be taken to ensure that turbulence resulting from the aeration does
not cause undue physical stress to the fish.
47
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11.5 FEEDING "
:-'":;. .:, •'•* -t
11.5.1 Since feeding 1s not required, It Is not necessary to clean the
test chambers daily.
11.6 TEST SOLUTION RENEWAL
11.6.1 The test solutions are renewed dally using freshly collected
samples, Immediately after cleaning the test chambers. During the dally
renewal process 7-10 mm of water Is left In the chamber to ensure that the
embryos and larvae remain submerged during the renewal process. New test
solution (400 mL) should be added slowly by pouring down the side of the
test chamber to avoid excessive turbulence for the larvae.
11.7 ROUTINE CHEMICAL AND PHYSICAL DETERMINATIONS
11.8.1 At a minimum, the following measurements are made:
11.8.1.1 DO Is measured at the beginning and end of each 24-h exposure
period at all test concentrations and In the control.
11.8.1.2 Temperature, pH, and conductivity are measured at the beginning
of each 24-h exposure period at all test concentrations and in the control.
11.8.1.3 Alkalinity and hardness are"measured at the beginning of each
24-h exposure period in 100% effluent and in the control.
r
TT.8 OBSERVATIONS DURING THE TEST
11.8.1 At the end of the first 24 h of exposure, before renewing the test
solutions, examine and count the embryos. Remove the dead embryos (milky
colored and opaque) and record the number. If the rate of mortality or
fungal infection exceeds 20% in the control chambers, or if excessive
non-concentration-related-mortality occurs, terminate the test and start a
new test with new embryos. If the above mortality conditions do not
occur, continue the test for the full eight days.
11.8.2 At 25°C, hatching begins on about the fourth day. After
hatching begins, count the number of dead and live embryos and the number
of hatched, dead, live, and deformed larvae, daily. Deformed larvae are
those with gross morphological abnormalities such as lack of appendages,
lack of fusiform shape (non-distinct mass), lack of mobility, a colored,
beating heart in an opaque mass, or other characteristics that preclude
survival. Remove dead embryos and larvae as previously discussed and
record the numbers for all of the test observations (see sample data
record in Table 1 below).
11.8.3 Protect the embryos and larvae from unnecessary disturbance during
the test by carrying out the daily test observations, solution renewals,
and removal of dead organisms, carefully. Make sure the test organisms
remain immersed during the performance of the above operations.
48
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n.9 TERMINATION OF THE TEST
11.9.1 The tist is terminated after eight days of exposure. Count the
number of surviving, dead, and deformed larvae, and record the numbers of
each. The deformed larvae are treated as dead. Keep a separate record of
the total number of deformed larvae for use in reporting the
teratogenicity of the test solution.
11.10 ACCEPTABILITY OF TEST RESULTS
11.10.1 For the test results to be acceptable, survival in the controls
must be at least 80%, except where survival in any test concentration is
80% or better.
11.11 SUMMARY OF TEST CONDITIONS
11.11..1 A summary of test conditions is listed in Table 1.
12. CALCULATIONS
12.1 The endpoints of this toxicity test are based on the effects on
survival and occurrence of terta. Table 2 shows the data on survival and
deformaties after eight days of exposure.to trickling filter waste.
12.2 The mortality data are analyzed-using Probit Analysis (Finney,
1971), Dunnett's Procedure (Dunnett, 1955), or Steel's Many-One Rank Test
(Steel, 1959; Miller, 1981).
12.3 The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent. Dunnett's
Procedure is used when the tests for the assumptions described in the
Appendix are satisfied. If the assumptions are not met, Steel's Many-One
Rank Test may be more appropriate, but the advice of a statistician should
be sdught.
12.4 Data Preparation
12.4.1 Total mortality (combined total number of dead embryos, dead
larvae, and deformed-larvae) has been found to be the most sensitive
endpoint. Tabulate and summarize the data and combine the number of dead
embryos, dead larvae, and deformed larvae at each concentration. Total
mortality is expressed as the mortality proportion, and can be determined
by dividing the total mortality by the number of live embryos at the
beginning of the test.
12.5 DATA ANALYSIS
12.5.1 Probit Analysis
12.5.1.1 Probit Analysis is used to determine the concentration causing
1% mortality. In this analysis, the mortality data from the test
replicates at a given concentration are combined (see Table 28).
49
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The program.transforms the concentration values to log-jo and the percent
mortality to probits, and then performs a regression analysis. A listing
of the computer program and an example of data input and program output
for the Probit Analysis are provided in the Appendix.
12.5.1.2 Report the LCI and its 95% confidence limits. The LCI is an
estimate of the threshold (chronically toxic) concentration. For the
sample data in Table 2, the LCI is 4.7% effluent, with upper and lower 95%
confidence intervals of 5.6% and 3.6% effluent, respectively.
12.5.1.3 If an LCI can not be calculated using Probit analysis, analyze
the data using Dunnett's Procedure, as described below.
12.5.2 Dunnett's Procedure
12.5.2.1 Dunnett's Procedure includes an analysis of variance (ANOVA)
followed by a comparison of each toxicant concentration mean with the
control mean. The error value calculated in the ANOVA is used in the
comparison of the control and treatment means. An example of Dunnett's
Procedure is included in the Appendix. The computer program listed in the
Appendix generates output that includes an ANOVA table, a statement about
each treatment mean that can be used to identify the NOEC, and the minimum
difference between treatment and control means that can be detected as
statistically significant.
12.5.2.2 It is necessary to have at least duplicate test chambers at each
treatment concentration to carry out Dunnett's Procedure. With Steel's
Many-One Rank Test,'it is necessary to have at least four replicates per
treatment.
12.5.2.3 The computer program makes the necessary transformation of the
survival data by converting the square root of the proportion of surviving
organisms to arc sine during the analysis, and includes a special
modification of the arc sine transformation which is required where the
proportion of surviving organisms is 0 or 1 (Bartlett, 1937). For a more
detailed information on the arc sine transformation see the Appendix.
12.5.2.4 The results of the computer analysis of the data in Table 2A
using by Dunnett's Procedure is provided below. The results of the
analysis of variance are shown in Table 3, and indicate a statistically
significant difference in survival among effluent concentrations. The
results of the comparisons of the control mean with the treatment means is
as follows:
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 2
(3% EFFLUENT) AND CONTROL.
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 3
(5% EFFLUENT) AND CONTROL.
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 4
(7% EFFLUENT) AND CONTROL.
50
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THERE IS A SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 5
(11X EFFLUENT) AND CONTROL.
THERE IS A SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 6
(16% EFFLUENT) AND CONTROL.
12.5.2.5 Report the NOEC and LOEC. For the set of data analyzed, the
NOEC is 7% effluent and the LOEC is 11* effluent.
12.5.2.6 For this set of data, the minimum difference that can be
detected as statistically significant is 0.2385. This represents a 28.7%
change in the response from the control.
12.5.3 The chronic value (ChV) is the geometric mean of the NOEC and LOEC
and is calculated as follows:
Log-jo NOEC * Logib 7.0 - = 0.8451
Log-|Q LOEC » Logio 11.0 * 1.0414
ChV » Antilog (0.8451 + 1.0414)/2 * Antilog 0.9432
ChV * 8.8% effluent
13. PRECISION AND ACCURACY ''"...-
13.1 PRECISION
13.1.1 Data shown in Tables 4 and 5 indicate that the precision-of the
embryo-larval survival and teratogenicity test, expressed as the relative
standard deviation (or coefficient of variation, CV) of the LCI values,
was 62% for cadmium (Table 4), and 41% for Diquat (Table 5).
13.1.2 Precision data are also available from four embryo-larval survival
and teratogenicity tests on trickling filter pilot plant effluent
(Table 6). Although the data could not be analyzed by Probit Analysis,
the NOECs and LOECs obtained using Dunnett's Test were the same for all
four tests, 7% and 11% effluent, respectively, indicating maximum
precision in terms of the test design.
13.2 ACCURACY
13.2.1 The accuracy of toxicity tests cannot be determined.
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TABLEMv SUMMARY OF RECOMMENDED TEST CONDITIONS FOR THE FATHEAD
MINNOW (PIMEPHALES PRQMELAS) EMBRYO-LARVAL SURVIVAL AND
— - TERATOSENICITY TEST
1. Test type:
2. Temperature:
3. Light quality:
4. Light Intensity:
5. Photoperiod:
6. Test chamber size:
7. Test solution volume:
8. Renewal of test concentration:
9. Age of test organisms:
10. No. of embryos/chamber:
11. Replicate test
chambers/concentrat1on:
12. Embryos per concentration:
13. Feeding regime:
14. Aeration:
15. Dilution water:
16. Effluent test concentrations:
17. Dilution factor:
18. Test duration:
19. Effects measured:
Static renewal
25 + 2°C
Ambient laboratory light
10-20 uE/ra2/s, or 50-100 ft-c (ambient
laboratory levels)
16 h light, 8 h dark
500- mL
400 mL
Daily
2- to 24-h old embryos; preferably less
than 12-h old
20 to 50
2
40 to 100
Feeding not required
None unless DO falls below 40% saturation
Hardness greater than 25 mg/L (CaCOs);
receiving water or other surface water,
ground water, or synthetic water if
similar to receiving water
5 and a control
Approximately 0.3 or 0.5
8 days
Percent hatch, percent larvae with terata,
percent of normal larvae surviving 4 days
post-hatch; surviving normal larvae from
original embryos
52
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TABLE 2. DATA FROM FATHEAD MINNOW EMBRYO-LARVAL TOXICITY
TEST WITH TRICKLING FILTER WASTE
A. REPLICATES A AND B (USED IN DUNNETT'S PROCEDURE)
Repl. Effl. No. Dead at
Cone. Eggs at Hatching
(51) Start No. (%)
A Cont.
3
5
7
. n
16
B Cont.
3
5
7
n
16
51
50
52
50
50
49
49
50
50
50
49
50
5
5
5
2
10
39
9
6
10
6
30
29
10
10
10
4
20
80-
18
12
20
12
61
58
Dead + Deform.
at hatching
No. (%)
6
5
6
8
25 .
39
9
6
10
10
37-
34
12
10
12
16
50
80
18
12
20
20
76
68
Dead at Test
Termination
No. (%)
6
5
5
9
17
49
10
9
10
16
33
45
12
10
10
18
34
100
20
18
20
32
66
90
Dead + Deform.
at termination
No. (%)
7
5
6
15
32
49
10
. 9
10
20
40
50
14
10
12
. 30
64
100
20
18
20
40
82
100
B. COMBINED DATA FROM REPLICATES A AND B (USED IN PROSIT ANALYSIS)
Repl.
A&B
Effl. No.
Cone. Eggs at
(%) Start
Cont.
3
5
7
n
16
100
100
102
100
99
99
Dead at Dead + Deform.
Hatching at hatching
' No. (X) No. (X)
14
11
15
a
40
68
14
11
15
8
40
69
15
11
16
18
62
73
15
11
16
18
62
74
Dead at Test Dead + Deform.
Termination at termination
No. (%) No. (%)
16
14
15
25
50
94
16
14
15
25 •
50
95
17
14
16
35
72
99
17
14
16
35
73
100
53
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TABLE 3. ANALYSIS OF VARIANCE TOTAL MORTALILTY DATA IN TABLE 2A, BASED
ON THE PROPORTION OF DEAD AND DEFORMED ORGANISMS IN REPLICATES
A AND B AT TEST TERMINATION
Source
Among
Within
Total '
OF
5
6
11
Sum of Squares
2.089
0.043
2.132 . -
Mean Square
0.418
0.007
-
Calculated Tabular
58.80* 4.39
Significant at P = 0.05.
54
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TABLE-4. PRECISION OF THE FATHEAD MINNOW EMBRYO-LARVAL
SURVIVAL AND TERATOGENICITY TEST, USING CADMIUM
AS A REFERENCE TOXICANT3*0
Test
1
2
• 3
4
5
N
Mean
SD
- CV(X)
LCic 95% Confidence
(mg/L) Limits
0.014 0.009 - 0.018
0.006 0.003 - 0.010
0.005 . 0.003 - 0.009
0.003 0.002 - 0.004
0.006 0.003 - 0.009
5
0.0068
0.0042
' 62
NOEC<*
(mg/L)
0.012
0.012
0.013
0.011
0.012
aTests conducted by Drs. Wesley Birge and Jeffrey Black,
University of Kentucky, Lexington, under a cooperative
agreement with the Aquatic Biology Section, Environmental
Monitoring and Support Laboratory, U.S. Environmental
Protection Agency, Cincinnati, Ohio (Cornelius I. Weber,
Project Officer).
^Cadmium chloride was used as the reference toxicant.
The nominal concentrations, expressed as.cadmium (mg/L), were:
0.01, 0.032, 0.100, 0.320, and 1.000. The water hardness was
100 mg/L as calcium carbonate.'
C0etenained by Probit Analysis.
dHighest no-observed-effect concentration determined
by independent statistical analysis (2x2 Chi-square Fisher's
Exact Test).
55
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TABLE 5. PRECISION OF THE FATHEAD MINNOW, EMBRYO-LARVAL,
SURVIVAL AND TERATOGENICITY TOXICITY TEST, USING
DIQUAT AS A REFERENCE TOXICANT*
Test
1
2
3 : , - .
. 4
5
N
Mean
SO
CV(*) ;
LClb
(mg/L)
0.58
2.31
1.50
1.71
1.43
"5
1.51
0.62
41.3
95% Confidence
Limits
0.32 - 0.36
c
1.05 - 1.87
1.24 - 2.09
0.93 - 1.83
aTests conducted by Drs. Wesley Birge and Jeffrey Black,
University of Kentucky, Lexington, under a cooperative
agreement with the Aquatic Biology Section, Environmental
Monitoring and Support Laboratory, U.S. Environmental
Protection Agency, Cincinnati, Ohio (Cornelius I. Weber,
Project Officer).
^Determined by Probit analysis.
cNot calculatable.
56
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TABLE 6. PRECISION OF FATHEAD MINNOW EMBRYO-LARVAL
SURVIVAL AND TERATOGENICITY STATIC-RENEWAL
TEST CONDUCTED WITH TRICKLING FILTER EFFLUENTa»b
Test
No.
1
2
3
4
NOEC
(% Effl)
7
. 7
7
7
LOEC
(X Effl)
11
11
11
11
aEffluent concentrations used: 3, 5, 7, 11
and 16%.
^Maximum precision achieved in terms of
NOEC-LOEC interval. For a discussion of the
precision of data from chronic toxicity tests
see Section 4, Quality Assurance.
57
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SECTION 13
TEST METHOD1.2
CERIOOAPHNIA SURVIVAL AND REPRODUCTION TEST
METHOD 1002.0
1. SCOPE AND APPLICATION
1.1 This method measures the chronic toxlcity of whole effluents and
receiving water to the cladoceran, Ceriodaphnia dubla. during a seven-day,
static renewal exposure. The effects include the synergistic,
antagonistic, and additive effects of all the chemical, physical, and
biological components which adversely affect the physiological and
biochemical functions of the test organisms.
1.2 Detection limits of the toxicity of an effluent or pure substance are
organism dependent.
1.3 Single or multiple excursions in acute toxicity may not be detected
using 24-h composite samples. Also, because of the long sample collection
period involved in composite sampling and because the test chambers are
not sealed, highly volatile and highly degradeable toxicants in the source
may not be detected in the test.
-f
1.4 This method should be restricted to use by or under the supervision
of professionals experienced in aquatic toxicity testing.
2. SUMMARY OF METHOD
2.1 Ceriodaphnia are exposed in a static renewal system for seven days to
different concentrations of effluent, or to receiving water. Test results
are based on survival and reproduction. If the test is conducted as
described, the control organisms should produce three broods of young
during the seven-day period-
3. DEFINITIONS
(Reserved for addition of terms at a later date.)
4. INTERFERENCES
4.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5,
Facilities and Equipment).
format used for this method was taken from Kopp, 1983.
2This method was adapted from Norberg and Mount, 1984.
58
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4.2 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling and Sample
Handling).
4.3 Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.
4.4 The amount and type of natural food in the effluent or dilution
water may confound test results.
4.5 Food added during the test may sequester metals and other toxic
substances and confound test results.
5. SAFETY
5.1 See Section 3, Health and Safety.
6. APPARATUS AND EQUIPMENT '
6.1 Laboratory Ceriodaphnia culture unit — See culturing methods
below. To test effluent or receiving water toxicity, sufficient numbers
of newborn (neonate) organisms must be available.
6.2 Samplers — Automatic sampler, preferrably with sample cooling
capability, capable of collecting a 24-h composite sample of 1 L.
6.3 Sample containers. — for sample shipment and storage (See Section 8,
Effluent and Receiving Water Sampling and Sample Handling).
6.4 Environmental chamber, incubator, or equivalent facility with
temperature control (25+ 1°C).
6.5 Water purification system — Millipore Super-Q or equivalent.
6.6 Balance — Analytical, capable of accurately weighing 0.0001 g.
6.7 Reference weights, Class S — for checking performance of balance.
6.8 Racks for test vessels — Racks approximately 8 cm x 40 cm, drilled
to hold 10 test vessels each.
6.9 Dissecting microscope — for examining organisms in the test
chambers.
6.10 Light box — for illuminating organisms during examination.
6.-11 Volumetric flasks and graduated cylinders — Class A, borosilicate
glass or non-toxic plastic labware, 10-1000 nl, for culture work and
preparation of test solutions.
6.12 Volumetric pipets— Class A, 1-100 mL.
59
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6.13 Serological plpets— 1-10 mL, graduated.
6.14 Pipet bulbs and fillers ~ PropipetR, 8r equivalent.
6.15 Disposable polyethylene pipets, droppers, and glass tubing with
fire-polished edges, 2-mm ID — for transferring organisms.
6.16 Wash bottles — for rinsing small glassware and instrument
electrodes and probes.
6.17 Glass or electronic thermometers — for measuring water temperatures.
6.18 Bulb-thermograph or electronic-chart type thermometers ~ for
continuously recording temperature.
6.19 National Bureau of Standards Certified thermometer — see EPA Method
170.1, USEPA 1979b. -
6.20 pH, .DO, and specific conductivity meters — for routine physical and
chemical measurements. Unless the test is being conducted to specifically
measure the effect of one of the above parameters, a portable, field-grade
instrument is acceptable.
6.21 Miscellaneous apparatus and equipment — transfer containers, pumps,
and automatic dilution devices should-be constructed of materials as
indicated in Section 5, Facilities and Equipment.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Reagent water — defined as activated-carbon-filtered distilled or
deionized water which does not contain substances which are toxic to the
test organisms. A water purification system may be used to generate
reagent water (see paragraph 6.5 above).
7.2 Effluent, surface water, and dilution water — see Section 7,
Dilution Water, and Section 8, Effluent and Surface Water Sampling and
Sample Handling. Dilution water that contains undesirable organisms, that
may attack the test organisms should be filtered through a fine mesh net
(30-um or smaller openings).
7.3 Reagents for hardness and alkalinity tests (see EPA Methods 130.2 and
310.1, USEPA 1979b).
7.4 pH buffers 4, 7, and 10 (or as per instructions of instrument
manufacturer) for standards and calibration check (see USEPA Method 150.1,
USEPA 1979b).
.7.5 Membranes and filling solutions for dissolved oxygen probe (see USEPA
Method 360.1, USEPA 1979b), or reagents for modified Winkler analysis.
7.6 Laboratory quality assurance samples and standards for the above
methods.
60
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7.7 Specific conductivity standards (see EPA Method 120.1, USEPA 1979b).
7.8 Reference toxicant solutions (see Section 4, Quality Assurance).
7.9 Test Vessels — 30-mL borosilicate glass beakers or disposable
plastic salad dressing cups (manufactured by Anchor-Hocking Plastic
Division, and supplied by Plastics Inc., 224 Ryan Avenue, St. Paul,
Minnesota, 55164) are recommended because they will fit in the viewing
field of most stereoscopic microscopes. Rinse thoroughly in distilled
water and then in dilution water before use. A volume of 15 ml of test
solution is adequate for the organisms, and will provide a depth in which
it is possible to count the animals with a minimum of re-focusing. Ten
test vessels are used for each effluent dilution and for the control.
