ซ>EPA
United States
Environmental Protection
Agency
Research And Development EPA-600-4-91-002
(MD-591) July 1994
Short-Term Methods For
Estimating The Chronic Toxicity
Of Effluents And Receiving Water
To Freshwater Organisms
Third Edition
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DISCLAIMER :
This document has been reviewed by the Environmental Monitoring Systems
Laboratory-Cincinnati (EMSL-Cincinnati), U.S. Environmental-Protection Agency
(USEPA) and approved for publication. The mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
The results of data analyses by computer programs described in the section on
data analysis were verified using data.commonly obtained from effluent
toxicity tests. However, these computer programs may not be applicable to all
data, and the USEPA assumes no responsibility for their use.
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FOREWORD
Environmental measurements! are required to determine the quality of ambient
waters and the character of waste effluents. The Environmental Monitoring
Systems Laboratory-Cincinnati (EMSL-Cincinnati) conducts research to:
Develop and evaluate analytical methods to identify :and measure the
concentration of chemical pollutants in drinking waters, surface waters,
groundwaters, wastewaters, sediments, sludges, and solid wastes.
Investigate methods for the identification and measurement of viruses,
bacteria and other microbiological organisms in aqueous samples and to
determine the response of aquatic organisms to water quality.
Develop and operate a quality assurance program to support the
achievement of data quality objectives in measurements of pollutants in
drinking water, surface water, groundwater, wastewater, sediment and
solid waste. .
Develop methods and models to detect and quantify responses in aquatic
and terrestrial organisms exposed to environmental stressors and to
correlate the exposure with effects on chemical and.biological
indicators.
The Federal Water Pollution Control Act Amendments of 1972 (PL 92-500), the
Clean Water Act (CWA) of 1977 (PL 95-217), and the Water Quality Act of 1987
(PL 100-4) explicitly state that it is the national policy that the discharge
of toxic substances in toxic amounts be prohibited. The detection of
chronically toxic effects, therefore, plays an important role in identifying
and controlling toxic discharges to surface waters. This manual is a third
edition of the freshwater chronic toxicity test manual for effluents first
published (EPA/600/4-85/014) by EMSL-Cincinnati in December 1985 and revised
(EPA/600/4-89/001) in March, 1989. It provides updated methods for estimating
the chronic toxicity of effluents and receiving waters to freshwater organisms
for use by the U.S. Environmental Protection Agency (USEPA).regional and state
programs, and National Pollutant Discharge Elimination System (NPDES)
permittees. '
Thomas A. Clark,, Director
Environmental Monitoring Systems
Laboratory-Cincinnati
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PREFACE
This manual represents the third edition of the Agency's methods manual for
estimating the chronic toxicity of effluents and receiving waters to :
freshwater organisms initially published by USEPA's Office of Research^ and
Development, Environmental Monitoring and Support Laboratory (EMSL-
Cincinnati) in December 1985. This edition reflects changes recommended by
the Toxicity Assessment Subcommittee of the Biological Advisory Committee,
USEPA headquarters, program offices, 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.
The membership of the Toxicity Assessment Subcommittee, USEPA's Biological
Advisory Committee is as follows:
William Peltier, Subcommittee Chairman, ;
Environmental Services Division, Region 4 . ;
Peter Nolan, Environmental Services Division, Region 1
Jim Ferretti, Environmental Services Division, Region 2 j
Ronald Preston, Environmental Services Division, Region 3 ;
Charles Steiner, Environmental Services Division, Region 5 '
Evan Hornig, Environmental Services Division, Region 6
Terry Hollister, Environmental Services Division, Region 6
Michael Tucker, Environmental Services Division, Region 7
Loys Parrish, Environmental Services Division, Region 8 ;
Peter Husby, Environmental Services Division, Region 9 ;
Joseph Cummins, Environmental Services Division, Region 10 .
Gretchen Hayslip, Water Monitoring and Analysis Section, Region 10 !
Bruce Binkley, National Enforcement Investigations Center, Denver ;
Wesley Kinney, Environmental Monitoring Systems Laboratory-Las Vega|s
James Lazorchak, Environmental Monitoring Systems Laboratory-Cincinnati
George Morrison, Environmental Research Laboratory-Narragansett
Douglas Middaugh, Environmental Research Laboratory-Gulf Breeze ;
Teresa Norberg-King, Environmental Research Laboratory-Duluth i
Donald Klemm, Environmental Monitoring Systems Laboratory-Cincinnati
Philip Lewis, Environmental Monitoring Systems Laboratory-Cincinnati
Cornelius I. Weber, Environmental Monitoring Systems Laboratory-
Cincinnati , !