7.10 Test Organisms. — Neonate Ceriodaphnia dubia 2- to 24-h old and
released during the same 4-h period.See information on culturing methods
below,
7.10.1 The test organism (species being used) cultures should be started
at least two weeks before the brood animals are needed, to provide an
adequate supply of neonates for the test. Only a few individuals are
needed to start a culture because of their prolific reproduction.
7.10.2 Ceriodaphnia may be shipped or otherwise transported in
polyethylene bottles. Several hundred animals will live as long as one
week in a 1-L.bottle filled three-fourths full with culture medium
containing the trout chow diet (Paragraph 7.10.6.2.4 below). Animals
received from an outside .source should be transferred to new culture media
gradually, over a period of 1-2 days, to avoid mass mortality.
7.10.3 It is best to start the culture from one animal, which is
sacrificed after producing young, embedded, and retained as a permanent
microscope slide mount.to facilitate identification and permit future
reference. The species identification of the stock culture should be
verified by preparing slide mounts, regardless of the number of animals
used to start the culture. The following procedure is recommended for
making slide mounts of Ceriodaphnia (Beckett and Lewis, 1982):
1. Pipet the animal onto a watch glass.
2. Reduce the water volume by withdrawing excess water with the
pipet. -
3. Add a few drops of carbonated water (club soda or seltzer
water)or 701 ethanol to relax the specimen so that the
post-abdomen is extended. (Optional: with practice,
extension of the postabdomen may be accomplished by putting
pressure on the cover slip.)
4. Place a small amount (one to three drops) of mounting medium
on a glass microscope slide. The recommended
61
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mounting medium is CMCP-9/9AF Medium^, prepared by
mixing two parts of CMCP-9 with one part of CMCP-9AF. For
more viscosity and faster drying, CMC-10 stained with acid
fuchsin may be used.
5. Using a forceps or a pipet, transfer the animal to the
drop of mounting medium on the microscope slide.
6. Cover with a cover slip and exert minimum pressure to
remove any air bubbles trapped under the cover slip.
Slightly more pressure will extend the postabdomen.
7. Allow mounting medium to dry.
8. Make slide permanent by placing CMC-10 around the edges of
the coverslip.
9. Identify to species (see Pennak, 1978, and Berner, 1985).
10. Label with waterproof ink or diamond pencil.
11. Store for permanent record.
7.10.4 One-litre glass beakers are recommended for use as culture
vessels. Use of aquaria (40- to 80-L, or 10- to 20-gal) and other types
of culture vessels may also be convenient. Maintain cultures in several
(four or more) separate vessels to provide back-up cultures in case one is
lost due to accident or other problems, such as low DO concentrations or
lack of food. Fill the 1-L culture vessels with 900 mL of medium.
7.10.5 A new culture is started each week, and the oldest culture is
discarded. Using this schedule, 1-L .cultures will provide 500 to 1000
neonate Ceriodaphnia per week for use in the tests.
7.10.6 Feeding • .
7.10.6.1 Feeding the proper amount of the right food is extremely
important in Ceriodaphnia culturing. The key is to provide sufficient
nutrition to support normal reproduction without adding excess food which
may clog the animal's filtering apparatus or greatly decrease the DO
concentration, and lead to the death of the animals. The suspension of
trout chow, yeast, and CEROPHYLR described below will provide adequate
nutrition if fed daily at the rate of 3 mL/L of medium.
7.10.6.2 The combined food is prepared from three ingredients as follows:
7". 10.6.2.1 Digested trout chow:
1. Add 5.0 g of No. 1 trout chow, U.S. Fish and Wildlife Service
Specification Diet SD9-30, to 1 L of distilled or deionized
water. This trout chow may be obtained from Ziegler Bros.,
Inc., P. 0. Box 95, Gardners, PA, 17324. Mix well in a
blender and aerate continuously (digest) for one week at
ambient laboratory temperature prior to use.
TCMCP-9 and 9AF are available from Polysciences, Inc., Paul Valley
Industrial Park, Warrington, PA, 18976. (215-343-6484).
62
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2. At the end of digestion period, place the mixture In a
refrigerator and allow to settle overnight. Decant 300 ml of
the supernatant and combine with equal volumes of supernatant
from CEROPHYLR and yeast preparations (below). Discard the
remainder.
7.10.6.2.2- Yeast:
1. Add 5.0 g of dry yeast, such as FLEISCHMANN'SR or ST.
REGISR, to 1 L of distilled water.
2. Stir with a magnetic stirrer until well dispersed or use a
blender at low speed for 5 min.
3. Place in a refrigerator overnite, mix well, and combine
300 mL with equal volumes of supernatant from the trout chow
(above) and CEROPHYLR preparations (below). Discard the
remainder.
7.10.6.2.3 CEROPHYLR (Powdered, Dried," Cereal Leaves) 1:
1. Place 5.0 g of CEROPHYLR powder in a blender.
2. Add 1 L of distilled water.
3. Mix at high speed for 5 min.
4. Place in a refrigerator overnite to settle, decant 300 mL of
the supernatant and combine with equal volumes of supernatant
from trout chow and yeast preparations (above). Discard the
remainder.
7.10.6.2.4 Combined trout chow-yeast-CEROPHYLR food:
1. Mix equal (300 mL) volumes of the three foods.
2. Place aliquots of the final mixture in small (50 mL to
100 mL) screw-cap plastic bottles and freeze until needed.
3. Fresh or thawed food is stored in the refrigerator between
feedings, and is used for a maximum of one week.
7.10.6.2.5 The quality of each batch of .food prepared with a new
supply of components should be determined by using the food in a
7-day reproduction test with control water (use culture medium, see
7.10.7, below).
7.10.7 Synthetic, moderately hard water (hardness of 80 to 100 mg/L as
CaCOs) is recommended as a standard culture medium (see Table 1, Section
7, Dilution Water). Other culture water, such as well water, pond water,
or dechlorinated tap water, also may be satisfactory.
1 Available from Sigma Chemical Company, P.O. Box 14508, St. Louis,
Missouri, 63178. (800-325-3010).
63
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7.10.8 Ceriodaphnia should be cultured at a temperature of 25 + 2°C No
water temperature control equipment is needed if the ambient laboratory
temperature remains in the recommended range, and if there are no
frequent, rapid, large, temperature excursions in the culture room.
7.10.9 Day/night cycles prevailing in most laboratories will provide
adequate illumination for normal growth and reproduction. A 16-h/8-h
day/night cycle is recommended.
7.10.10 Ceriodaphnia cannot survive DO concentrations below 5 mg/L for
extended periods.However, aeration is generally not needed unless the
cultures are overfed. DO should be measured when the cultures are first
started and weekly thereafter. Aerate if the DO concentration drops below
5 mg/L.
7.10.11 Suspend a clear glass or plastic panel over the cultures to
exclude dust and dirt. There should be space for circulation of air over
the vessels to provide oxygen_for the cultures.
7.10.12 Ceriodaphnia are eaten by many species of copepods and shrimp,
and cultures must be maintained free of preditors. Natural waters used as
culture media should be filtered through a plankton net with 30 urn mesh
openings. .
7.10.13 Ceriodaphnia have been reported to be very sensitive to sudden pH
and temperature changes, and care should be taken to limit rapid changes
in pH to less than 0.5 units and rapid changes in water temperature to
less than 5°C.
7.10.14 The test organisms should be handled carefully and as little as
possible so that they are not unnecessarily stressed. They should be
transferred from cultures to test vessels, and between test vessels, with
a pipet of approximately 2-mm bore, taking care to release the animals
under the surface of the water. Any organism that is injured during
handling should be discarded.
7.10.15 Culture Maintenance
7.10.15.1 Cultures should be fed daily to maintain the organisms in ,
optimum condition so as to 'provide maximum reproduction in the toxicity
tests. Stock cultures which are stressed because they are not adequately
fed may produce large number of males and ephippial females. Also, brood
females and their offspring may produce few young when used in the test.
1. If food is frozen, remove a bottle of food from the freezer. 1 h
before feeding time, and allow to thaw.
2. Shake thoroughly.
3-. Feed daily at the rate of 4 mL/L of medium.
4. Return unused food mixture to the refrigerator. Do not re-freeze.
Discard unused portion after one week.
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7.10.15.2 Careful culture maintenance is essential. The population
should not be allowed to exceed 1000 animals/L of medium. If
necessary, thin the cultures every four or five days after changing
the medium to prevent crashes and discourage gametogenesis.
7.10.15.3 The culture medium in each culture vessel should be
replaced with fresh medium weekly as follows:
1. Pour about one half (450 mL of the 900 mL) of the contents of
a culture vessel into a shallow vessel. A large finger bowl
works well.
2. Discard the remainder of the medium and animals unless needed
for a test or to start a new culture.
3. Clean the culture vessel by brushing the sides and bottom, or
wiping with a clean sponge or paper towel, and rinsing with
distilled or deionized water. Each month, the culture
. vessels should be washed as described in Section 5,
(Facilities and Equipment), and air dried.
4. Place about 100 ml of fresh medium in the clean culture
vessel.
5. Remove about 100 Ceriodaphnia from the holding vessel (finger
bowl) with a pipet, plastic tubing, or by dipping with a
small beaker, and transfer them to the fresh medium, along
with a small amount of the old.medium to provide seed
bacteria for the new culture.
6. Carefully add sufficient additional fresh medium to fill the
culture vessel.
8. SAMPLE COLLECTION, PRESERVATION AND HANDLING
8.1 See Section 8, Effluent and Receiving Water Sampling and Sample
Handling.
9. CALIBRATION AND STANDARDIZATION
9.1 See Section 4, Quality Assurance.
10. QUALITY CONTROL
10.1 See Section 4, Quality Assurance.
11. PROCEDURE
11.1 TEST SOLUTIONS
11.1.1 Surface Waters
11.1.1.1 Surface water toxicity is determined with samples used directly
as collected.
65
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11.1.2 Effluents
11.1.2.1 Thft selection of the effluent test concentrations should be
based on the objectives of the study. One of two dilution factors,
approximately 0.3 or 0.5, is commonly used. A dilution factor of
approximately 0.3 allows testing between 100% and 1% effluent using only
five effluent concentrations (100%, 30%, 10%, 3%, and 1%). This series of
dilutions minimizes the level of effort, but because of the wide interval
between test concentrations provides poor test precision (+ 300%).
A dilution factor of 0.5 provides greater precision (+ 100%*), but requires
several additional dilutions to span the same range oF effluent
concentrations. Improvements in precision decline rapidly as the dilution
factor is increased beyond 0.5
11.1.2.2 If the effluent is known or suspected to be highly toxic, a
lower range of effluent concentrations should be used (such as 10%, 3%,
1%, 0.3%, and 0.1%). If a high rate of mortality is observed during the
first"1 to 2 h of the test, additional"dilutions at the lower range of
effluent concentrations can be added.
11.1.2.3 The volume of effluent required for daily renewal of 10
replicates per concentration, each containing'15 ml of test solution, is
approximately 1 I. Prepare enough test solution (approximately 600 mL) at
each effluent concentration to provide 400 mL additional volume for
chemical analyses.
11.2 OBTAINING NEONATES FOR THE TEST
11.2.1 This test method requires neonates 2- to 24-h old (all within 4 h
of the same age) to begin the test. To obtain a sufficient number of young
which are all less than 4-h old, brood animals containing eggs are placed
singly in 30-mL beakers containing 15 mL of media (using the same source
of dilution water that will be used in the test), four or five days prior
to the initiation of the test. One brood animal is needed for each test
vessel that will be used in the test. For example, if five concentrations
and a control will be used in a test to begin on a Friday, 60 brood
animals are placed in individual beakers on Monday and fed daily, as in
the test (see Paragraph 11.6). The brood stock are transferred to fresh
media daily, and the young are discarded with the old media. Four hours
before the test is to begin, the young are removed from the brood beakers
and discarded. In this way, all the young in the brood beakers when the
test is ready to start will be less than 4-h old, and are used in the
test. Some workers prefer to begin removing and saving young from the
brood chambers at 2- or 4-h intervals beginning 8 h before the test is-
scheduled to start. This makes it possible to use older, but similar
aged, young for beginning the test, thus improving chances of good
survival of test animals.
66
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11.3 START OF THE TEST
11.3.1 The test should begin as soon as possible, preferably within 24 h
of sample collection. In no case should tn"e test be started more than 72
h after sample collection. Just prior to testing, the temperature of the
sample should be adjusted to 25 + 1°C and maintained at that temperature
until portions are added to the dilution water.
11.3.2 Begin the test by randomly placing one neonate in each test
beaker. Because of their small size and difficulty in handling, the test
chambers are usually placed in racks, 10 to a rack. The position of the
test chambers is randomized in the racks at the beginning of the test. On
following days, the positions of the racks are randomized.
11.4 LIGHT, PHOTOPERIOD AND TEMPERATURE
11.4.1 The light quality and intensity should be at ambient laboratory
levels, approximately 10-20 u£/m2/s, or 50 to 100 foot candles (ft-c),
with a photoperiod of 16 h of light and 8 h of darkness. It is critical
that the test water temperature be maintained at 25 + 1°C to obtain
three broods in seven days. ~"
11.5 DISSOLVED OXYGEN .
11.5.1 Low DO concentrations may be important when running effluent
toxicity tests. However, aeration is not practical for the Ceriodaphnia
test. If the DO in the effluent and/or dilution water is low, aerate
before preparing the test solutions.
f
11.6 FEEDING
11.6..1 During the test, the Ceriodaphnia are fed the same diet as used
for the cultures. The organisms in the test vessels are fed digested
trout chow-yeast-CEROPHYLR diet daily at a rate of 0.1 mL food
suspension/15 mL of test solution.
11.7 TEST SOLUTION RENEWAL
11.7.1 Using a glass or polyethylene dropper, or pipet, transfer each
test organism daily to a new test vessel containing 15 mL of
freshly-prepared test solution and 0.1 mL of the food suspension. The
animals should be released under the surface of the water so that air is
not trapped under the carapace.
11.8 ROUTINE, CHEMICAL AND PHYSICAL DETERMINATIONS
11.8.1 At a minimum, the following measurements are made:
11.8.1.1 DO is measured in each test solution at the beginning of each
24-h exposure period, and at the end of the exposure period in one test
vessel at each test concentrations and in the control, after the adult
67 '
-------
has been removed and the young have been counted. (Acid must not be added
to count the young in that chamber.)
11.8.1.2 Temperature, pH, and conductivity are measured at the beginning
of each 24-h exposure period at all test concentrations and in the control.
11.8.1.3 Alkalinity and hardness are measured at the beginning of each
24-h exposure period in 100X effluent and in the control.
11.9 OBSERVATIONS DURING THE TEST
11.9.1 Three broods are usually obtained in the controls in a seven-day
test conducted at 25°C. The first brood of two to five young is usually
released on the third day of the test, soon after the adults are
transferred to fresh test solutions. Successive broods are released every
36 to 48 h thereafter, and may contain 10 to 15 young.
11.9.2 Each day, at the time the organisms are to be transferred to fresh
test solutions, determine adult survival and count and record the number
of young. First remove the adult to the new test solution. Count any
dead young, and then add two drops of IN hydrochloric acid to the vessel
(except the vessel used for DO measurements). The living young die
quickly and settle to the bottom of the test vessel where they may be
counted with a minimum of effort and error. The young are discarded after
counting.
11.9.3 The young are best counted with the aid of a stereomicroscope.
The organisms are more easily seen if viewed against a black background.
If counts are made without the aid of a stereomicroscope, place the test
vessels on a black strip of tape on a light box.
11.9.4 In the absence of toxic substances, young production may exceed 30
per adult. If toxic substances are present, young may develop in the
brood pouch of the adults, but may not be released during the exposure
period.
11.10 TERMINATION OF THE TEST
11.10.1 Because of the rapid rate of development of Ceriodaphm'a, the
test must be terminated and all observations completed within + 2 h of
exactly seven days" after the initiation of the test. The seveF-day test
period was selected because each control animal will normally produce
three broods, totaling 10 to 30, at a test temperature of 25°C. An
extension of more than a few hours in the test period would be a
significant part of the brood production cycle of the animals, and could
result in additional broods.
11.11 SUMMARY OF TEST CONDITONS
11.11.1 A summary of test conditions is listed in Table 1.
68
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12. CALCULATIONS .
12.1 DATA PREPARATION , ?
12.1.1 The number of young produced per adult female Is determined by
taking the total number of young produced until either the time of death
of the adult or the end of the experiment, whichever comes first. An
animal that dies before producing young would be included in the analysis,
with zero entered as the number of young produced. The subsequent
calculation of the mean number of young produced per adult for each
toxicant concentration provides a combined measure of the toxicant's
effect on both mortality and reproduction.
12.1.2 Tabulate and summarize the data. A sample set of test data are
listed in Table 2. Using these data, the total number of young produced
per adult during the test is shown in Table 3.
12.2. DATA ANALYSIS
12.2.1 Fisher's Exact Test (Finney, 1948; Pearson and Hartley, 1962) and
Dunnett's Procedure (Dunnett, 1955) or Steel's Many-One Rank Test (Steel,
1959; Miller, 1981) are used to analyze the data. The first step.is to
test for a significant difference in the survival of the original test
organisms in the various effluent concentrations and control, using
Fisher's Exact Test. Dunnett's Procedure is then used to analyze the
reproduction data from only those effluent concentrations where mortality
was not significantly different from the controls.
i»
12.2.2 For the data in Table 3, reference to the tabulated values in
Appendix E show that the survival in 25% effluent is significantly
different from the control survival. Therefore, the 25% effluent
concentration will not be included in the analysis of the reproduction
data.
12.2.3 Since it is border-line as to whether the remaining data meet the
assumptions for normality, both Dunnett's Procedure and Steel's Many-One
Rank Test can be used to analyze the reproduction data.
12.2.4 The safe concentration derived from the test is reported as the
NOEC. •
12.2.5 Dunnett's Procedure
•'ty>& <•- • '
12.2.5.1 Dunnett's procedure includes an analysis of variance (ANOVA)
followed by a comparison of each toxicant concentration mean with the
control mean. The error value calculated in the ANOVA is used in the
comparison of the control and treatment means. An example of a Dunnett's
calculation is included in the Appendix. The computer program listed in
the Appendix generates output which includes an ANOVA table, a statement
about each treatment mean that can be used to identify the NOEC, and the
minimum difference between treatment and control means that can be
detected as statistically significant.
69
-------
12.2:5.2 The analysis is carried out using the total number of live young
produced by each adult female during the the test (Table 3).
12.2.5.3 The output from the analysis of variance is shown in Table 4,
and indicates that the effects of the various treatments on reproduction
were significant at P * 0.05.
12.2.5.4 The computer output for the comparison of the control mean with
the treatment means is shown below:
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 2
(IX EFFLUENT) AND CONTROL.
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 3
(3X EFFLUENT) AND CONTROL.
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 4
(6X EFFLUENT) AND CONTROL. , ' • .
THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 5
(12X EFFLUENT) AND CONTROL.
12.2.5.5 The NOEC and LOEC, as determined from the analysis, were
12% and 25% effluent, respectively. Note that the Dunnett program
considers the control as Concentration 1. Thus, Concentration 5 in
the output from Dunnett's test is effluent Concentration 4
(12% effluent) in the test.
12.2.5.6 For this set of data, the minimum difference that can be
detected as statistically significant is 6.28 young per adult. This
represents a 34.5% reduction in the response from the control.
12.2.6 Steel's Many-One Rank Test
12.2.6.1 Steel's Many-One Rank Test compares several treatments
with a control by analyzing the ranks of the data, thereby
eliminating the necessity that the data meet the normality
assumptions. The calculations for Steel's Many-One Rank Test of the
data in Table 3 are detailed in the Appendix. The NOEC determined
from Steel's Test was 12X, which agrees-with the NOEC from Dunnett's
Procedure above.
12.2.7 The chronic value (ChV) is the geometric mean of the NOEC and
LOEC and is calculated as follows:
Log.io NOEC » Logic 12 * 1.0792
Logic LOEC = Logic 25 * 1.3979
ChV * Antilog (1.0792 + 1.3979)/2 -• Antilog 1.2386
ChV » 17.3% effluent
70
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13. PRECISION AND ACCURACY
13.1 PRECISION v
13.1.1 Infonnatldh on the single laboratory precision of the
Ceriodaphnia reproduction test based on the NOEC and LOEC values
from nine tests with the reference toxicant NaPCP is provided in
Table 5.
13.2 ACCURACY
13.2.1 The accuracy of toxicity tests cannot be determined.
71
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TABLE U SUMMARY OF RECOMMENDED TEST CONDITIONS FOR CERIODAPHNIA
SURVIVAL AND REPRODUCTION TEST
1. Test type:
2. Temperature (°C):
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test vessel size:
m • '
7. Test solution volume:
8. Renewal of test concentrations:
9. Age of test organisms: - -
10. Number of test organisms
per chamber:
11. Number of replicate
chambers per treatment:
12. Feeding regime:
13. Aeration:
14. Dilution water:
15. Dilution factor:
16. Test duration:
17. Effects measured:
Static renewal
25 i 1°C
Ambient laboratory light
10-20 uE/m2/s, or 50-100 ft-c
(ambient laboratory levels)
16 h light, 8 h dark
.30 mL
15 mL
Daily
Less than 24 h; and all released
within a 4-h period
1
10
Feed 0.1 mL food suspension/15 mL,
daily
None
Moderately hard standard water,
receiving water, other surface
water, or ground water with hardness
similar to receiving water
Approximately 0.3 or 0.5
7 days
Survival and reproduction
72
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TABLE 2. DATA FROM CERIODAPHNIA EFFLUENT TOXICITY TEST
Effl Day
Replicate
Total No. Most Young
Live Live By Any
Cone..
Cont
1.0%
3.0%
6.0%
12.0%
25.0%
No.
3
4
5
6
7
3
4
5
6
7
3
4
5
6
7
3
4
5
6 .
7
3
4
5
6
7
3
4
5
6
• 7
. A
0
2
9
5
6
TZ
0
2
9
6
27
0
2
3
10 .
10
TS
0
2
2
9
10
2T
0
0
1
8
11
m
0
X
-ff
8
0
2
2
6
8
Tff
0
4
5
3
TT
0
i
4
8
12
2T
0
4
3
2
2
Tff
0
4
8
4
2T
0
0
X
IT
c
0
4
9
9
5
IT
0
i
2
6
12
FT
0
2
2
6
»
0
2
0
2
3
0
2
0
8
if
0
0
1*
T
0
0
0
0
6
10
Tff
0
0
.0
2
4
T
0
4
9
9
2
IT
0
2
2
3
6
TT
0
2
2
3
6
TT
0
0
X
"o1
E
0
6
9
0
1
TC
0
4
8
3
3
27
0
4
n
•o
0
17
0
0
4
10
12
is
0
2
3
10
10
I?
0
X
IT
F
X
~s
0
2
.13
8
8
3T
0
8
6
3
6
IT
0
1
2
6
13
IF
0
0
3
10
12
•"25*
0
X
~Q
G
0
2
0
3
"1
0
2
2
10
9
IT
•o
Z
2
6
n
FT
0
2
2
8
9
IT
0
1
0
5
10
TF
0
0
X
"o1
H
0
6
6
5
8
TS
0
3
" 8
0
0
TT
0
0
4
10
8
IF
0
0
4
6
11
IT
d
2
2
10
n
•US
0
X
-^
I
0
1
2
11
10
2T
0
2
2
6
13
If
0
4
8
8
0
Iff
0
4
6
2
9
IT
0
1
3
6
8
Iff
0
0
X
IT
J
0
4
9
10
if
0
2
2
6
12
2?
0
3
6
3
6
TS
3
2
8
4
4
IT
0
4
0
7
7
Tff
0
0
X
IT
Young
0
27
46
55
54
187
0
22
51
55
69
197
0
30
55
63
65
2TJ
3
19
38
52
79
19T
0
18
22
71
87
13S
0
0
1
0
0
Adults
9
9
9
9
9
10
10
10
10
10
10
10
10
10
10.
10
10
10
10
10
10
10
10
10
10
10
6
1
0
0
One Adult
0
6
9
11
10
0
4-
13
10
13
0
8
11
10
12
3
4
8
10
13
0
4
3
10
12
0
0
1
0
0
x 3 dead adult, no young produced before death.
1X s Dead adult; one young produced before death.
Note: Days 1 and 2 are not included because young were not produced until
the third day. Adult mortality was not recorded for days 1 and 2.
73
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TABLE 3. NUMBER OF YOUNG PRODUCED PER CERIQDAPHNIA
Effluent Concentration (%)
Replicate
(Organism)
A
B
C
0
E
F
G
H
I
J
Control
22
18
27
16
16
0
a
25
24
26
1
22
15
21
6
23
31
23 ,
11.
23
22
3
25
25
20
24
15
23
21
22
20
18 .
6
23
16
7
13
26
22
21
21
21
21
12
20
21
17
13
25
25
16
25
18
18
25
0
0
1
0
0
0
0
0
0
0
TABLE 4. ANALYSIS OF VARIANCE OF CERIODAPHNIA REPRODUCTION DATA
FROM CONTROL AND FIRST FOUR. EFFLUENT CONCENTRATIONS (1-12*)
Calculated Tabular
Source DF Sum of Squares Mean Square F FQ.QS
Among
Within
4
- 45 '
51.480
1664.300
12.870
36.984
0.35 2.58
TOTAL
49
1715.780
74
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TABLE 5. PRECISION OF THE CERIODAPHNIA REPRODUCTION TEST,
USINfrNAPCP AS A REFERENCE TOXICANT*
Test -
lb
2C
3
4d
5
6
7
8
• 9
NOEC
(mg/L)
0.25
0.20
0.20
0.30
0.30
0.30
0.30
0.30
0.30
LOEC
(mq/L)
0.50
0.60
0.60
0.60
0.60
0.60
0.60
0.60
- 0.60
Chronic
Value
(mg/L)
0.35
0.35
0.35
0.42
0.42
0.42
0.42
0.42
0.42
aFor a discussion of the precision of data from chronic toxicity
tests see Section 4, Quality Assurance.
bConcentrations used in Test 1 were; 0.03, 0.06, 0.12, 0.25, 0.50,
1.0 mg NaPCP/L.
concentrations used in Tests 2 and 3 were, 0.007, 0:022, 0.067,
0.20, 0.60 mg NaPCP/L.
dConcentrations used in Tests 4 through 9 were, 0.0375, 0.075,
0.150, 0.30, 0.6Q mg NaPCP/L.
75
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SECTION 14-
TEST METHOD!.2
ALGAL (SELENASTRUM CAPRICORNUTUM) GROWTH TEST
METHOD 1003.0
1. SCOPE AND APPLICATION
1.1 This method measures the chronic toxicity of whole effluents and
receiving water to the fresh water alga, Selenastrum capricornutum, during
a four-day, static exposure. The effects include the synergistic,
antagonistic, and additive effects of all the chemical, physical, and
biological components which adversely affect the physiological and
biochemical functions of the test organisms.
1.2 Detection limits of the toxicity of an effluent or pure substance are
organism dependent.
1.3 Single or multiple excursions in acute toxicity may not be detected
using 24-h composite samples. Also, because of the long sample collection
period involved in composite sampling, and because the test chambers are
not sealed, highly volatile and highly degradeable toxicants in the source
may not be detected in the test.
1.4 This-test is very versatile because it can also be used to identify
wastewaters which* are biostimulatory and may cause nuisance growths of
algae, aquatic weeds, and other organisms at higher trophic levels.
1.5 This method is restricted to use by or under the supervision of
professionals experienced in aquatic toxicity testing.
2. SUMMARY OF METHOD
2.1 A Selenastrum population is exposed in a static system to a series of
concentrations~~of" effluent, or to receiving water, for 96 h. The response
of the population is measured in terms of changes in cell density (cell
counts per mL), biomass, chlorophyll content, or absorbance. By extending
the test to 14 days, it may be used to measure the algal growth potential
of wastewaters and surface waters.
3. DEFINITIONS
(Reserved for addition of terms at a later date.)
format used for this method was taken from Kopp, 1983.
2This method was adapted from Miller et al, 1978.
76
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4. INTERFERENCES.
4.1 Toxic substances may be Introduced by contaminants in dilution
water, glassware, sample hardware, and testing equipment (see Section 5,
Facilities and Equipment).
4.2 Adverse effects of high concentrations of suspended and/or dissolved
solids, and extremes of pH, may mask the presence of toxic substances.
4.3 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling and Sample
Handling).
4.4 Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival, and confound test results.
4.5 The amount of natural nutrients in.the effluent or dilution water -
may confound test results.
5. SAFETY
5.1 See Section 3, Safety and Health.
6. APPARATUS AND EQUIPMENT
6.1 Laboratory Selenastrum culture unit — See culturing methods below.
To test effluent toxicity, sufficient numbers of log-phase-growth
organisms must be available.
6.2 Samplers — Automatic sampler capable of collecting a 24-h composite
sample of 1 L.
6.3 Sample containers — for sample shipment and storage see Section 8,
Effluent and Receiving Water Sampling and Sample Handling.
6.4 Environmental chamber, incubator, or equivalent facility — with
cool-white fluorescence illumination (60 uE/m2/s, or 400 ^ 40 ft-c) and
temperature control (24 + 2°C).
6.5 Mechanical shaker — Capable of providing orbital motion at the rate
of 100 cycles per minute (cpm)
6.6 Light meter — with a range of 0-200 uE/m2/* (0-1000 ft-c).
6.7 Water purification system — Millipore Super-Q or equivalent;
6.8 Balance — Analytical, capable of accurately weighing 0.0001 g.
6.9 Reference weights, Class S — for checking performance of balance.
6.10 Glass or electronic thermometers — for measuring water
temperatures.
77
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6.11, Bulb-thermograph or electronic-chart type thermometers — for
continuously recording temperature.
6.12 National Bureau of Standards Certified thermometer (see EPA Method
170.1, USEPA 1979b).
6.13 Meters: pH and specific conductivity — for routine physical and
chemical measurements. Unless the test is being conducted to
specifically measure the effect of one of the above parameters, a
portable, field-grade instrument is acceptable.
6.14 Fluorometer (Optional) — Equipped with chlorophyll detection light
source, filters, and photomultiplier tube (Turner Model 110 or
equivalent).
6.15 UV-VIS spectrophotometer — capable of accommodating 1-5 cm
cuvettes.
6.16 Cuvettes for spectrophotometer — 1-5 cm light path.
6.17 Electronic particle counter (Optional) — Coulter Counter, ZBI, or
equivalent, with mean cell (particle) volume determination.
6.18 Microscope — with 10X, 45X, and 100X objective lenses, 10X ocular
lenses, mechanical stage, substage condenser, and light source (inverted
or conventional microscope).
6.19 Counting chamber — Sedgwick-Rafter, Palmer-Maioney, or
hemocytometer.
6.20 Centrifuge — with swing-out buckets having a capacity of 15-100 mL.
6.21 Centrifuge tubes,— 15-100mL, screw-cap.
6.22 Filtering apparatus — for membrane and/or glass fiber filters.
6.23 Volumetric flasks and graduated cylinders — Class A, 10-1000 ml,
borosilicate glass, for culture work and preparation of test solutions.
6.24 Volumetric pipets— Class A, 1-100 ml.
6.25 Serological pipets— 1-10 ml, graduated.
6.26 Pipet bulbs and fillers — PropipetR, or equivalent.
6.27 Wash bottles — for rinsing small glassware, instrument electrodes,
and probes.
6.28 Culture flasks — 1-4 L borosilicate, erlenmeyer flasks.
6.29 Test flasks — 125 or 250 ml borosilicate, erlenmeyer flasks.
78
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6.30 Preparation of glassware — prepare all graduated cylinders, test
flasks, bottles-; volumetric flasks, centrifuge tubes and vials used in
algal bioassays as follows: •
6.30.1 Wash with non-phosphate detergent solution, preferably heated to
50°C or hotter. Brush the inside of flasks with a stiff-bristle brush
to loosen any attached material. The use of a commercial laboratory
glassware washer or heavy-duty kitchen dishwasher (under-counter type) is
highly recommended.
S.30.2 Rinse thoroughly with tap water, and drain well.
6.30.3 All new test flasks, and all flasks which through use may become
contaminated with toxic organic substances, must be rinsed with acetone
or heat-treated before use. To thermally degrade organics, place
glassware in a high temperature oven at 40QQC for 30 min. After
cooling, proceed with the next step.
6.30.4 If acetone is used in 6.30.3, rinse thoroughly with tap water.
If the heat treatment is used, go directly to 6.30.5.
6.30.5 Carefully rinse with a 10% solution (by volume) of reagent grade
hydrochloric acid (HC1); fill vials and centrifuge tubes with the 10% HC1
solution and allow to stand a few minutes; fill all larger containers to
about one-tenth capacity with HC1 solution and swirl so that the entire
surface is bathed.
6.30.6 Rijise with'tap water and drain well.
6.30.7 To neutralize any residual acid, rinse with a saturated solution -
of
6.30.8 Rinse five times with tap water and then five times with
deionized or distilled water.
6.30.9 Dry in an oven, cover the mouth of each vessel with aluminum foil"
or other closure, as appropriate, before storing.
6.31 Use of sterile, disposable pipets will eliminate the need for pipet
washing and minimize the possibility of contaminating the cultures with
toxic substances.-
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Reagent water — defined as carbon-filtered distilled or deionized
water which does not contain substances which are toxic to the test
organisms. A water purification system may be used to generate reagent
water (see paragraph 6.7 above).
7.2 Effluent, surface water, and dilution water — see Section 7,
Dilution Water, and Section 8, Effluent and Receiving Water Sampling and
Sample Handling.
79 '.
-------
7.3 Reagents for hardness and alkalinity tests (see EPA Methods 130.2
and 310.1, USEPA 1979D).
7.4 pH buffers 4, 7, 8 and 10 (or as per instructions of instrument
manufacturer) for standards and calibration check (see USEPA Method
150.1, USEPA 1979b).
7.5 Laboratory quality assurance samples and standards for the above
methods.
7.6 Specific conductivity standards (see EPA Method 120.1, USEPA 1979b).
7.7 Reference toxicant solutions (see Section 4, Quality Assurance).
7.8 Acetone — pesticide quality or equivalent.
7.9 Dilute hydrochloric (or nitric) acid — carefully add 10 mL of
concentrated HC1 to 90 'mL of reagent water.
7.10 Test Organisms — log-phase-growth Selenastrum capricornutum. See
information on culturing methods below.
7.10.1 Culture Medium
7.10.1.1 The culture medium is used to maintain the stock cultures of
the test organisms, for the control flasks in each test, and as a.diluent
in tests to measure the toxicity of effluents and receiving waters.
7.10.1.2 Prepare five stock nutrient solutions using reagent grade
chemicals as described in Table 1.
7.10.1.3 Add 1 mL of each stock solution, in the order listed in
Table 1, to approximately 900 mL of distilled or deionized water. Mix
well after the addition of each solution. Dilute to 1 L, mix well, and
adjust the pH to 7.5 £0.1, using 0.1N sodium hydroxide or hydrochloric
acid, as appropriate. The final concentration of macronutrients and
micronutrients in the culture medium is given in Table 2.
7.10.1.4 Immediately filter the pH-adjusted medium through a 0.45um pore
diameter membrane at a vacuum of not more than 380 mm (15 in.) mercury,
or at a pressure of not more than one-half atmosphere (8 psi). Wash the
filter prior to use by passing 500 mL of distilled water through it.
7.10.1.5 If the filtration is carried out with sterile apparatus,
filtered medium can be placed immediately into sterile culture flasks,
and-no further sterilization steps are required before the inoculation of
the medium. The medium can also be sterilized by autoclaving after it is
placed in the culture flasks. However, the pH should be checked after
autoclaving to determine if it was changed.
80
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TABLE T. NUTRIENT STOCK SOLUTIONS FOR MAINTAINING ALGAL STOCK CULTURES
AND TEST CONTROL CULTURES.
Nutrient
Stock
Solution
Compound
Amount dissolved in
500 mL Distilled Water
£
3_
4_
5
FeCl3*6H26
CoCl2'6H20
Na2EDTA'2H20
NaNOs
K2HP04
NaHC03
6.08 g
2.20 g
92.8 mg
208.0 mg
1.64 rag*
79.9 mg
0.714 mgb
3.63 mgc
0.006 mgd
150.0 mg
12.750 g
7.350 g
0.522 g
7.50 g
aZnCl2 - Weigh out 164 mg and dilute to 100 mL. Add 1 mL of this
solution to Stock II.
bCoCl2 '6H20 " Wel9h out 71.4 mg and dilute to 100 mL. Add 1 mL of
this solution to Stock #1.
- Weigh out 36.6 mg and dilute to 10 mL. Add 1 mL
of this solution to Stock #1.
dCud2 -2H20 ,'•- Weigh out 60.0 mg and dilute to 1000 mL. Take 1 mL
of this solution and dilute to 10 mL. Take 1 mL of the second dilution
and add to Stock #1.
81
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Table 2. FINAL CONCENTRATION OF MACRONUTRIENTS AND MICRONUTRIENTS
IN THE CULTURE MEDIUM
Macronutrient
NaN03
MgCl2-6H20
CaC12-2H20
MgS04.7H20
K2HP04
NaHC03'
-
Micronutrient
H3B03
MnCl2-4H20
ZnCl2
CoCl2-6H20
CuCl2*2H20
Na2Mo04«2H20
FeCl3.6H20
Na2EDTA*2H?b
Concentration
(mq/L)
25.5
12.2
4.41
14.7
1.04
: is.o .
.• -•
Concentration
(uq/L)
185
416 •
3.27
1.43
0.012
7.26
160
300
Element
N
Mg
Ca
S
P
Na
K
C
Element
B
Mn
Zn
Co
Cu
Mo
Fe
— —
Concentration
" (mg/L)
4.20
2.90
1.20
1.91
0.136
11.0
0.469
2.14
Concentration
(uq/L)
32.5
115
1.57
0.354
0.004
2.88
33.1
....
82
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7.10.1.6 Unused sterile medium should not be stored In the (250 ml) test
culture flasks more than one week prior to use, because there may be
substantial Toss of water by evaporation.
7.10.2 Algal Cultures
7.10.2.1 Test organisms — Selenastrum capricornutum, a unicellular
coccoid green alga. See Section 6, Test Organisms, for information on
sources of "starter" cultures.
7.10.2.2 Stock algal cultures
7.10.2.2.1 Upon receipt of the "starter" culture (usually about 10 ml), a
stock.culture is initiated by aseptically transferring 1 ml to a culture
flask containing control algal culture medium (prepared as described
above). The volume of stock culture medium initially prepared will
depend upon the number of test flasks to be inoculated later from the -
stock, or other planned uses,, and may range from 25 ml in a 125 ml flask
to 2 L i.n a 4-L flask. "The remainder of the starter culture can be held
in reserve for up to six months in a refrigerator (in the dark) at 4°C.
7.10.2.2.2 Maintain the stock cultures at 24 + 2°C, under continuous
"Cool-White" fluorescent lighting of 86 + 8.6 u£/m2/s, or 400 + 40
ft-c. Shake continuously at 100 cpm or once daily by hand. "~
7.10.2.2.3 Transfer 1 to 2 mL of stock culture weekly to 1 L of new
culture medium to maintain a continuous supply of "healthy" cells for
tests. Aseptic techniques should be used in maintaining the algal
cultures, and extreme care should be exercised to avoid contamination.
7.10.2.2.4 To maintain unialgal culture material over a long period of
time, it is advantageous to use a semi-solid medium containing 1.0%
agar. The medium is placed in sterile Petri dishes, and a 1-mL portion
of a liquid algal culture is streaked onto it and incubated as described
above. Place rubber bands around the petri dishes to reduce evaporation
loss of the medium. Fresh (liquid) stock cultures may be started at four
week intervals by transfer of cells from a single clone in a petri dish
to an appropriate volume of liquid medium.