Richard Swartz, Environmental Research Laboratory-Newport \
Margarete Heber, Human Health and Ecological Criteria Division,
Office of Science and Technology (OST), Office of Water (OW)
Bruce Newton, Assessment and Watershed Protection Division, Office bf
Wetlands, Oceans, and Watersheds, OW
Christopher Zarba, Human Health and Ecological Criteria Division, Office
of Science and Technology, OW ;
Daniel Rieder, Hazard Evaluation Division, Office of Pesticides Programs
Jerry Smrchek, Health and Environmental Review Division, Office of ;
Toxic Substances I
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Gail Hansen, Office of Solid Waste
Royal Nadeau, Emergency Response Team, Edison
Teresa J. Norberg-King
Chairman, Biological Advisory Committee
Regulatory Ecotoxicology Branch, ERL-Duluth
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ABSTRACT j
i
This manual describes four short-term (four- to seven-day) methods for
estimating the chronic toxicity of effluents and receiving waters to three
freshwater species: the fathead minnow, Pimephales promelas, a daphnid,
Ceriodaphnia dubia, and a green alga, Selenastrum capricornutum. The methods
include single and multiple concentration static renewal and non-renewal
toxicity tests for effluents and receiving waters. Also included are
guidelines on laboratory safety, quality assurance, facilities, equipment and
supplies; dilution water; effluent and receiving water sample collection,
preservation, shipping, and holding; test conditions; toxicity test data
analysis; report preparation; and organism culturing, holding, and handling.
Examples of computer input and output for Dunnett's Procedure, Probit
Analysis, Trimmed Spearman-Karber Method and the Linear Interpolation Method
are provided in the Appendices.
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CONTENTS
: ! Page
Foreword ! iii
Preface ., .; . iv
Abstract ; vi
Figures , . xii
Tables . . . ; xv
Acknowledgments . : ........ xx
Section Number '< Page
1. Introduction 1
2. Short-Term Methods for Estimating Chronic Toxicity 3
Introduction : 3
Types of Tests : . . 5
Static Tests 6
Advantages and Disadvantages of Toxicity Test Types 7
3. Health and Safety 8
General Precautions 8
Safety Equipment 8
General Laboratory and Field Operations 8
Disease Prevention . 9
Safety Manuals ; 9
Waste Disposal ..... 9
4. Quality Assurance ; 10
Introduction . 10
Facilities, Equipment, and Test Chambers . 10
Test Organisms .'. ; 11
Laboratory Water Used for Culturing and
Test Dilution Water ; 11
Effluent and. Receiving Water Sampling and
Handling \ .......; 11
Test Conditions 11
Quality of Test Organisms . 12
Food Quality . 12
Acceptability of Short-Term Chronic Toxicity Tests 13
Analytical Methods 13
Calibration and Standardization . : 14
Replication and Test Sensitivity ........ 14
Variability in Toxicity Test Results 14
Test Precision . . . \. . 14
Demonstrating Acceptable Laboratory Performance 16
Documenting Ongoing Laboratory Performance .! 16
Reference.Toxicants 17
Record Keeping 19
Video Tapes of USEPA Culture and Toxicity ,
Test Methods 19
Supplemental Reports for Training Video Tapes: 20
vn
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CONTENTS (CONTINUED)
Section Number Page
5. Facilities, Equipment, and Supplies .... . . . I . 21
General Requirements ............ 21
Test Chambers '<. . 22
Cleaning Test Chambers and Laboratory Apparatus ......... 22
Apparatus and Equipment for Culturing and Toxicity Tests '. . 23
Reagents and Consumable Materials :. . 24
Test Organisms . 24
Supplies 24
6. Test Organisms 26
Test Species . . I . 26
Sources of Test Organisms '. . 27
Life Stage 28
Laboratory Culturing ;. . 28
Holding and Handling Test Organisms I . 28
Transportation to the Test Site I . 29
Test Organism Disposal ;. . 30
7. Dilution Water |. . 31
Types of Dilution Water . 31
Standard, Synthetic Dilution Water ....... 31
Use of Receiving Water as Dilution Water .......... 32
Use of Tap Water as Dilution Water . 34
Dilution Water Holding ........ i . 34
8. Effluent and Receiving Water Sampling, Sample Handling, ;
and Sample Preparation for Toxicity Tests i. . 35
Effluent Sampling ......... 35
Effluent Sample Types . 35
Effluent Sampling Recommendations . . 36
Receiving Water Sampling . . 37
Effluent and Receiving Water Sample Handling,
Preservation, and Shipping . 38
Sample Receiving . 39
Persistence of Effluent Toxicity During Sample i
Shipment and Holding . i . 39
Preparation of Effluent and Receiving Water Samples
for Toxicity Tests .....;. 39
Preliminary Toxicity Range-Finding Tests I . 42
Multi-concentration (Definitive) Effluent
Toxicity Tests i . 42
Receiving Water Tests ; . 43
9. Chronic Toxicity Test Endpoints and Data Analysis i . 44
Endpoints . 44
Relationship Between Endpoints Determined by
Hypothesis Testing and Point Estimation Techniques . . ; . 45
Precision j . 47
Data Analysis I . 47
Choice of Analysis i . 49
Hypothesis Tests . . ; . 52
Point Estimation Techniques i . 53
viii
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CONTENTS (CONTINUED) I