8. SAMPLE COLLECTION, PRESERVATION AND HANDLING
8.1 See Section 8, Effluent and Receiving Water Sampling and Sample
Handling.
9. CALIBRATION ANO STANDARDIZATION
9.1 See Section 4, Quality Assurance.
10. QUALITY CONTROL
10.1 See Section 4, Quality Assurance.
83
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11. PROCEDURES
11.1 TEST SOLUTIONS
11.1.1 Surface Waters
11.1.1.1 Surface water toxicity is determined with samples used directly
as collected.
11.1.2 Effluents
11.1.2.1 The selection of the effluent test concentrations should be
based on the objectives of the study. One of two dilution factors,
approximately 0.3 or 0.5, is commonly used. A dilution factor of
approximately 0.3 allows testing between 100% and 1% effluent using only
five effluent concentrations (100%, 30*, 10%, 3%, and 1%). This series
of dilutions minimizes the level of effort, but because of the wide
Interval between test concentrations provides poor test precision
(j^300S). A dilution factor" of 0.5 provides greater precision (£ 100X),
but requires several additional dilutions to span the same rangeTof
effluent concentrations. Improvements in precision decline rapidly as
the dilution factor is increased beyond 0.5
11.1.2.2 If the effluent is known or.suspected to be highly toxic, a
lower range of effluent concentrations should be used (such as 1051, 3%,
1%, 0.3S, and 0.1%). If a high rate of mortality is observed during the
first 1 to 2 h of the test, additional dilutions at the lower range of
effluent concentrations can be added.
11.1.2.3 The volume of effluent required for the test is 1 L. Prepare
enough test solution at each effluent concentration (approximately 700
mL) to provide 100 mL of test solution for each of three replicate test
chambers and 400 mL for chemical analyses.
11.1.3 Dilution water may consist of stock culture medium without the
EDTA, or other water such as surface water, depending on the objectives
of the test. However,-if water other than the stock culture medium is
used for dilution water, -1 mL of each stock nutrient solution (except for
EDTA) should be added per litre of dilution water. Surface waters used
as dilution water must be filtered through a 0.45 urn pore diameter
filter, such as a-GF/A, GF/C, or equivalent filter.
11.1.4 Effluents may be toxic and/or nutrient poor. "Poor" growth in an
algal toxicity test, therefore, may be due to toxicity or nutrient
limitation, or both. To eliminate false negative results due to low
nutrient concentrations, 1 mL of each stock nutrient solution (except
EDTA) is added per litre of effluent prior to use in preparing the test
dilutions. Thus, all test treatments and controls will contain at least
the basic amount -of nutrients.
11.1.5 If the growth of the algae in the test solutions is to be
measured with an electronic particle counter", the effluent and dilution
84
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water must brfiltered through a GF/A, GF/C, or equivalent pore diameter
filter, and checked for "background" particle count before it is used in
the test.
11.1.6 If samples contain volatile substances, the test sample should be
added below the surface of the dilution water towards the bottom of the
test container through an appropriate delivery tube.
11.2 PREPARATION OF INOCULUM
11.2.1 The inoculum is prepared no more than 2 to 3 h prior to the
beginning of the test, using Selenastrum capricornutum harvested from a
four- to seven-day stock culture! Each mi Hi liter of inoculum must
contain enough cells to provide an initial cell density of 10,000
cells/ml in the test flasks. Assuming the use of 250 mL flasks, each
containing 100 mL of test solution, the inoculum must contain 1,000,000
cells/mL. Estimate the.volume of stock, culture required to prepare the
inoculum as described in the following example:
If the seven- to 10-day stock culture used as the source of the
inoculum has a cell density of 2,000,000 cells/mL, a test
employing 25 flasks, each containing 100 mL of test medium and
inoculated with a total of 1,000,900 cells, would require
25,000,000 cells or 12.5 mL of stock solution
(25,000,000/2,000,000) to provide sufficient inoculum. It is
advisable to use a volume 20 to 50% i-n excess of the minimum-
volume required, to cover accidental loss in transfer and
handling.
1. Determine the density of cells (cells/ml) in the stock
culture (for this example, assume 2,000,000 per mL).
2. Calculate the required volume of stock culture as follows:
Volume (mL) of Number of flasks X Volume of Test X 10,000 cells/mL
Stock Culture s to be used Solution/Flask
Required Cell density (cells/mL) in the stock culture
= 25 flasks X 100 nt/flask X 10.000 cells/Hi
. '' 2,000,000 cells/mL
« 12.5 mL Stock Culture
3. Centrifuge 20 mL of stock culture at 1000 x g for 5 rain.
This volume will provide a 50% excess in the number of cells.
4. Decant the supernatant and resuspend the eel Is-in 15 mL of
distilled or deionized water.
5. Repeat the centrifugation and decantation step, and resuspend
the cells in 15 mL distilled or deionized water.
6. Mix well and determine the cell density in the makeup water.
Some cells will be lost in the concentration process.
85
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Dilute the cell concentrate as needed to obtain a cell density of
1,000,000 cells/ml, and check the cell density In the final Inoculum.
11.3 START OF THE TEST
11.3.1 Tests should begin as soon as possible, preferably within 24 h of
sample collection. If the persistence of the sample toxicity 1s not
known, the maximum holding time should not exceed 36 h. In no case should
the test be started more than 72 h after sample collection. Just prior to
testing, the temperature of the sample should be adjusted to that of the
test (24 +• 2°C) and maintained at that temperature until portions are
added to the dilution water.
11.3.2 The test begins when the algae are added to the test flasks.
1. Mix the inoculum well, and add 1 ml to the test solution in each
. flask.
2. Make a final check of the cell density in three of the test
,solutions at time "zero * (within 2 h of the inoculation).
11.4 LIGHT, PHOTOPERIOO, AND TEMPERATURE
11.4.1 Test flasks are incubated- under continuous illumination at
86 + 8.6 uE/m2/s (400 + 40 ft-c), at 24 > 2°C, and should be shaken
coritinously at 100 cpoTon a mechanical shaker or twice daily by hand.
Flask positions in the incubator should be randomly rotated each day to
minimize possible spatial differences in illumination and temperature on
growth rate. If it can be verified that test specification are met at all
positions, this need not be done.
11.5 ROUTINE CHEMICAL AND PHYSICAL DETERMINATIONS
11.5.1 Measure the pH.and specific conductivity of the highest, midrange,
and lowest effluent concentrations and the dilution water when the test
dilutions are prepared. Additional measurements may be appropriate
depending on the test objectives.
11.6 OBSERVATIONS DURING THE TEST
11.6.1 Toxic substances in the test solutions may degrade or volatilize
rapidly, and the inhibition in algal growth may be detectable only during
the first one to two days in the test. It may be desirable, therefore, to
determine the algal growth response daily.
11.7 TERMINATION OF THE TEST
11.7.1 The test is terminated 96 h after initiation. The algal growth in
each flask is measured by one of the following methods: (a) cell counts,
(b) chlorophyll content, or (3) turbidity (light absorbance). Regardless
of the method used to monitor growth, the algae in the test solutions
should be checked under the microscope to detect abnormalities in cell
size or shape.
86
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11.7.2 Cell Counts
11.7.2.1 Automatic Particle Counters
11.7.2.T.1 Several types of automatic electronic and optical particle
counters are available for use in the rapid determination of cell density
(cells/mD-and mean cell volume (MCV) in um3/cell. The Coulter Counter is
widely used and is discussed in detail by Miller et al., 1978.
11.7.2.1.2 If biomass data are desired for algal growth potential
measurements, a Model ZBI or ZB Coulter Counter is used. However, the
instrument must be calibrated with a reference sample of cells of known volume.
11.7.2.1.3 When the Coulter Counter is used, an aliquot (usually 1 mL) of the
test culture is suspended in a 1% sodium chloride electrolyte (such as
IsotonK), in a ratio of 1 mL of test culture to 9 ml (or 19 mL) of 0.22-ura
filtered saline solution (dilution of 10:1 or 20:1). The resulting dilution is
counted using an aperture tube with a 100-urn diameter aperature. Each cell
(particle) passing through the aperture causes a voltage drop proportional to
its volume. Depending on the model, the instrument stores the information on
the number of particles and the volume of each, and calculates the mean cell
volume.
The following procedure is used: -
1. Mix the algal culture in the flask thoroughly by swirling the contents
of the flask approximately six times in a clockwise direction, and then
six times in the-reverse directionj repeat the two-step process at
least once.
2. At the end of the mixing process, stop the motion of the liquid in the
flask with a strong brief reverse mixing action, and quickly remove
1 mL of cell culture from the flask with a sterile pipet.
3. Place the aliquot in a counting beaker, and add 9 mL (or 19 mL) of
electrolyte solution (such as Coulter ISOTONR).
4. Determine the cell density (and MCV, if desired).
11.7.2.2 Manual microscope counting methods
11.7.2.2.1 Cell counts may be determined using a Sedgwick-Rafter,
Palmer-Maloney, hemocytometer, inverted microscope, or similar methods. For
details on microscope counting methods, see APHA 1985 and Weber 1973
Whenever feasible, 400 cells-per replicate are counted to obtain + 105S
precision at the 95* confidence level. This method has the advanTage of
allowing for the direct examination of the condition of the cells.
11.7.3 Chlorophyll Content
11.7.3.1 Chlorophyll may be measured in-vivo fluorometrically, or in-vitro
either fluorometrically or spectrophotometrically. In-vivo fluorometric
measurements are recommended because of the simplicity and sensitivity of the
technique and rapidity with which the measurements can be made (Rehnberg et
al., 1982) ,-
87
-------
The measurements are made as follows:
1. Adjust the "blank" reading of the fluorometer using the filtrate from
an equivalent dilution of effluent filtered through a 0.45 urn particle
retention filter.
2. Mix the contents of the test culture flask by swirling successively in
opposite directions {at least three times), and remove 1 ml of culture
from the flask with a sterile pipet.
3. Place the aliquot in a small disposable vial and record the
fluorescence as soon as the reading stabilizes. (Do not allow the
sample to stand in the instrument more than 1 min.)
4. Discard the sample.
11.7.4 Turbidity (Absorbance)
11.7.4.1 A second rapid technique for growth measurement involves the use of
a spectrophotometer to determine the turbidity, or absorbance, of the cultures
at a wavelength of 750 rim. Because absorbance is a complex function of the
volume, size, and pigmentation of the algae, it would be useful to construct a
calibration curve to establish the relationship between absorbance and cell
density.
The algal growth measurements are-made as follows:
1. A blank is prepared as described for the fluorometric analysis.
2. The culture is thoroughly mixed as described above.
3. Sufficient sample is withdrawn from the test flask with a sterile pipet
and transferred to a 1- to 5-cm cuvette.'
4. The absorbance is read at 750 nm and divided by the light path length
of the cuvette, to obtain an "absorbance-per-centimeter" value.
5. The 1-cm absorbance values are used in the same manner as the cell
counts.
11.7.5 Biomass
11.7.5.1 The results of algal growth potential tests are commonly expressed
in terms of biomass (mg dry wgt organic matter/L). Algal biomass can be
calculated from cell counts and mean cell volumes, or can be measured directly
by gravimetric methods,
11.7.5.2. Biomass calculated from Cell Counts and Mean Cell Volumes
Algal biomass is calculated as follows:
Dry Weight (mg/L) 3 Cells/L X MCV(um3) X mg organic matter/um3
Where: MCV a either:
1. The "measured" or "actual" cell volume, such as determined with
a Coulter Counter or determined by microscopic examination, or
88
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2. When actual measurements are not available, a value of 60 uro3
Is Used as an estimate of the MCV. A standard value of 3.6 X
10-10mg is used for the weight of organic matter/un»3.
If the cell count is 1,000,000 cells/ml:
Bioraass (rag/L) » 1,000,000 cells/ml. X 1000 mL/L X 60 um3/cell X
3.6 X 10-10 mg/um3
* 21.6 mg/L
11.7.5.3 Biomass by Direct, Gravimetric, Dry Weight Measurements
11.7.5.3.1 Direct, gravimetric methods of measuring biomass are
appropriate where the harvestable biomass per test flask is greater than
10 mg dry weight. This condition is met where the cell density is equal
to or exceeds 5,000,000'cells/ml. This cell density would not ordinarily
be achieved in a 96-h test, but would be applicable with a 14-day test.
11.7.5.3.2 If the cell density is large enough to warrant use of the
gravimetric method, proceed as follows, treating each flask separately:
1. Centrifuge the entire contents-of each flask at 1000 X g for 5 min.
2. Decant the supernatant (do not retain more than 10 mL of culture
medium with the cells). Note: Caution must be exercised to avoid
disturbing the sedimented cells when decanting the supernatant.
3. Resuspend the cells in 10 ml distilled water.
4. Centrifuge and decant, as in (2) above.
5. Transfer the sedimented cells from each flask to a separate weighed
crucible or weighing pan.
6. Dry overnight at 70°C.
7. Cool in a desiccator and weigh to the nearest 0.1 mg.
8. Report the dry weight in mg/L.
11.7.5.3.3 The cells may .also be concentrated and dried on 0.45-um pore
diameter membrane filters or on equivalent glass fiber or other filters,
as follows:
1. Dry the filters for 2 h at 70°C (temperatures above 75°C may
cause the pores to close in membrane filters.
2. Allow the filters to cool in a desiccator for at least 1 h before
weighing.
3. Filter the contents of the test flask using a vacuum of 380 mm of
- mercury, or a pressure not to exceed one-half atmosphere.
4. Rinse the filter funnel with 50 mL of distilled water using a wash
bottle, and allow the rinsings to pass through the filter. The
rinse serves to transfer all the algae to the filter and washes the
nutrient salts through the filter.
5. Dry the filters overnight at 70°C (to constant weight), cool in a
desiccator for 1 h, and weigh to the nearest 0.1 mg.
89
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11.8 SUMMARY OF TEST CONOITONS
11.8.1 A suriaary of test conditions is listed in Table 3.
11.9 ACCEPTABILITY OF TEST RESULTS
11.9.1 The test results are acceptable if the algal cell density in the
control flasks exceeds 106cells/mL at the end of the test, and does not vary
more than 10X among replicates.
12. CALCULATIONS
12.1 TOXICITY DATA
12.1.1 The data can be analyzed by Dunnett's Procedure or Probit Analysis.
The results of the toxicity test are expressed as an NOEC and/or EC!
(threshold effect concentration). The chronic value can also be determined if
desired. ; . - .
12.1.2 The NOEC and LOEC (MATC), and chronic value are based on comparisons
of the growth of the control organisms with each of the toxicant
concentrations, using Dunnett's Test.
12.1.3 The statistical tests described here must be used with a knowledge of
the assumptions upon which the tests are contingent, tests for normality and
homogeneity of variance are included in the Appendix. The assistance of a
statistician is recommended for analysts who are not proficient in statistics.
12.1.3 Dunnett's Procedure:
12.1.3.1 Dunnett's Procedure (Dunnett, 1955) includes an analysis of variance
(ANOVA) followed by a comparison of each toxicant concentration mean with the
control mean. The error value calculated in the ANOVA is used in the
comparison of the control and treatment means. The computer program listed in
the Appendix generates output which includes an ANOVA table, a statement about
each treatment mean that can be used to identity the NOEC and LOEC, and the
minimum difference between treatment and control means that can be detected as
statistically significant. Dunnett's Procedure is used when the assumptions
of normality-and homogeneity of variance are met. If they are not met,
Steel's Many-One Rank Test is used.-
12.1.3.2 The sample set of cell counts shown in Table 4 were transformed to
logiO to help meet the assumptions for normality and homogeneity of
variance. The logs were input to the Dunnett's program listed in the
Appendix.
12.1.3.3. The results of the analysis of variance of the log transformed data
from Table 4 are shown in Table 5, and indicate a statistically significant
difference in survival among cadmium concentrations. The computer output for
the -comparison of treatment means with the control mean is shown below:
90
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THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 2
(5 US CO/I) AND CONTROL.
THERE IS A SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 3
(10 US CD/L) AND CONTROL.
THERE JS A SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 4
(20 UG CD/L) AND CONTROL.
THERE IS A SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 5
(40 US CD/L) AND CONTROL.
THERE IS A SIGNIFICANT DIFFERENCE BETWEEN CONCENTRATION 6
(80 UG CD/L) AND CONTROL.
12.1.3.4 The NOEC and LOEC, as determined from the Dunnett computer
analysis, were 5 ug Cd/L and 10 ug Cd/L, respectively. (Note that the
Ounnett program considers the control as Concentration 1, so that
Concentration 2 in the output is 5 ug Cd/L in the test). The MATC was
5 - 10 ug Cd/L
12.1.3.5 For this set of data, the minimum difference between the control
mean and a treatment mean that can be detected as statistically
significant is 225,339 cells/mL. This represents a 18.151 reduction in the
response from the control.
12.1.3.6 The chronic value is determined by calculating the geometric
mean (GM) of the NOEC and LOEC, as follows:
Logic NOEC = Logic 5.0 = 0.69897
Logio LOEC » Logic 10.0 = 1.00000
ChV = Antilog (0.69897 + 1.0000)/2 = Antilog 0.8495
ChV = 7.07 ug Cd/L
12.1.4 Probit Analysis
12.1.4.1 Although the assumptions for Probit Analysis are not met in the
classical sense because of the very nature of the algal growth data, the
analysis is used to obtain an estimate of the EC1. To carry out the
Probit Analysis, use the growth response data, such as cell counts,
in-vivo chlorophyll fluorescence, or absorbance, for each set of three
replicate flasks in a given treatment. The special Probit program listed
in the Appendix uses the algal count data, determines the mean, and
converts it to an "inhibition proportion" using the following formula:
Where: C = The mean growth in the control flasks
T = The mean growth at a given effluent
concentration.
91 :
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12.1.4.2 The cell counts were entered Into the "Algal" Probit program
listed 1n th« Appendix. The EC1 was found to be 2.27 ug Cd/L, with upper
and lower confidence limits of 3.42 and 1.20 ug Cd/L, respectively, at
P * 0.05. An example of how the data are entered in the program and the
program output are provided In the Appendix.
12.2 BIOSTIMULATION
12.2.1 Where the growth response in effluent (or surface water) exceeds
growth In the control flasks, the percent stimulation, S(X), 1s calculated
as shown below. Values which are significantly greater than the control
indicate a possible degrading enrichment effect on the receiving water
(Walsh, et al., 1980b):
S(X) - T - C
X 100
13. TEST PRECISION AND-ACCURACY
13.1 PRECISION
13.1.1 Data from repetitive 96-h toxicity tests, conducted with three
reference toxicants, are shown in Table. 6. The relative standard
deviation (coefficient of variation) of the LCls ranged from 47% to 83%.
13.2 ACCURACY
13.2.1 The accuracy.of toxicity tests can not be determined.
92
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TABLE 3. SUMMARY OF RECOMMENDED TEST CONDITIONS FOR THE ALGAL GROWTH TEST
1. Test type:
2. Temperature:
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test flask size:
7. Test solution volume:.
8.. Age of stock culture
used for inoculum:
9. Initial cell density:
10. Number of replicates
per concentration:
11. Shaking rate:
12. Dilution water:
13. Dilution factor:
14. Test duration:
15. Effect measured:
16. End point(s):
Static
24 + 2°C
"Cool white" fluorescent lighting
86 + 8.6 uE/m2/s (400 + 40 ft-c)
Continuous illumination
125 mL or 250 mL
50 mL or 100 mL
4 to 7 days
10,000 cells/mL
3
100 cpm continuous, or twice daily
by hand
Algal stock culture medium without
EDTA, or surface water
Approximately 0.3 or 0.5
96 h
Growth (cell counts, chlorophyll
fluorescence, absorb.ance, biomass)
EC1, NOEC, S(»)
93
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TABLE 4. SAMPLE DATA FROM ALGAL TOXICITY TEST WITH CADMIUM CHLORIDE
Toxicant
Concentration
(uq Cd/L)
0 (Control)
•
5
10
20
40
80
Growth
Response:
Cells/mL
ToTSo"
1209
1180
1340
1212
1186
1204
826
628
816
493
416
413
127
147
147
49.3
40.0
44.0
Cells/mL
1000
(Logic)
3.082
3.072
3.127
3.084
3.074
3. .081
2.917
2.798
2.912
2.693
2.619
2.616
2.104
-2.167
2.167
1.693
1.602
1.643
Percent
Inhibition
of Growth
!(*)
0
3.4
39.1
64.5
88.7
96.4
TABLE 5. ANALYSIS OF VARIANCE OF CELL COUNT DATA IN TABLE 4
TRANSFORMED TO
Source
Among
Within
Total
OF
5
12
17
Sum of Squares
4.995
0.021
5.016
Mean Square
0.999
0.002
Calculated Tabular
F PQ.05
560.183* 3.11
Significant at P » 0.05.
94
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SELECTED REFERENCES
Akasenova, Y. I.* 6. I. Bogucharskova, and M. I. Zozulina.. 1969. The
role of phytoplankton and bacterloplankton In the food of the
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104
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APPENDIX
A. Validating Normality and Homogeneity of Variance
Assumptions 106
1. Introduction 106
1. Test for Normal Distribution of Data . 106
2. Test for Homogeneity of Variance 109
8. Arc Sine Square Root.Transformation Ill
C. Ounnett's Procedure ....... 113
1. Manual Calculations . . . . «, 113
2. Computer Calculations .-,-.". 118
D. Steel's Many-one Rank Test 132
E. Fisher's Exact Test ...... 135
F. Probit Analysis 144
1, Probit Analysis of Fathead Minnow Embryo-larval
Test Data 144
2. Probit Analysis of Algal Growth Test Data 154
105
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APPENDIX A
VALIDATING NORMALITY AND HOMOGENEITY OF VARIANCE ASSUMPTIONS1
1. INTRODUCTION
1.1 Ounnett's Procedure 1s a parametric procedure and 1s based on the
assumptions that the observations are Independent and normally
distributed, and that the variance of the observations is homogeneous
across all toxicant concentrations and the control. These assumptions
should be checked prior to using Dunnett's Procedure, to determine if they
have been met. Tests for validating the assumptions are provided in the
following discussion. If the tests fail (if the data do not meet the
assumptions), a non-parametric procedure such as Steel's Many-One Rank
Test may be more appropriate. However, the decision on whether to use
parametric or non-parametric tests may "be a judgement call, and a
statistician should be consulted in selecting the analysis.
2. TEST FOR NORMAL DISTRIBUTION OF DATA
2.1 A formal test for normality is the Chi-Square Sdodness of Fit Test.
This test compares the observed sample distribution with a normal
distribution. An example of the test -is provided below.
2.2 The example uses mortality data from the fathead minnow larval
survival and growth test with sodium pentachlorophenate (NaPCP) listed in
Section 10, and is the same data used in the discussion of the homogeneity
of variance determination in Appendix A.3 and the Dunnett's Procedure in
Appendix C. The data used in the Dunnett's Procedure has been arc sine
square root transformed, and it is the transformed data which will be
tested for normality. The transformed data and the mean and standard
deviation of the observations at each toxicant concentration, including
the control, are listed in Table A.I.
2.3 The first step of the Chi-Square Goodness of Fit Test is to
standardize the observations by subtracting the mean from each observation
and dividing the difference by the standard deviation. The standardized
observations are listed in Table A.2.
2.4 Form five cells as follows: < -1.5; -1.5 to < -0.50; -0.50 to
0.50; > 0.50 to 1.5; >1.5. Tabulate the number of standardized
observations which fall into each of the five cells. These are the
observed frequencies, f-j. The expected frequency, FT, is found by
multiplying the area under the standard normal curve over the ith cell
limits by the total number of standardized observations, N.
^Prepared by John Menkedick and Florence Kessler, Computer Sciences
Corporation, 26 W. St. Clair St., Cincinnati, Ohio 45268; Phone
513-568-7968.
106 -;
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TABLE A.T. ARC SINE SQUARE ROOT TRANSFORMED DATA FOR CHI-SQUARE TEST
NaPCP Concentration
Replicate
1
2
3
4
Total -.
Mean
Si
i
Control 3
1.412
1.412
1.249
1.249
5.322
1.330
0.0941
1
TABLE A.2.
1.107
1.107
1.412
1.107
.4.733
1.183
0.1524
••' 2
5
1.249
1.412
1.412
1.412
•5.485
1.371
0.0815
3
EXAMPLE OF CHI-SQUARE TEST
7
1.249
1.249
1.107
1.412
5.071
1.254
0.1246
4
(ug/L)
11
0.991
1.249
1.412
0.785
4.437
1.109
0.2769
5
16
0.685
0.580
0.685
0.464
2.414
0.604
0.1054
6
FOR NORMALITY:
STANDARDIZED OBSERVATIONS
NaPCP Concentration
Replicate
1
2
3
4
Control
0.8714
0.8714
- Q.8608
• 0.8608
3
- 0.4987 -
- 0.4987
1 .5026
- 0.4987
5
1.4969 -
0.5031
0.5031 -
0.5031
7
0.0401
0.0401
1.1798
1.2680
(ug/L)
11
- 0.4261
0.5056
1 .0942
- 1.1701
16
0.7685
- 0.2277
0.7685
- 1.3283
107
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2.5 For this exauple, N » 24. The areas for each cell, the observed
frequencies, and the expected frequencies are given in Table A.3.
TABLE A.3. EXAMPLE OF CHI-SQUARE TEST FOR NORMALITY:
ACTUAL AND EXPECTED FREQUENCIES
Cell
Interval
i
Area
Fi
f' '
<-1.5 -1.5 to <-0.5
1 2
0.067 0.242
1.608 5.808
0 6.
-0.5 to 0.5
3
0.382
9.168
7
>0.5 to 1.5 :
4
0.242
5.308
10
>1 5
5
0.067
1.608
1
2.6 The Chi-Square Goodness of Fit Test statistic (X2) is calculated as
follows: .
2s V
X2 - S (f, - FJ2
-
2.7 For the data in the example:
2 (0 - 1.608}2 + (6 - 5. 80S)2 + (7 - 9.168)2 + (10 - 5. 80S)2 + (1 - 1.608)2
X * 1.608 5.808 9.168 5.308 TISS
* 5.3825
2.8 The decision rule for this test is to compare the critical value,
X2, with four degrees of freedom ( No. of cells - 1) and an alpha of
0.01, to the computed X2. If the computed value exceeds the critical
value, conclude that the data are not normally distributed. For this
example, the critical value is 13.28. The calculated value, 5.3825, does
not exceed th€ critical value. Thus, the conclusion of the test is that
the data are normally distributed.
2.9 In general, if the data fail the test for normality, a transformation
such as to log values may normalize the data. After transforming the
data, repeat the Chi-Square Goodness of Fit Test for normality.
108
-------
3. TEST FOR HOMOGENEITY OF VARIANCE .
3.1 For Qunnett*s Procedure, the variances of the data obtained from each
toxicant conctntration and the control are assumed to be equal.
Bartlett's Test is a formal test of this assumption. In using this test,
it is assumed that the data are normally distributed. An example of how
this test is performed is provided below:
3.2 The data used in this example are mortality data from a fathead
minnow larval survival and growth test, and are the same data used in
Appendix C. Since Dunnett's Procedure is performed with arc sine
transformed data, Bartlett's Test is performed with the same transformed
data. These data are listed in Table A. 4, together with the calculated
variance for the control and each toxicant concentration.
3.3 The test statistic for Bartlett's Test (Snedecor and Cochran, 1980)
is as follows:
v[a(ln S2) - £ In sf]
Where: v * Degrees of freedom for. each toxicant concentration and control
a » Number of levels of tox-icant concentration including the
control
32 s The average of the individual variances
C » 1 +C(*H)/3av].
In = Loge
3.4 For the data in this example, v = 3, a = 6, U2 » 0.0236, and
C = 1.1296. The calculated B value is:
3 [6(ln 0.0236) - Z In S?]
1.1296
3[6(-3.7465) - (-24.7529)]
1.1296
6.0390
109
-------
3.5 Since Bis approximately distributed as Chi Square with a - 1 degrees
of freedom whin the variances are equal, the appropriate critical value
for the ttst is 15.09 for a significance level of 0.01. Since B < 15.09,
the conclusion is that the variances are equal.
TABLE A.4 DATA USED IN BARTLETT'S TEST FOR HOMOGENEITY OF VARIANCE
NaPCP Concentration (ug/L)
Replicate
1
2
3
4
Total
Mean
Si 2
i
Control
1.412
1.412
1.249 1
1.249
5.322
1.330
0.0088
1
3
1.107
1.107
' -1.412
1.107
4.733-'
1.183
0.0232
2
5
1.249
1.412
"1.412
1.412
5.485'
." 1.371
0.0066
3
7
1.249
1.249
1.107 '
1.412
5.071
1.254
0.0155
4
11
0.991
1.249
1.412
0.785
4.437
1.109
0.0767
5
16
0.685
.0.580
0.685
0.464
2.414
0.604
o.om
6
no
-------
b. For RP * 0 (no mortality), the following adjustment is made:
Angle (in radians) » Arc Sine (l/4N)0-5
Where: N * Number of animals/treatment
Example; If 20 animals are used, .
"Angle « Arc Sine (1/80)0-5
- Arc Sine 0.1118
» 0.1120 radians
c. For RP - 1.0 (10031 mortality):
Angle * 1.5708 radians - (radians for RP » 0)
Example: Using above value
Angle - 1.5708:- 0.1120
* 1.4538 radians
112
-------
APPENDIX B
ARC SINE SQUARE ROOT TRANSFORMATION1
1. Arc Sine transformation consists of determining the angle (in radians)
represented by a sine value. In the case of arc sine square root
transformation of mortality data, the proportion of dead (or affected)
organisms is taken as the sine value, the square root of the sine value is
determined, and the angle (in radians) for the square root of the sine
value is determined. Whenever the proportion dead is 0 or 1, a special
modification of the arc sine square root transformation must be used
(Bartlett, 1937). An explanation of the arc sine square root
transformation and the modification is provided below.
2. Calculate the response proportion (RP) at-each effluent concentration,
where:
RP « (number of dead or "affected" organisms)/(initial number).
Example; If 8 of 20 animals in a given treatment die:
• RP - 8/20
.- 0.40
3. Transform each RP to Arc Sine, as follows:
a. For RP * > 0 to < 1:
Angle (radians) * Arc Sine
Example: If RP » 0.40,
Angle » Arc Sine (0.40)°-5
* Arc Sine 0.6325
* 0.6847 radians
Vrora Peltier and Weber (1985).
m
-------
APPENDIX C
OUNNETT'S PROCEDURE
1. MANUAL CALCULATIONS1
1.1 Ounnett's Procedure is used to compare each concentration mean with
the control mean to decide if any of the concentrations differ from the
control. This test has an overall error rate of alpha, which accounts for
the multiple comparisons with the control. It is based on the assumptions
that the observations are independent and normally distributed and that
the variance of the observations is homogeneous across all concentration
and control. (See Appendix A for a discussion on validating the
assumptions). Ounnett's Procedure uses & pooled estimate of the variance,
which is equal to the error value calculated in an analysis of variance.
1.2 The data for this example are mortality data from a fathead minnow,
larval survival and growth test. Since the data are expressed in
proportions, a transformation of the square root of the proportion to an
arc sine value is required (See Appendix 8.) A table of the raw data
(Table C.I) and transformed data (Table C.2) are provided below.
. TABLE C.I. PROPORTIONS OF SURVIVING TEST ORGANISMS.
Concentration i Proportion Surviving
(ug NaPCP/L°) in Replicate Chambers
Control
3
5
7
11
16
1
2
3
4
5
6
A
1.00
0.30
0.90
0.90
0.70
0.40
3
1.00
o.ao
1.00
0.90
0.90
0.30
C
0.90
1.00
1.00
0.80
1.00
0.40
0
0.90
o.ao
1.00
1.00
0.50
0.20
^Prepared by John Menkedick and Florence Kessler, Computer Sciences
Corporation, 26 W. St. Clair St., Cincinnati, Ohio 45268; Phone
513-568-7968)
113 :•
-------
TABLE C.2. ARC SINE TRANSFORMED DATA FOR OUNNETT'S PROCEDURE
NaPCP
(ug/L)
Control
3
5
7
n
16
Cone
(D
1
2
3
4
5
6
Transformed Data
(Arc Sine In Radians))
Replicate
A
1.412
1.107
1.249
1.249
1 0.991
0.685
B
1.412
1.107
1.412
1.249
1.249"
0.580
C
1.249
1.412
1.412
1.107
1.412
0.685
0
1.249
1.107
1.412
1.412
0.785
0.464
Total
Ti
5.322
4.733
5.485
5.017
4.437
2.414
Mean
Y1
1.330
1.183
1.371
1 .254
1.109
0.604
1.3 One way to obtain an estimate of-.the pooled variance is to construct
an ANOVA table including all sums of squares, using the following
formulas:
Total Sum of Squares: TSS • 2 Y2. - G2/N
ij 7J
Between Sum of Squares: SSB 3 2 T2/n. - G /N
Within Sum of Squares: SSW * TSS - SSB
Where: G » The grand total of all sample observations; G » 2 T^
N » The total sample size; N = 2 n. 1
*
The number of replicates for concentration "1".
T.I » The total of the replicate measurements for concentration "i".
Y.J.J * The jth observation for concentration "i".
' j
1.4 Calculations:
Total Sum of Squares: TSS » 2 Y?. - G2/N
33.302 - (27.408)/24
2.002 ;".
114
-------
Between Sum of Squares: SS8 » E T?/n. - G2/N
1
- 32.875 - (27.408)2/24
* 1.S75
Within Sura of Squares: SSW » TSS - SS8,
» 2.002 - 1.575
- 0.427
1.5 Prepare the ANOVA table as follows:
Source
Between
Within
Total
b
N
N
OF
*-l
- b
- 1
Sum of Squares (SS)
SSB
SSW
TSS .-
Mean Square(MS)
.(SS/DF) .
s| » SSB/(b-l)
S2 » SSW/(N-b)
F
S2/S2
W
*Where b » Number of different concentrations, including the control,
1.6 The completed ANOVA table for this data is provided below:
TABLE C.3. COMPLETED ANOVA TABLE FOR OUNNETT'S PROCEDURE
Source OF
Between . 6 -- 1 » 5
Within 24 - 6 » 18
SS
1.575
0.427
MS
0.315
0.024
F
13.125
Total 23 ' • . 2.002
115
-------
1.7 To perfora the individual comparisons, calculate the t statistic
for etch concentration and control combination, as follows:
•Where: ?i * Mean for each concentration
TI * Mean for the control
Sw * Square root of the within mean square
ni » Number of replicates in the control.
n^ » Number of replicates for concentration "1".
1.8 Table C.4 includes the calculated t values for each concentration
and control combination.
TABLE C.4. CALCULATED T VALUES.
Concentration
(ug NaPCP/L)
3
5
7
11
16
2
3
4
5
6
1.342
- 0.374
0.694
. 2.108
6.630
1.9 Since the purpose of the test is only to detect a decrease in
survival frora the control, a one-sided test is appropriate. The
critical value for the one-sided comparison, with an overall alpha level
of O.OS, 18 error degrees of freedom, and 5 concentrations excluding the
control, 1s 2.41 (See Table C.S). Comparing each of the calculated t
values in Table C.4 with the critical value, the 16 ug NaPCP/L
concentration is found to have significantly lower survival than the
control (6.630 > 2.41). Thus the NOEC is 11 ug NaPCP/L.
116
-------
1.10 To quantify the sensitivity of the test, the minimum significant
difference (MSO) may be calculated. The formula is as follows:
Where: -d
S*
n
For example:
MSO »
MSD » d 3^(17^) + (1/n)
Critical value for the Qunnett's Procedure
The square root of the within mean square
The number of replicates at each concentration,
assuming an equal number of replicates at all
treatment concentrations
Number of replicates in the control
2.41 (0.1S5)[V(l/4)
2.41 (0.155)(0.707)
0.264 ;
(1/4)1 a 2.41 (0.155)(
1.11 The MSD (0.264) is in transformed units. To determine the MSO in
terms of percent survival, carry out the following conversion:
1.11.1. Subtract the MSO from the transformed control mean.
1.330 - 0.264 * 1.066
1.11.2; Obtain untransformed values for -the control mean (1,3305) and the
difference (1.0665) obtained in Step 1.
[Sine (1.330)]2 = 0.943
[Sine (1.066)]2 * 0.766
1.11.3. The untransformed MSD (MSDU) is determined by subtracting the
untransformed values obtained in Step 2.
MSDU * 0.943 - 0.766 = 0.177
1.11.4 For this set of data, the minimum difference between the control
mean and a concentration mean that can be detected as statistically
significant is 0.177. This represents a decrease in survival of 19% from
the control. " •
117
-------
2. COMPUTER CALCULATIONS
2.1 Tli1s computer program incorporates two analyses: an analysis of
variance (ANOVA), and a multiple comparison of treatment means with the
control mean (Ounnett's Test). The ANOVA indicates whether there is a
significant difference between the results obtained at different toxicant
concentrations. The Dunnett Test indicates which toxicant concentration
means (if any) are statistically different from the control mean. The
program also gives the minimum difference between the control and
treatment means that could be detected as statistically significant. The
multiple comparison is based on Ounnett, C. W., 1955, "Multiple Comparison
Procedure for Comparing Several Treatments with a Control," J. Araer.
Statist. Assoc. 50:1096-1121. Tables for one-tailed "F" and Dunnett's "t"
(P » 0.05) are included in the program, but are limited to values for
eight concentrations, including the control, and six replicates per
concentration. If the test design exceeds these limits, it is necessary
to consult the "t" and "F" tables listed following the program.
2.2 The program was written in FORTRAN PLUS IV v.3, by James Dryer, and
runs on the Digital Equipment Corporation, POP 11/70 computer under IAS
version 3.1. Machine readable copies of the program, and assistance in
running it, can be obtained by contacting James Dryer, EMSL-Cincinnati
Newtown Facility (FTS 778-8350; comm'l 513-527-8350).
2.3 Description,of Data Input and Output from the Computer Program
2.3.1 Data Input ' •
2.3.1.1 Cell growth data from a Selenastrum toxicity test, listed in
Table 4, Section 14, were used to illustrate the data input process and
the output provided by the computer program. As shown below, the program
prompts the operator for the following information:
1. The type of transformation to be used with the data.
2. The number of replicates at each concentration.
3. The values (cell growth) obtained from each replicate toxicant
concentration.
2.3.1.2 Several transformations are available in the program.
Transformation option "3", conversion to log-jo, was used in the example
provided. Six concentrations, each with three replicates, were used in
the test. The control is considered as Concentration 1.
2.3.2 Program Output
2.3.2.1 The output from the analyses includes:
1. A table of the original data (cell growth) input to the program.
2. An ANOVA table listing the calculated "F" and tabular "F" values.
3. A series of statements regarding the results of each sequential
comparison of a toxicant concentration mean with the control mean.
118
-------
2.3.2.2 The calculated F from the ANOVA j,s compared with the tabular F
value where P * 0.05, and OF » 5,12. The" calculated F (560.183) is
larger than the tabular F (3.11), indicating that the survival is
significantly different at the different toxicant concentrations.
2.3.2.3 The output from the Qunnett sequential comparisons indicates
that there is a significant difference between Concentration 3 and the
control (Concentration 1). On the basis of these results, the NOEC is
Concentration 2. •
2.3.3 Computer Generated Listings of Input and Output from the Computer
Program for Dunnett's Test ana Analysis of Variance
BUNNETTS TEST
IF TOO MEED INSTRUCTIONS»TYPS 1. OTHCRyiSS.TTPF 0-1
ANOVA-4IITH gUNNCTTS COMPARISON OP EACH CONCENTRATION (ISAM
V3 THE CONTROL MEAN*
A ONE-SIDED »T« AT *SZ IS USED IN ALL "CASES.
THE FOLLOWING TRANSFORMATIONS ARE AVAILABLE TO USE ON
THE VALUES:
NUNBER TRANSFORMATTON
0 NONE
1 SQUARE ROOT(X)
3 LOO 10
3 4
ARCSINOO. RTCX+1)/CN+1>J>/7
6 ARC3ZN((X>nO.S>
TRANSFORMATION NUMBER • 3
AVERAGE NUMBER OF TFST ORGANISMS AT WGINNTNG PER CONCENTRATION « 100,
INCLUDING CONTROLf NUMBER OF CONCENTRATIONS * 4
NOTE: BESTN WITH CONTROLCCONCENTRATION NUMBER i> AND ENTER IN ASCENDING ORDER.
NUMBER OF REPLICATES IN CONCENTRATION NUMBER 1
* 3
NUMBER OF REPLICATES IN CONCENTRATION NUMBER ?
- 3
NUMBER OF REPLICATES IN CONCENTRATTON NUMBER 3
- 3
NUMBER OF REPLICATES IN CONCENTRATION NUMBER 4
• 3
NUMBER OF REPLICATES IN CONCENTRATION NUMBER S
- 3
NUMBER OF REPLICATES IN CONCENTRATION NUMBER *
- 3
119
-------
2.3.3 Cosput**" generated Listings of Input and Output from the Computer
FrogrM for Dunnttt's Procedure (Continued)
TEST OATA FWH IW1UIIUM. RtPUCATEJ.
MTEl KCXMM. MIHT HtST BE USED IN
OF ancarnunw unto t -
120f. . , ' '.'
•VMOJT* tf CONCSHTHMIOI MJNtER .1 -
tilt.
•VMJUC- tF COKBtmtnw MJNIER 2
1212.
"V*UJC- ^ COHCEHIIWriOl KU1IER 2
tit*. •
•VMJK" IF COKZrmATiaN ItJHISt 2
1204.
•UILUC- IF COKStTMTtUN HJHISt 3~
' 82*. '
-V«JJ£" IF CONCS4TRITHW IU11ER 3
421.
-VMJJC" IF CJWSHTRiTtW MJNIER 3
- OF coNcemwrioN KUHISI 4
4t3.
*VMJJ£M IF eaicsfTiMriaN IUIIER 4
41*.
"VtLUt" IF COKeiTRATZBN RMIG? 4
413.
IF CONCStTRATIOl HU1IER S
127.
-v*LUt" jf eoNCENTmrioi IUMIER s
•VUJUl" IF COKElTOmiN HHISt S
147.
-VtUJt" V C9NOKT1ATION MJUEX 6
44. ' ' .
-VALUE" IF coNcamwnoi HWIIER 6
4».
•VMOJE- IF CGNCEHTMTItH IUUER "4
4».3 —
120
-------
2.3.3 Computer Generated Listings of Input and Output from Computer
Program for Qunnett's Procedure (Continued)
tESILTS OF 36 I Ue I T I * I COMPARISONS
II S I N • THE D U » » E T I S TEST
FBI THIS SET W MTA, TIE IIMIKM OtFFERElCE THAT CAI BE
JETECTEB AS STATISTICAL!.! S1S1IITICJWT IS 223.33* .
THIS REFftESEftTS A IS.tZ KESUCTION IN THE MEAN
lEsrtmse nan THE antTsou.
T • 2.39
TWW3 NO SIBNiriONT OIFFCREICE 3ETWEN COKSITfttnON Z MO GOITRIL.
-> TUEU IS $I8«FICJ»*T 1IFFOEXCE BETUEEN COKartMnB* 1 US COITlttU
» THERE IS SI6KIFICMT 3IFFE8EXCE BETUKN COMCENTRrtTIOH 4 MO COITBB..
«> THEM IS SI8MIFICAHT BIFfESENCS IETUEEN CONCENT!MTIQI 3 AMI CONTROL.
» THERE IS SIBNIFICANT DIFFEREHCE lETBEEl CONCENTfMTIOl 4 Mil CONTtOL.
I1PUT DATA TABLE
. tZOf.DM
1180.900
1340.000
1212.000
11 8*. 000
1204.000
82i.«06
428.000
814.000
493.800
414.0*0
413.08O
107.100
147.000
147.000
44.00O
40.010
49.30*
THANSFdRlEO DATA TASL.E
NOTE: TRAMSF08MTION HUMEX 3 UAS USES FOX THIS SET OF OAtA
3.012 3.014 2.917 2.J93 2.104
3.072 3.074 2.7f« 2.419 ' 2.147 !.
-------
2.4 Listing of Coaputar Program for Ounnett's Procedure
1,23)^131(30,22)
C
,43)
c
3,1.73,2.03,2.2,2.31,2.4,2.47,
1,1. 73.3.03,2.13,2.3,2.3»,2.4«,3. 31,1. 72.2.03,2.1S,2.3,2.3»,3.
*4«,2.31,1.72,2.03 ,2.1>,2.3,2.3«,3.43,2.3,1.71,2.a2,2.U,2.2»,2.3I
•,2.43,U,1.71,2.al.2.17,ZK2I,2J«,2.43,2.4«,l. 71,2.01, 2.17,2.27,
«3J«,3.43,2.4«fl. 71,3.01.2.17,2.27,2.33,2.42.2.47,1.71.2. ,2.14,2
•.27,2.3S,2.41^.4«,1.71,2.,2.K.2.2«,2.34,2.41.2.43,1.7,2.,2.H,
«2J*,2,3«,2.41,2.43,1.7,1.»,2.I3,2.23,2.33,2.4,2.43,1..7,1.»,2.
«13,3>21,3.33>3.4(3«43f1.7,i.3>,2.13,3.23,2'.33,3.4,2.43/
C . - " •
•3.M,3.«,3.2I,3.3,3.U,4.2<,3.M,3.C3,3.4«,3.37,3.29,4.9«.4.1,3.7
•l,3.4»,3.33,3.22,3.14,4,«4,3.9«,3.5»,3.3«,3.2,3.a»,3.01.4.73,3.W,
•3.«,3^2«,3.11.3..2.S2.4.«7,3.I,3.41.3.U,3.a2,2.32,2.«4,4.«,3.7«,
«3,3.24,3.Ql,2.««,2.74,2.«,4.43,3.3»,3^,2.9«,2.ai,2;7,2.«2,4.41,
•3.S,3.1«,3.»3,2.77,2.«4,2.5I,4J«,3.52,3.I3,2.9,2.74,2.«3,3.5S,
*4.33t3.49.3.1,3.*7.2.71.2,«,2.32.4.32,3.*7,3.a7,2.S4,2.S»,2.37,
«3,2.43,4.2«,3.4,3.01,2.7«,2.«2,2^S1,2.43,4.24,3.3«,2.M,2.7«,2.«,
•7.4S,2.41.4^3,3.37,2.M,2»74,2.»,2.47,2,3S,4.21,3.3S,2.3«,i.73,
«2.37,2.4«,2. 37,4.2,3.04,2^5,2. 71,2.3S,2.44,3.3«,4.1I,3.33,2.93,
n.7,2.34,2.43.2.33,4.17,3.32,2.32,2.«»,2.a3,2.«,2.34,4.1«,3.31,2.
•«l,2.«i,2ja.2.41.2^3,4.13,3.3,2.3,2.S7,2.31.2.4,2.32,4.14,3.3S,
«3.2«,2.»4,2.«3,i.4«,X.3«,2.2I,4.1,3.23f2.«3,2. 42,2.47,2.33,2.27,
•4.1,3.23,2.«3,2.a,2.4«,2.33,2.2«,4.0» ,3.24,2.84,2.41.2-43,2.34,
*7.23,4.0«,3.23.2.*4,2.«l,2.4S,2.J4,2.23,4.C»,3.22.2.a3,2.*,2.44,
«3.33, 2.24,4.07,3.22,2. *3,2.S9,2.44,2.32,2.24, 4. 0«,3.21,2.83,3.i»,
•J.43,2.33, 2.23, 4.0*,3.21,2-I2,2.M, 2.43, 2.31,2.23, 4.03,3.2.2.»1.
^.S1^2.42,2.3,2.23,4.03,3.2,2.«1.3.S7,2.42,2.3,2.22.4.04,3.2,2.«
: oaeset, axt. n? TO rssar onczxnnasaa
S '*•"-'
•I" AT 95% IS ""« Bf All, CASZS. '
! HttlCWIOT tSMBZOMOZCa ARE AWHABtZ TO OSE 3t>
1 TRASSK
0 HENS1
1 SJOHZ
3 ICC 1000'
122
-------
2.4 Listing of Program for Ounnett's Procedure (Continued)
s
•
MOf* J'
'OX UHCS CBP SOt JSB *•!• AMD +1* r
»nm 13 ____
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ta wnz(3,i3),i
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123
-------
— - 2.4- Listing of Program for Dunnett's Procedure (Continued)
VUSE (XBCSV tSMBKRODEII) •'
m ts.i-L.a3ta.
TWMCfn
IS ' **»f 1MIB
_
CD 90.I-l.CaCl
ig>*(i)
90 «_l_IIHli_B_
-i
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17(<3.CT.71
-------
2.4 Listing of Program for Ounnett's Procedure (Continued)
. i«ejs*)«)
s» **•&• '*••,*)
ss» icaitttr4.a)
* TOX stt
' r- •,»
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890 KMX ' T* MS.3)
a..'3 •
TOT*,1 KR TOO SET OF CM3L SB MDOHK BQIBOBiaiE 1HW OH S*
914
913 RtMKTC RXSdLTS OF SZgOZHTIXI. O O K P A
IRZSOV3M '
oo 4tt.z^.aaia
7(AaB(lCCX)-M
-------
2.4 Llstinf of Program for Dunnett's Procedure (Continued)
TJtAXSTOXMXO 0 A g A TABU'
_ _ ____. ._
TO* jcMec JOBS awnoHoaEH noon*,*),
1 SB
•
I •
.1
MlOMfff ««.«.•!»«,«, •««*!.•,3C,1 MDBCSJ.'
1.3X.1 OtfCr 917(0.09)')
u name HrnBr,43c.n,jg.2(n4.3,i»i)
T
1-
J54 tOC*,' '
i •
434 SOT
126
-------
2.5 Table of Dunnett's "t" values.
TABLE C.5. DUNNETT'S "T" VALUES1
^^w*
18
It
12
a
14
a
it
a
»
is
20
24
JO
4t
tc-
ii*
m
UM
U«4
UM
UM
US!
UH
l.M
UTS
UTT
UTS
1.75
UTS
UT4
UTS
UTS
UTS
UTI
UTO
UM
u«
UM
l.M
1.44
1.34
U2T
2.8:
1.1*
2.»
2.13
2.11
1.M
2.M
•S.OT
•2.M
!.«
1.04
2.01
2. OS
2.01
UM
un
UM
un
UM
1.M
2.U
1.U
2.41
1.2T
2.14
2.31
2.2S
2.1T
2. IS
2.14
1.29
2.21
2.21
2.20
tit
un
2. IS
2.13
2.U
2.M
2.M
(0*r
2.M
UT1
UM
ua
u»
2. IT
2.44
2.41
Ui»
U3T
2.3S
2.34
2.33
U3Z
U21
UJO
U2S
2.3S
2.2S'
2.12
2. IS
U14
•MUA4
• «.M
UM
i.n
2.71
UM
UM
2.M
2.11
UM
Ut*
U14
U~44
UU
2.41
U41
UM
U3t
2.3*
U33
U3I.
. *•**
*2.2S
ua
k.»
UM
un
UM
U74
UM
UM
UM
UM
UM
UU
UM
UM
U4S
U4«
U4T
U4«
UU
U40
U37
2.3S
U»
UM
2. IS
uos
UM
UU
un
UTS
un
UM
Ufl
UM
UJT
UM
UM
UU •
Utt
UJ1
UM
U4S
Utt
U3t
UJT
U34
U24
UOT
UM
UfT
uu
UTS
UTS
UM
UM
UM
un
Uft
UM
UM
UJT
UM
ua
UM
U4T
U44
U4I
UM
UM
U12
uot
un
UM
uat
' UTT
UT4
UTt
UM
UfT
UU
UM
Utt
Uil
UM
UJT
U14
UU
U4S
UU
ua
Vrom: Miller, 1981,
127
-------
U U.
06*
UJ
••• <§•• ^^ *»W W«W
s
ss. ss s:
*•* «•• »^
;s ss ss
•a2 ss" •£ •? 's '- r|
s= ss 22 S 5* S
53 S3 SS SS S3 ^S
S3 SS. S3 S^ «• =»
4*V «•* "t1* Wl* '•^ ****
S3 S3 SS S5 S3 SS
fiV •» •" •»•• *" ——
SS SS =3 S3 S3 SS
a
co
03
V.
U
(3
o
u
-------
LU *
uu.
a; a
LU
1:
o<
oi t—
UJ
osu.
UJ
a. (/i
ll
S5SS 5 2 3 S S3SS8
S3 SS SB S3.
33 SS SB SS
ss ss ss ss ss
SS S3 IS SS
!S SS SS SS
8S 2= S; SS
S3 SS S3 SS
SS SS SS SS SS
ss ss ss ss ss
jjj,- ,^ ,,-,-; jjfj
SS S3 SS 3B
S3 S3 IS 89
2B S3 SS SS
SS SS SS 5* BS
SS SS SS SS 28
SS S3 S3 SS
ss as -" ss
mm mm ^*> *•*•
SS SS SS S3
ss ss i ss ss ss
«»^ «n — J~* ^** *•*"*
SS SS 3S 55 SS
ss ss ss ss
«wt m*i ««•• «•••
SC SS SS SS
=s ss SB ss
«t*« «••• •«* «"••
SS =S SS SS
ss ss ss ss ss
•"* • •• "*• *•** "" *"
SB 3.B S3 S3 SS
ss ss ss ss
**+ 33 M** «•«••
«••» OT«* «•»» *•*%
SS SS S3 SS
••«• MM •••« ••••
S^ ^S» ••«• •««
5 -v*« .•««• ••••»
«••• «•«* «••* «tW
SS SB 33 SS
SS 33 S3 SS
S3 SS SB 33
SS S3* SS 3S
•iW «W W«* «•«•
SS S3 SS SS
' WW mm mm mm
ss ss ss ss
SS SB SB SB S3
mm mm mm mm ~m
ss ss ss ss SB
SS SS 2= SS S3 35 SS =S 2S
w« ww «•; «wi •»;«;«»•»••»•
-.» —• •« ••» **" «~ «2 "2 22
«•«• •»•! «?•• »r— ••-• ••• —• —• — •
W« M« «U «••• «i« •«» •« «w» «w»
K: ss ss ss ss ss ss ss ss
W-i •-» «w «i» •« <•« ••" "« «••«
?S SS SS 55 SS SS 52 SS SS
M« MM 1M W* «M •>» ••>•• «m IM
S3 55 3S SS SS SS SS SS KS
SS S3 SS SS 25 S? SS m S2
W»iW« WW W« •»••««•<
S«» "•• ••«• •«• ft* 22 SS Z± SS
•• *•* "t T^ T^ "^^ ^^ ^^ ^^
••*« «••• *••< «»^ i«^ m^ n^ «^ *i^
SS 3S 35 SS SS 3S SS S3 SS
S3 SS_ SS SS S3 S2 SS SS SS SS S3 SS
=3 SS SB SS SS S3 S3 SS SS. S4 SS SS
SS SS 8S'3=
2S 25 2S SS SS W SS 8
WW ••«• «t*W W^ m* *r* w» «r
••"• «•*• *•* *^^ ?^ 2!S SST 5
*« ^*» T^* T^ ^^ ^^ ^"* .
SS S3 SS
129
-------
U u.
ui
O.UU
I
oi t—
LU (/)
S
ocu.
LU
LU
S 3 g a g S 8 S 3 5 3 5 g
SS. 33 S3 S3 as SS SS SS SS SS 55 SS SS
SS S3 33 5
as
SS 35 SS S3
3S 33
cs ss ss 33 S 55 55 55 55 55 55 55 55
SS SS SS SS S3 55 S3 3S 33 SS SS 53 SS
S3 SS SS SS SS S3 35 SS SS 33 SS 53 S3
as ss
S3 S3 S3 SS S3 SS SS SS
S3 SS S3 SS SS
SS SS S3 S3 5S
35 SS 35 S3
SS SS SS SS
ss as ss ss
35 SS S3 SS
*m^ ss' ss ss ss
^^ ^^ ^*^ ^^ * •
SS SS SS 33 S3 .SS S3 33
•9^ ••«« aiB •<«
«r> QV> 3« S«
3** «• «*• <•«
• 3m ••• OK
••«• «'•• «M «•"
SS SS SS 35
S SS 8=
ss ss. ss as ss
a; ss ss sc ss
••• $•* ••• 22
«r* a^ a^ A*^
W«« ••« •** •^
SS SS 3S SS
«•«•• W« «**• «i^
£3 SS SS SS
S3 SS SS S3 25
f3 S3 SS S3 S3
SS SS 33 83 35
ss as ss ss as
ss ss ss as ss
SS SS SS SS
S SS
S= 83 S3 SS
2- 33 S3 SS
S.SS SS 33
5S 33 SS 35
•••% w^
-------
LU3
U U.
as 3
UJ
°-&
UJ (/»
-a.—
O.V)
UJ
UI59
o
UJ
03
*l«
333
8 8 § 5 S S § I
S3
*«•
SS
ss
••«*
«••
53 as ss
ss ss as
SS 53 S3
38 SS SS
3S 33 SS
•• «*. M
ss as
53 SS
*»^ *••
•••
as
as
«•<•
««
__
as
ss
s*s
as
as
mm
53
*•*
mm
ss
S3
st
•a M mfi mfi «••
S3 32 S3 S3 s;
S3 S3 S3 S5 S3 55
22 22 22 S3 S3
S3
n'W
33
33 SS S3
=S =3 S
«•» •»»
S3 83
S SS 38
'
S^* V«* *»*«
«• f»«* *•*• .
3 S3 SS 38
K 33 S3 83
3333 2383 S||8
131
-------
-------
APPENDIX 0
STEEL'S MANY-ONE RANK TEST^
1. Steel's Many-One Rank Test is a nonparametric test for comparing
treatments with a control. This test is an alternative to the Ounnett's test,
and may be applied to the data when the normality assumption has not been
met. Steel's test requires equal variances across the treatments and the
control, but it is thought to be fairly insensitive to deviations from this
condition (Steel, 1959). The tables for Steel's Test require an equal number
of replicates at each concentration. If this is not the case, critical values
can be approximated by interpolation.
Z. An example of the use of this test is provided below. The test employs
reproduction data taken from Table 2, Section 13, Ceriodaphnia survival- and
reproduction test. The reproduction data for 25X effluent were omitted from
the analysis because the results of Fisher's Exact Test indicated that a
significant proportion of the organisms died at that concentration. Since
mortality is a significant effect, the remaining task was to determine if
there were significant differences in reproduction at the lower effluent
concentrations.
3. For each control and concentration conbination, combine the data and
arrange the observations in order of size from smallest to largest. Assign
the ranks (1,2,3, ... 20) to the ordered observations (1 to the smallest, 2
to the next smallest, etc.). If ties occur in the ranking, assign the average
rank to the observation.
4. An example of assigning ranks to the combined data for the control and
concentration IX effluent is given in Table 0.1 below. This ranking procedure
is repeated for each control and concentration combination. The complete set
of rankings is listed in Table 0.2. The ranks are then summed for each
effluent concentration, as shown in Table 0.3.
5. For this set of data, we wish to determine if the reproduction in any of
the effluent concentrations is significantly lower than the reproduction by
the control organisms. If this occurs, the rank sum at that concentration
would be significantly lower than the rank sum of the control. Thus, we are
only concerned with comparing the rank sums for the reproduction of each of
the various effluent concentrations with some "minimum" or critical rank sum,
at or below which the reproduction would be considered to be significantly
lower than the control. At a probability level of 0.05, the minimum rank sum
in a test with four concentrations and 10 replicates per concentration, is 76
(see Table 0.4).
6. None of the rank sums in Table 0.3 is less than or equal to 76.
Therefore, none of the four effluent concentrations reduced reproduction
significantly below that of the controls, and the NOEC is assumed to be 12%
effluent..
^Prepared by John Menkedick and Florence Kessler, Computer Sciences
Corporation, 26 W. St. Clair St., Cincinnati, Ohio 45268; Phone 513-568-7968)
132
-------
TABU 0.1. EXAMPLE OF STEEL'S MANY-ONE RANK TEST: ASSIGNING
RANKS TO THE CONTROL AND IX EFFLUENT CONCENTRATION
Rank Number of Young
Produced
1
2
3
4
5
6.
6.
8
' 9
11
11
11
14
14
14
16
17
18
19
20
0
6
3
11
15
.5 16
.5 16
18
21 - .
22 .
22
22
23
23
23
24
25
26
27
31 '
Control or X Effluent
Control
IX
Control
IX
Control
Control
Control
IX
IX
IX
Control
IX
IX
-. • ' 1*
Control
Control
Control
Control
IX
TABLE 0.2.
Replicate Control
(Organism)
A
8
C
0
E
F
G
H
I
J
22 (11,11.5,13.5,13)
18 (8,6.5,8,9,)
27 (19,20,20,20)
16 (6.5,4.5,6,5)
16 (6.5,4.5,6,5)
0 (1,1,1,1)
8 (3,2,3,2)
25 (17,17,17,16.5)
24 (16,14.5,16,14)
26 (18*19,18.5,19)
22
15
21
6
23
31
23
11
23
22
TABLE
OF RANKS
Effluent Concentration
1%
(11)
(5)
(9)
(2)
(14)
(20)
(14)
(4)
(14)
(11)
3% 5&
25
25
20
24
15
23
21
22
20
18
(17)
(17)
(8.5)
(14.5)
(3)
(13)
(10)
(11.5)
(8.5)
(6.5)
23
16
7
13
26
22
21
21
21
21
(15)
(6)
(2)
(4)
(18.
(13.
(10.
(10.
(10.
(10.
5)
5)
5)
5)
5)
5)
12%
20
21
17
13
25
25
16
25
18
18
(ID
(12)
(7)
(3)
(16.
(16.
(5)
(16.
(9)
(9)
5)
5)
5)
Control ranks are given in the order of the concentration with which they
were ranked.
133
-------
TABLE 0.3. RANK SUMS
Effluent
Concentration
(X).
Rank Sum
1
3
6
12
104.0
109.5
101.0
105.5
TABLE 0.4. SIGNIFICANT VALUES OF RANK SUMS: JOINT CONFIDENCE
COEFFICIENTS OF 0.95 (UPPER) and 0.99 (LOWER) FOR
ONE-SIDED ALTERNATIVES
*
• ' 4
5
. 6
7
3
9
10
11
12
13
14
13
IS
.-.•••33' •
18
10
20
2
11
IS
f «
id
27
23
'37
32
49
43
03
50
79
71
97
37
110
105'
138
125
161
147
-ISO
170
213
193
241
223
272
252
304
282
339
315
ft -
3
10
17
26
22
30
31
43
42
02
53
77
69
95
35
114
103
133
123
•153-
144
182
167
209
192
237
219
267
243
299
278
333
310
number of treatmtnts (excluding control)
4
10
17
25
21
35
30
47 '
41
01
54
76
63
93
34
112
102
133
121
155
142
180
165
203
190
234
217
234
245
298
275
330
307
5
10
1G
25
21
: 35
30
46
40
60,.
53-
75
97
92
83
111
100
132
120
154
141
178
1G4
204
183
232
215
262
243
294
273
327
305
6
10
•- 10
-
24
_
34
29
46
40
. 59
52
74
66
91
32
110
99-
130
119
153
140
177
162
203
187
231
213
260
241
292
271
325
303
7
-
IS
24
—
34 .
29
45
40
59
52
74 "
68
90
31
109
99
129
118
152
139
178
161
201
186
229
212
259
240
2SO
270
323
301
3
—
16
24
—
33
29
45 .
39
58
51
73
65 •
90
81
103
98
129
117
151
133
175
160
200
185
228
211 .
257
239
288
268
322
300
9
—
15
23
—
33
29
44
39
53 .
51
72
65
39
80
103
98
123
117
150
137
174
160
199
184
227
210
250
233
287
2C7
320'
299
From Steel, 1959.
134
-------
APPENDIX E
FISHER'S EXACT TEST^
1. Fisher's Exact Test (Finney, 1948; Pearson and Hartley, 1962) is a
statistical method based on the hypergeometric probability distribution
that can be used to test if the proportion of successes is the same in
two Bernoulli (binomial) populations. When used with the Ceriodaphnia
data, it provides a conservative test of the equality of any two survival
proportions assuming only the independence of responses from a Bernoulli
population.
2. The data for this example (Table E.I) are mortality data from the
Ceriodaphnia survival and reproduction test, discussed in Section 13 of
this manual.
3. For each control and effluent concentration construct a 2x2
contingency table as follows:
Number of Number of
Observations
Successes Failures
Condition 1 a A - a A
Condition 2 b B - b B
Total a + b [(A+B) - a - b] A * B
4. Arrange the table so that A >. B. Categorize a success such that
a/A £ b/B. For this data, a success may be 'alive' or 'dead1 whichever
causes a/A >. b/B. The test is then conducted by looking up a value in
the table of significance levels of b and comparing it to the b value
given in tht contingency table. The table of significance levels of b is
included in this example, Table E.4. Enter Table E.4 in the section
for A, subsection for 8, and the line for a. If the b value of the
contingency taole is equal to or less than the integer in the column
headed 0.05, a/A is significantly greater than b/B. A dash or absence of
entry in Table E.4 indicates that no contingency table in that class is
significant*.
5. For the control and effluent concentration of IS the appropriate
contingency table for the test is given in Table E.2.
^Prepared by John Menkedick and Florence Kessler, Computer Sciences
Corporation, 26 W. St. Clair St., Cincinnati, Ohio 45268; Phone
513-568-7968.
135
-------
TABLE E.I. EXAMPLE OF FISHER'S EXACT TEST:
CERIOOAPHNIA MORTALITY DATA
Effluent
Concentration (%)
Control
1
3
6
• 12
25
No. Dead
1
0
0
0
0
10
Total*
9
10
10
10
10
10
''Total number of live adults at the beginning of the test.
TABLE E.2. 2X2 CONTINGENCY TABLE FOR CONTROL AND 1% EFFLUENT
1% Effluent
Control
Total -
Number-
A 1 Ti/a
A nve
10
3
18
of ...
ueaa
0
1
1
Number of
Observations
10
9.
19
6. Since 10/10 218/9, the category 'alive1 is regarded as a success.
For A = 10, B * 9 and, a = 10, under the column headed 0.05, the value
from Table E.4 is b * 5. Since the value of b (b = 8) from the
contingency table (Table E.2), is greater than the value of b (b = 5)
from Table E.4, the test concludes that the proportion of survival is not
significantly different for the control and 1% effluent.
136
-------
7. The contingency tables for the combinations of control and effluent
concentrations of 3%, 6S, 12% are identical to Table £.2. The conclusion
of no significant difference in the proportion of survival for the
control and the level of effluent would also remain the sane.
8. For the combination of control and 25% effluent, the contingency
table would be constructed as Table E.3. The category 'dead' is regarded
as a succes's, since 10/10 > 1/9. The b value (b * 1) from the
contingency table (Table E.3) is less than the b value (b » 5) from the
table of significance levels of b (Table E.4). Thus, the percent
mortality for 25% effluent is significantly greater than the percent
mortality for the control.
Table E.3. 2X2 CONTINGENCY TABLE FOR CONTROL AND 258 EFFLUENT
Number- of • Number of
Observations
Dead .• Alive •
25J6 Effluent 10 "- 0 10
Control 1 8 9
Total 11 8 '19
137
-------
TABLE :E-.4* SIGNIFICANT LEVELS OF B AND CORRESPONDING
PROBABILITIES1
-
A«3 B«3
A-41-4
3
A-3 B«3
4
3
2
A-d 3«4
S
4
3
2
A. 7 B-7
i
S
4
a
•
*
3
4
4
3
4
3
4
3
i
„
3
4
3
4
4
3
0
3
7
$
5
7
3
4
7
4
3
7
4
3
7
4,
7
9«J
94W
9 -m
9 «
1 4»
9-4M
* ^^
94*1
9«t
9-Mt
2 4»
1 «•
9 •*»
9 411
9 MfT
't 4a
9 -a*
9 4"
9 «•
9 4*
3
•^B
9 •««•
_
^ ^
—
1 «a
9 on
___
9 •»
— »
9-OM
"^
^
0 -Mi
^~
•^M
«.
^
«•»
A-« a.<
7
*
3
4
3
2
A-9 B-9
I
7
4
<
f
7
4
3
4
1
3
7
3
f
7
3
S
7
5
I
7
1
9
S
7
J
4
9
S
7
6
3
9
t
7
S
9
S
7
4
3
093
4-0*
2 -at
t -at
9«u
9 -at
3-ai
t -m
9 4I»
1 «*
9«
2 -a-
1 4B
9-«*
9 -ft
I •«•
9 •«••
9 •«*
9 4M
9-a»
9 -a
5-rn
2 -as
9 4U*
0 -*H
4 4V
3 •«>
2 *•
I -a*
9-Oi
3 «*
2 -a*
I -a*
9 ••»
3 -M*
I -ur
9 -M7
9 -M
ftab
O025
3 -MI
2 -a>
1 «•
9 4O
•^
2 4V
1 40
9 4M
9
—
2 4»
1 4^
9 •«•
^*
1 4in
9 ••»
94W
I 40t
9 4W*
9 4«
— •
9 4M
—
— .
3 -M-
1 -w
9 «*•
...
^—
3 -••
1 -on
9
—
_
3 •<«•-
t «
9 -m
9 «r-
^^
24H
1 -OB
9 4B
«••
2 4tr-
9 -an
9 40
—
1 IB
9 -M
_
"•
table shows:(l) In bold type, for given a, A and 8, the
value of b (
-------
TABLE E.+. SIGNIFICANT LEVELS OF 8: VALUES OF 8 (LARGE TYPE
AND CORRESPONDING PROBABILITIES (SMALL TYPE) (CONTINUED)
A-9B-5
4
3
2
A-'0-'°
4
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*7 t .
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TABLE £.4."^ SIGNIFICANT LEVELS OF 8: VALUES OF 8 (LARGE TYPE
AND CORRESPONDING PROBABILITIES (SMALL TYPE) (CONTINUED).
A-ll B»«
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140
-------
TABLE E.4, SIGNIFICANT LEVELS OF 8: VALUES OF 3 (LARGE TYPE
AND CORRESPONDING PROBABILITIES (SMALL TYPE) (CONTINUED),
\-U 1-13
12
11
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1 40
-------
TABLE 6**V:." SIGNIFICANT LEVELS OF 8: VALUES OF 3 (LARSE TYPE
AND CORRESPONDING PROBABILITIES^SMALL TYPE) (CONTINUED)
-
a
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142
-------
TABLE E.4. SIGNIFICANT LEVELS OF B: VALUES OF 3 (LARGE TYPE
AND CORRESPONDING PROBABILITIES (SMALL TYPE) (CONTINUED),
A«U 1-14
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*****
-------
APPENDIX F
PROSIT ANALYSIS^
1. -PROSIT ANALYSIS OF FATHEAD MINNOW EMBRYO-LARVAL DATA
1.1 This program calculates the LCI value and associated 95* confidence
limits using Probit Analysis. The program, written in FORTRAN PLUS IV
v.3, was adapted by James Dryer, EMSL-Cincinnati, from a program obtained
from Ors. Wesley Birge and Jeffrey Slack, Graduate Center for Toxicology,
University of Kentucky, Lexington.
1.2 The program runs on the Digital Equipment Corporation, PDP-11/70 -
computer under IAS version 3.1. Machine readable copies of the program
and assistance in running it can be obtained by contacting James Dryer,
EMSL-Cincinnati Newtown Facility (FTS 778-8350; comm'l 513-527-8350).
1.3 Sample Data Input and Output from the Computer Program
1.3.1 The data input process and the output provided by the computer
program are illustrated with a set of mortality data from a fathead
minnow embryo-larval survival and teratogen.icity test.
The program begins with a request for the following information:
1. The number of concentrations.
2. The number of test organisms exposured in the control.
3. The number of test organisms that died in the control.
The program then requests information on each toxicant concentration:
1. The concentration value.
2. The total number that died at each toxicant concentration.
3. The number of test organisms exposed at each toxicant
concentration.
Probit statistics are then listed for the analysis, including a table
which provides the LCI value and confidence limits.
1Prepared by James Dryer, Aquatic Biology Section, EMSL-Cincinnati
Newtown Facility, U. S. Environmental Protection Agency, 3411 Church.
Street, Newtown, Ohio
144
-------
1.3.2 Sa«pl« Oita Input and Output from the Computer Program for Probit
Analysis of Fathead Minnow Embryo-larval Data
PROBIT ANALYSIS***MAXIMNM LIKELIHOOD SOLUTION
USED FOR CALCULATING LCI S» . ,. . LC99 S
MAXIMUM NUMBER OF POINTS IS 20.
NUMBER OF CONCENTRATIONS = 5
'•CONTROL SAMPLE SIZE » 100.
NUMBER OF HEAD IN THE CONTROL GROUP17.
8ESIN INPUTTING OF DATA WITH LOWFST CONCENTRATION
CONCENTRATION - 3.
NUMBER RESPONDING (BEAD) = 14.
NUMBER TREATED =* 100.
CONCENTRATION =5.
NUMBER RESPONDING (HEAD) = 1/5.
NUMBER TREATED = 102.
CONCENTRATION = 7.
NUMBER RESPONDING (DEAD) = 35.
NUMBER TREATED - 100.
CONCENTRATION =11.
NUMBER RESPONDING (HEAD) = 72.
NUMBER TREATED =99. '
CONCENTRATION = 1<5.
NUMBER RESPONDING (DEAD) = 99.
NUMBER TREATED = 99.
INPUT CONCENTRATION SCALE IS TRANSFORMED TO LOG(10)
145
-------
1.3.2 Sample;data Input and output from the computer program (continued),
LOS
CONCENTRATION SAMPLE
0.4771
0.8451
1.0414
1.2041
CONTROL
10O.
102.
100.
99.
99.
100.
PROSIT TABLE..
OBSERVED EXPECTED
14. .
35.
72.
99.
17.
DEVIATION PROBABILITY
15.38.
17.79
31.32
74.43
9A.37
15.57
-1.58
-1.79
3.48
-4.45
2.43
1.43
0.1558
0.1744
0.3132
0.7720
0.9734
0.1557
THP CONSTANTS USED IN THIS PROBLEM WERE5
HETEROGENEITY FACTOR » 1.5884
NUMBER OF POINTS - 5
DEGREES OF FREEDOM - 3
DEVIATE * 1.9400
8 > 0.0515
THE TOTAL NUMBER OF CYCLES- 2.
THE STATISTICS ARE}
' AVG Y 5.3497
. . ..AVG X 1.0059 "
AVG T 1.4983
NATURAL MORTALITY 0.1557
SLOPE 9.0354
INTERCEPT -2.7134
CHI SQUARED 4.7458
S£
SE
0.0229
0.93O4
POINT CONCENTRATION
LCI 4.4817
LOWER UPPER
9SZ CONFIDENCE LIMITS
3.4430
5.5047
NOTE:IF THE LCI is ABOVE/BELOW THE HIGHEST/LOWEST CONCEN-
TRATION TESTED* EXTREME CAUTION SHOULD BE EXERCISED IN
REGARD TO USING SUCH VALUE BECAUSE IT LIES OUTSIDE THE
KNOWN RANGE A*0 IS ONLY AN EXTRAPOLATED ESTIMATED VALUE.
SIMILAR CAUTIONS SHOULD ALSO BE USED IN REGARD TO THE
UPPER AND LOUER 9SZ CONFIDENCE LIMITS FOR LIKE REASONS.
FOR FURTHER CLARIFICATION. PLEASE CONSULT YOUR QUALIFIED
STATISTIC!***. .
146
-------
1.3..3 Listing of Computer Program for Probit Analysis of Fathead Minnow
Embryo-larval Data
SGSZCXF SSffi* (A-a.O-Z)
nQEBiar JMT(U) ,S3(20) ,aW3ES(20) ,SWSI(20) ,VEE(13)
ansfflzar acpz(20) ^993(20) ,^999(20) ,214(20) ,ros(is)
BHS>(20),(2132(18),
X(20),»(20)1
c
c
c
c
c
IHfUHTlH OF(1) ,02(1)
OP,OI/0.01,2.6737/
-**! VMUES 9SCB&D.
HOTS; THIS VERSION WRITTBK 9/16/83
CMDk TOS,CS1SO/12.70«,*.3CO,3.132, 2.776, 2.571,
* 2.447,2.353,2.306,2.262,2.228,2.201,2.179,2.160,
* 2.143,2.131,2.120,2.110,2.101,3.341,3.391,7.313,
* 9.483,11.07,12.592,14.067,13.307,15.913,13.307,
* 13. 673,21.026,22.362, 23. 633,24.99«,26.a9«r27.5a7,
* 23.3fi9/
100 TDK*,
T5SB*,
TXEC*/
T2ES 2
2 Pdaflfff' 2CHSES OF
ACCSPI 3,N
3 KSSOI(I2)
THE 4
4 IQEKXCC Gd/iHGLi SSMHE
10
WMSCR OF KOftS IS 20.
',$)
TO 1
1TOB 3
5 BJgag (' HOMEEaOFCEAO IK OB CCMIEGL GBOOF « ',$)
03 ID 200
192 SO ID 9999
200 HCG- 1
OO TO 260
230 03 250 J - 1,
H(J) --
230 X(J) " ESOS10WJ))
,280,270
JB 2
280 ICZCI^-1
lMf-2
270
GOTO 290
280
BIGCXI.CO
147 .
-------
1.3.3 Ltstlnig of Program for Probit Analysis of Fathead Minnow
Bnbryo-larval Data (Continued)
290 00 TO (300,320), XCOS
300 TSB*, ' ' _ _
TOE*, ' "o uiujt nocniHs 07 DMA HUH THE IOOST
03 330 J-1,H
TZ8B 313,
311 B3WKTC OSHCnnHMnar * ',$}
£0399 312,X((oasL.zz.o ) .OR. (icrct.Gz.of ) 0010320
CEOKKOOO
03 313 I-1,H
S3 TO 313
315 "*"''"'*
rF(nQc«.aa.o) GO TO 320
320 IPCOID.IX'.l) 03 TO 230
DO 400 I-l.K
6
IP (ICZtt) 350,390,390
350 3?(SMtt»(D .12.0.00) GO TO 330
13"(afltt3?(I) .GS.1.DO) O3 TO 340
03 TO 380
330 SMttS(I)«.
03 TO 380
340 SMOS(D-.9999EO
330 <
. 03 TO 400
3se>r-
400 <
C
C
C
c
S»0>O.DO
snoaiM.oo
SWOOO.DO
148
-------
-------
1.3.3 Listing of Program for Probit Analysis of Fathead Minnow
Embryo-larval Data (Continued)
SMflM.GO
. oo 470 X-1,H
S>OSSZ(X) -5.BO
nr
GO ID 431
430 »•( (0.000043063aPO**gH}.000276SS72PO) *»M. 000332014300) *Mft-O.Q09
A&-.5DO-.5IX/ ( ( (AI*AHK 042282012300) **». 070S230784CO) *A»1.DO) *•!
16 _
431 BBSP(I)».3DO+CSIGS(AI,£D)
440
!?( (BES?(Z) .ZZ.1.00) ~AMD. (DRBS(H5) .12.13.00) ) GO TO 441
WM^J.OO . • '
00 TO 442 -
441
442
HfflDWOCK(I)
sar -
3U&&K&BIHX
I? (ZCZCO 470,450,450
450
460
470 CCNCfflOS
IT(S»l.aj.O.DO) CD TO 520
471
&((atOX.ZQ.O).CR.(S»OGr.SQ.O)} GO TO 520
EHEW>€f*Qa/SltCCC
SB-OKEX
IFOflttO 471,472,472
149 :-
-------
1.3.3 Listing of Program for Probit Analysis of Fathead Minnow
Efflbryo-larval Data (Continued)
0*5.03
CZ2*>0«00
472 If (1CXCL) 480,500,500
490 00 490 1-1,21
490
GO TO 320
500 XF(satq 520,510,510
510
GUI-LOO/ (swcc-saonysanr*swca)
502
520 00 530
530
g(TgSr)540,550,550
540 fSSX^^CEStS
550 IF(XCSe&-100) 560,580,580
561 IF(TZ3T.IZ.CQ001EO) GO TO 610
GO TO 570
565 X?((TE5T.XZ..01DO).aR.(CaBS(BNE»-B8).£r..OOSQO)) GO TO 610
570 &BKSSI
GO TO 320
580
590
600 BSSBXC49H 100 CK32S HAVE BEEN CCMETEIED. 3(100) - 3(99) -,F14.7)
C
C
C rXTCTTy* r~"'*^i 2HD •'•"«'• FOR »«i-iTMiS%i'.i-'iV-
c
610 D-3
X7(2CaS3*Q) GO TD 320
zr ( sso.flR.nrreoxK)) GO TO 650
649 GO TO 820
650 W03B(5,660) XSQ
____ _ _ ^^^
640 JCBHK (47H
-------
1.3.3 'Ltsffiiif o? Program for Probit Analysis of Fathead Minnow
Data (Continued)
C
C SB? OP C9SS9NIS AHD ULMHUS. S393XSCCC3.
rFdCRIrfSM?) 830,330,330
330 WH2{5,340)
340 £CRKC( 26H NOdQUi'ICAOT HBSBESSSCN/)
350
DO 980
g(CBKCT .Z2.0.00) 00 TO 360
g(QB8Bf (CT) .CS.1.00J 00 TO 370
GO ID 920
860
_ GO TO 920
370
920
'
TOB*,' •
TBB*,'
XFCHOG.aa.2)
929 B3RaZ(fiC,3HC
W03S(5, 330)
1 ggg(J) ^j.,^
930 futouJCUC, l.lff ,tt!LiLMiliAlIIlJT,I^,SHSflMRZ>gyf
ID ,7X,?aa
PRO BIT TA-BI,a«
oa ro 940
_
T3Q3B(S , 933) GZISIi, COO, "SBSX, 3,5X£G
1
935
_
950 PCBMKT(4iaOIHB OONSBanS- IMfel) IN '1H13 ESOHtaC
«O3Z(5f360)HSr
960 ler^>nnt(*y, J JUUMI ym iJi.'Mgr'ffy FACTOR »,J9.4)
WaZE(5,970)H
970 !aWKP(iar,19HHCKBCEl 0? FOZHS - ,13)
!XQ!B(5,980)S
980 FOHJKTO^iaBnEXSEES OF ESEED3C - ,13)
990 B3WW(13X,3H3E7iaiE -,£9.4)
* ~ «B3B(5,1000)G
uoa
-' •- - •
wia KsaKctsaa IBE rorai. NUMBER or cxci2s-,i4,iH.)
940 nrd-GT.O) WRECE(5,94S)
945 ZCROS(3aO,48Xr '*-£XI®Cm3 I2SS ISftH 5 '/49X, <**>£S&CTED tCRS
1020 FOraai(22B3IHE S
151
-------
1.3.3 Listing of Program for Probit Analysis of Fathead Minnow
Embryo-larval Data (Continued)
1030 icaag(2af7gBic T -,212.4)
mis(5,io40)xsxac
1040 ICSKM(213C,7HXVG X - ,112.4)
10SO KHflg(23X,7HM?S T - ,512.4)
HQ2E(5,1060)BXBe,SEC
loco IOWMOX, isaoacsxL icRjacnz -,512.4, 4x,4HSB - ,Pio.4)
HBnE(5,1070)BKar,SEB
1070 KR9S(21X,7E5USS - ,F12.4,4iC,4BSB -,510.4)
W03S(5,10SO)A.
1080 IOH«C(I7X,llSniTEBC2E!? -,F12.4)
303E(5,1090)3SQ
1090
_
1095 FCE39£C( 'Q*G3£ aquflRED IS CTQOjTQHr. ^i-i-i.nnJMM||i|il|y E&JIUK IS
1 HOT I KID. CSS
TXH*,'
1
iioo
c _
13025(5,387)
987 rcswxrr • 95*
_
OLSUU1Z J;lHX.'LATi
GO ID 1150
U10
GO TO (1130,1120)
1120
1130 I?(HI.GI.X1*(II))A999(J)«3P(1)*100
1140 HS«XC(S IC1,4X,1 ',FlS.4,5XfFlS.4r3X,?16.4)
GO TO nso
1150 GO TO ,(1170,1160) ,HSS
1160 aMSO?(2.30253SOa*EH)
1170 wnas(5,nso) CP(D*IOO.,EH
LUM ICKBOrC tC',23.0,iaC,nS.4,aX, • aSCALCUL
lisa Tgs», '
1— - '
ins*,1 •
BOB*, ' • _ _
T&fi*, 'NOIKIF *Pffl! Tm XS ABCVE/EEICW THE HKjHEST/ICSffigr
'i'iltB*, "CaZECM 'in.vivj>f EOEEME cai/riLH *iHniJIJ) BE EXERCISED DT
T2HE*, 'BSSKEO TO US2HB ^.^^ VMDE HECKDSE TT TilTB W»JITI 1 1 f. TBS*
TXPE*, "SNCMJ SSNCSB AND IS CttlX AH SCTRAPOraiCED SSTIMRISO VAZDE. '
n CBDnCKS aCULD AtSO BE USED IH REGSVBD TO THE'
MO ICWER 95* CCNFUENCS TTHTT3 FOR USE SEASONS. '
152
-------
1.3.3 Listing of Progran for Probit Analysis of Fathead Minnow
Bafcrya-larval Data (Continued)
TSFE*, 'SCR gOigSgt QiSSKXCSSS, EUaSB tlHSU'luT YQCR
,
T2BB*,1 •
• HRnZ<3,9991)
9991 FCEHK
9999 STOP
HMD
i IHK'I'll H iMJfiill*8vii«jitJ
153
-------
2. PROBIT ANALYSIS OF ALGAL GROWTH TEST DATA
2.1 This prograa calculates the EC! value and associated 95% confidence
limits using Probit Analysis. The program, written in FORTRAN PLUS IV
v,3, was adapted by James Dryer, EMSL-Cincinnati, from a program obtained
from Ors. Wesley Birge and Jeffrey Black, Graduate Center for Toxicology,
University_of Kentucky, Lexington.
2.2 The program runs on the Digital Equipment Corporation, POP-11/70
computer under IAS version 3.1. Machine readable copies of the program
and assistance in running it can be obtained by contacting James Dryer,
EMSL-Cincinnati Nevrtown Facility (FTS 778-8350; comm'l 513-527-8350).
2.3 Sample Data Input and Output from the Computer Program
2.3.1 The cell counts are input to the program. (Counts obtained with
the Coulter Counter, must first be "corrected" for coincidence.). The
program determines the mean count for each concentration, divides each of
the treatment means by the control mean, and subtracts the quotient from
1.0 to form an inhibition proportion for each concentration. The
proportions are arbitrarily based on a population size of 100.
154
-------
2.3.2 SMffi- Data Input And Output From The Computer program for Prooit
Analysis f£jft1gaiT Growth Test Data
PROSIT ANALYSIS-USED FOR CALCULATING THE ECU (A.1 GAL TEST)
EXCLUDING CONTROL* NUMBER OF CONCENTRATIONS » 3
NUMBER OF VALUES FOR CONTROL » 3
PLEASE ENTER THE 3 CORRECTED COUNTS IN CONTROL.
CONTROL COUNT » 1209*
CONTROL COUNT * 1180.
CONTROL COUNT » 1340.
BEGIN INPUTTING OF DATA WITH LOWEST CONCENTRATION
CONCENTRATION » 5*
PLEASE ENTER THE 3 CORRECTED COUNTS IN CONG 5.00
COUNTS «1212.
COUNTS »1136.
COUNTS » 1204.
CONCENTRATION a 10.
PLEASE ENTER THE 3 CORRECTED COUNTS IN CONC 10.00
COUNTS »S26. , .
COUNTS =628.
COUNTS
CONCENTRATION = 20.
PLEASE ENTER THE 3 CORRECTED COUNTS IN CONC 20.00
COUNTS -493.
COUNTS »416.
COUNTS »413.
CONCENTRATION * 40.
PLEASE ENTER THE 3 CORRECTED COUNTS IN CONC 4O.OO
COUNTS i!27. •
COUNTS »147. .
COUNTS »147» v ;
80.
PLEASE ip THE 3 CORRECTED COUNTS IN CONC, 30.00
COUNTS «*?t»3
COUNTS' *4
-------
2.3.2 Safflplt'Oata Input And Output From The Computer Program for Probit
Analysis of Algal Growth Test Data (Continued)
NOTE» CHI SQUARED ESTIMATE QBTAINEDJY SUBTRACTION
PROBIT
CONCENTRATION PERCENT
5.0000
10.000O
20.0000
40.0000
SO.0000
3.41
39.13
64. SS
88.71
96.43
TABLE
PROBABILITY
O.OSS1
0.3090
0.6337
0.8866
0.9804
THE CONSTANTS USEB IN THIS PROBLEM WERE?
HETEROGENEITY FACTOR
NUMBER OF POINTS
DEGREES OF FREEDOM
DEVIATE
NUMBER OF CYCLES
THE STATISTICS ARE»
AUG X
AVG T
SLOPE
INTERCEPT
G
CHI SQUARED
2.7298
5
• 3
3.1820
'• 9.
S.0917
1.2082
•1.9104
2.8363
1.6650
0.0555
8.189?:
SE
0.2100
POINT ' CONCENTRATION
EC1 2.2681
LOUER
932
1.1959
UPPER
LIMITS
3.41S1
r - SETS
STATISTICIAN.
156
-------
2.3.3 Listing Of Computer Program For Probit Analysis Of Algal Growth
Test "-"
BXPg(30) ,A998(20) ,A999(20) ,3X4(20) ,103(13)
HB»(20) ,0130(13), OBSf (20) ,2B>(20)
CttHBSHnr X(20) ,27(20) ,3(20) ,3012(20)
sc(20) ,21(20) ,ss<20) ,
c _ _
C T AIB < HTSU 7UQES U1UMEL).
C-
C KOTB; THIS 7BR3IOM VTaiTtSH 9/1S/3S
C
c •
ami T05,CaiSO/13.706,*.303,3.132,2.T7(S,2.571r
* 2.*47,2.3ffl,2.30«,2.2a2,2.22a,2.201,2ol79,2.160,
* 2.143,2.131,2.120r2.UO,2.101,3.a41,5«991,7.3ia,
* 9.438, 11.07,12.392, 14. 087, 13.5O7,lS.9i9,lS.307,
* 19.S7S,21.02e,22.3S2,23.S8a,24.S9«,26.29«,27.S87,
* 28.369/
100 rag*, • • _ __
11! 'SBOBZC AtOffiZSXS^SSD ICR
TSE 2 _ _ .
2 JdMKTC BXtHTUIlHB OUMUUL, NDMBHR OF CTNCNtSMlCBS » ',$!•
MTfflT 3,H
3 IdSai(12)
T2BE*,1 '
TOES
5 K3MKCC MOHBEa.OF VamS BQR 03nBOL - ',$)
ACCSET S,itt
S 5T3IWM(13)
TSSB*,' ''
_
srsass ansa isB',13, ' m^""*'1"' OSOHIS DT
TSSB*,1 *
03 10 a*i,ia • .
TSE 501
501 KEMXTC aanoL asmzr - (,S)
10
TSSB*, ' •
200 nos - 1
GO TO 260
230 T2EB*, ' '
• SUM
see -2
260 XG2O>-1
SOtttXi.
521
(33 10(300,320) ,gQS _
300 THE*,* BEBOf IKtUl'illHB OF QXA HUB ICWEST
157
-------
2.3.3 Listing of Computer Program for Proo it Analysis of Algal Growth
Test Data (Continued)
00310
TH5311
3U KHflttC oaKsanaasar • ',$)
tcesss 3U,z(j)
312 KSK«(?33.13)
XL4(J)-X(J)
X(J)-£OS10(X(J))
_
314 JCSKHC HZRSS UEBSt CSS' /X3 , ' "mm"-!i'»in uJUWiS IN OCHC* ,F7.2)
DO 313 I»»1,KL
TOB 316 '
316 PCRflSC 032ns -',$)
JfXSSS 322,H7(S)
313
TZEE*,' '
DO Z31 I-1,H
TZHS*,' !
231 (INITHOS
320 1F(11O.IS.1)GO TO 230
DO 400 I-1,N
,390,390
350 U(£ZnU>(I).I2.Q.) GO TO 330
IF(SKMP(D.Gg.l.) <30 TO 340
GO TO 380
330 sax?(X)-.aooi
GOTO 330
340
380
GO TO 400
390
400
C
C
C
C
SNDM.
at«-o.
SWE-0.
swnx.
DO 470 3
IF(AB3(lD)-ia.) 401-,401,402
158
-------
2.3.3 Listing of Computer Program for Proolt Analysis of Algal Srowth
Test
.'•,:.;. QQTD 403
403 a».H>13
403 XT (ZC3CL} 410,420,420
410 Bffi? (I}-Sai»<(0.000043063a*«HW.000276S«72)*MW.0001S20143)*»H).OOS
1270073
AI-.5-.S/ ( ( (AI*A». 0422820123) *ABK 07QS230784) *Jfflfl. } **lfi
431
440
) 00033441
-
00 TO 442
441
442
SS*f • SHK 4- WT
- SRHO; *
ZT (1OCD 470,450,450
450 g(SHKtC) 470, 460, 460
460
SHHTNSMIPW5W
470
IT(S(«.aQ.O.) GO TO 520
IT((SStOOCJQ.O).CR.(SSMXZ.aQ.O)} GO TO 520
471,472,472
471
472 IT OCSffi) 480,500,500
4aOD0490>l,M
490
159
-------
2.3.3 Listing of Computer Program for Probit Analysis of Algal Growth
Test Data (Continued)
00 TO 320
500 XF(SOXC) 520,510,510
510
512
c __
C* QuKCtTOB ZSQ^SCZSO
C TEST ABS(B(I+11-B(I)-.OCXM1)
C TUST* T^P^Tr ** 100
c
520 DO 530
530
,550,550
540
550 iy(IC£CL-100)5«Or530,5aO
560 r?(SMMC5581,5iS5,565
551 XFCBSr.ZZ. 00001) C» TO 610
GO TO 570
563 TF((T25T.I2.. 00001) .CR.(ABS
-------
*»•«,•- '
2.3.3 Listing of Computer Program for Probit Analysis of Algal Growth
Test Data (Continued) •
8SO-
00 930
.Z8.o.) aa TO 8«o
IT(CBSZ(J} .08.1.) CO TO 870
00 TO 930
980 OBSr980)K _
980 reBMM?(8X,aiHISBaES OF numm** » ,13)
990
940 WffiB(5,10XO)IC2CL
1003
1010 PCraar(3X,2CH NDMEER OP G2OE3 -,I4,E.)
1020 KBtfljCC^HJIBK S33KCESXXCS US' }
1030 Kagg(21X,7HKBC 7 -,J12.4)
1040 CTBjgtaa^7H3E8B X « ,F12.4)
r(2JX,7H«« T - ,312.4)
,H2.4,4X,4ffiB -,F10.4)
3,2QSO)A
5,1000)G
1X380 IC««(17X,llHINIH?CEPr -,£12.4)
1090
.
1
________
1105 *uuaii(5,liOO)
1100 ICEH»(5X,£HB30OT,fiX,13SaXC3mtaZtCH,12X,SH]X»^
161
-------
2.3.3 Listing of Computer Program for Probit Analysis of Algal Growth
Test Data (Continued)
c .
wosE(5,9«7}
9*7 KSaOTC 95* OCNRD
• Tag*,' ' _
C G3SSOIK KM ' F ' f7ff!f
OENJ.OI
IFOSHn.IS.0.) GO TO 1120
1110
CO 7X3 (1130,1220) ,HCG
1120
U30
1140
1220 GO ID (1170,1160) ,1X05
1170 ra035(5,iiaO) EM
nao
12SO «KHBV-
1
TZIBV '
TOPS*, 'IIQIE: n* IBS EX3. •gams IS ABOVg/BEZCW TSB
, BflBEMS Cai/l!iLM SHXHD BE
, '127 KEGRED ID SOS VAIOE BEOnSS IT IHS (JUIMIIK IBS
TSE», •IONIS AND IS COS AM SOSABOLftlZD ESTmJOED VMBE.STMHAR1
TZHE*, 'aurrcKS saroojvcso SB HSEP nr SExaso TO IHE crest AND'
T2EB*, 'ICHSH. 95> CLttflTEMCE UHHS FOR UTO HEftSQS. SHJJID EUR-'
BE u»jiim.nrgrgagg qjfcjJU TCOR
.-1-i.-fi-ir-i -
TZIS5*/1
9991 TCBSKSV/////////////////////////-)
9399 STOP
BESL
(A-H,0-Z)
1EEMJWIHfiu:(2.S15517-«EMP»(0.3028S34lEEHP»0.010328))/
(l.-rtZ!ffi»(1.432788«H!P*(0.189269«H
-------
TECHNICAL REPORT DATA
(flea* rttd iHsaucriara on rfit reverse lit fort committing)
1. REPORT NOv
EPA-60Q/4-35-Q14
3. RECIPIENTS ACCESSIONINO.
4. T1TUZ ANDSurriTCK
SHORT^EMlsiCtHODS FOR ESTIMATING THE CHRONIC
TOXieiT£0£-EFFLUENTS ANO RECEIVING WATERS TO
FRESHWATER ORGANISMS
8. REPORT DATE
1985
. PCRPORMINQ ORGANIZATION CODE
7. AUTHORiB)
8. PERPORMINa ORGANIZATION REPORT NO
William B. Horning, II and Cornelius I. Weber
9. PSRPORMINQ ORGANIZATION NAMI AND ADDRESS
Environmental Monitoring & Support Laooratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
10. PROGRAM CUEMfNf N6.
AAPB1A
11. CONTRACT/QHANT NO.
12. SPONSORING AOSNCV NAMC AND ADDRESS
Environmental Monitoring & Support Laooratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPf O* PWORT ANO ^tRIOO COVgH6O
Innouse
14. SPONSOMINQ AGINCY COOC
EPA/600/06
15* 3UP~U«M*NTAnY NQTIS
Companion document to EPA/4-85-013, Methods for Measuring the Acute Toxicity
of Effluents to Freshwater and Marine Organisms.
This manual describes snort-term (four- to eight-day) methods for estimating
the chronic toxidty of effluents and receiving waters to a freshwater fish, an
invertebrate, and an alga. Also Included are guidelines on laboratory safety,
quality assurance, facilities and equipment, dilution water, effluent sampling
and holding, data analysis, and organism culturing and handling-. Listings of
computer programs for Ounnett's Procedure and Prooit Analysis are provided in
the Appendix.
7.
KIY WORDS ANO DOCUMENT ANALYSIS
OMCRirroRS
b.lDCNTtFISRS/OPeN ENDED TERMS
c. COSAT) Field/Group
Ecotoxicology
Effluents
Receiving Water
Tox lefty
Bioassay
Industrial Waste
Domestic Waste
Fish
Invertebrates
Algae
Freshwater Biology
Marine Biology
6C
3. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This JteponT
UNCLASSIFIED
21. NO. OP PACES
161
2O. ScCUmTT CUAS5 (TrtU
UNCLASSIFIED
EPA Form 1220-1 73)
163
-------
;'* "=* *S
------- |