Section Number Page
10. Report Preparation .; ' 55
Introduction . ; . . . 55
Plant Operations . . 55
Source of Effluent, Receiving Water, and Dilution Water ... 55
Test Methods . 56
Test Organisms 56
Quality Assurance 56
Results 56
Conclusions and Recommendations 57
11. Test Method: Fathead Minnow, Pimephales promelas, Larval
Survival and Growth Test Method 1000.0 . 58
Scope and Application ...,. 58
Summary of Method 1 58
Interferences 58
Safety I- 59
Apparatus and Equipment 59
Reagents and Consumable Materials ..... 60
Effluent and Receiving Water Collection, Preservation,
and Storage 68
Calibration and Standardization , 68
Quality Control : . 69
Test Procedures 69
Summary of Test Conditions and Test Acceptability
Criteria 76
Acceptability of Test Results 81
Data Analysis 81
Precision and Accuracy ; 108
12. Test Method: Fathead Minnow, Pimephales promelas, Embryo-larval
Survival and Teratogenicity Test Method 1001.0 114
Scope and Application 114
Summary of Method : 114
Interferences i 114
Safety . . . : , . . ' 115
Apparatus and Equipment , 115
Reagents and Consumable Materials 116
Effluent and Receiving Water Collection, Preservation,
and Storage : 118
Calibration and Standardization 118
Quality Control ......... 118
Test Procedures 118
Summary of Test Conditions and Test Acceptability
Criteria : . 126
Acceptability of Test Result 126
Data Analysis 130
Precision and Accuracy ; 139
IX
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CONTENTS (CONTINUED)
Section Number
Page
13. Test Method: Daphnid, Ceriodaphnia dubia, Survival and :
Reproduction Test Method 1002.0 ..... . . 144
Scope and Application . 144
Summary of Method j. . 144
Interferences ........... 144
Safety :. . 145
Apparatus and Equipment . i. . 145
Reagents and Consumable Materials ;. . 147
Effluent and Receiving Water Collection, Preservation,
and Storage 158
Calibration and Standardization . 158
Quality Control . 158
Test Procedures 158
Summary of Test Conditions and Test Acceptability ;
Criteria .........;.. 165
Acceptability of Test Results i. . 165
Data Analysis 170
Precision and Accuracy 189
14. Test Method: Green Alga, Selenastrum capn'cornutum, Growth
Test Method 1003.0 196
Scope and Application 196
Summary of Method 196
Interferences i . 196
Safety 197
Apparatus and Equipment 197
, Reagents and Consumable Materials 198
Effluent and Receiving Water Collection, Preservation,
and Storage '. . 203
Calibration and Standardization :. . 203
Quality Control 203
Test Procedures . 203
Summary of Test Conditions and Test Acceptability
Criteria . 209
Acceptability of Test Results ;. . 209
Data Analysis 212
Precision and Accuracy i. . 225
Cited References !. . 229
Bibliography , . 239
Appendices ;. . 246
A. Independence, Randomization, and Outliers ;. . 248
B. Validating Normality and Homogeneity of Variance
Assumptions 254
C. Dunnett's Procedure 271
D. T test with Bonferroni's Adjustment 283
E. Steel's Many-one Rank Test i. . 289
F. Wilcoxon Rank Sum Test 293
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CONTENTS (CONTINUED)
Section Number ; Page
G. Fisher's Exact Test . . ,' 299
H. Single Concentration Toxicity Test - Comparison
of Control with 100% Effluent or Receiving Water 308
I. Probit Analysis 312
J. Spearman-Karber Method 315
K. Trimmed Spearman-Karber Method . 320
L. Graphical Method 325
M. Linear Interpolation Method 329
Cited References 340
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FIGURES '
SECTIONS 1-10 ;
Number ! page
1. Control (cusum) charts !. . is
2. Flowchart for statistical analysis of test data ...... I . 50
SECTION 11
Number Page
1. Data form for the fathead minnow, Pimephales promelas,
larval survival and growth test. Routine chemical and
physical determinations , . 73
2. Mortality data for the fathead minnow, Pimephales promelas, -
larval survival and growth test 75
3. Weight data for the fathead minnow, Pimephales promelas,
larval survival and growth test 77
4. Summary data for the fathead minnow, Pimephales promelas,
larval survival and growth test 78
5. Flowchart for statistical analysis of fathead minnow,
Pimephales promelas, larval survival data by hypothesis ^
testing i . 83
6. Flowchart for statistical analysis of fathead minnow,
Pimephales promelas, larval survival data by point :
estimation 84
7. Plot of survival proportion data in Table 3 i . 86
8. Output for USEPA Probit Analysis program, Version 1.5 . . . ; . 95
9. Flowchart for statistical analysis of fathead minnow,
Pimephales promelas, larval growth data . . 96
10. Plot of weight data from fathead minnow, Pimephales
promelas, larval survival and growth test for point
estimate testing '. . . . J . 98
11. Plot of raw data, observed means, and smoothed means for ' .
the fathead minnow, Pimephales promelas, growth data ;
in Tables 2 and 18 ; 106
xn
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FIGURES (CONTINUED)
SECTION 11 (Continued) j
Number ; Page
12. ICPIN program output for the IC25 109
13. ICPIN program output for the IC50 110
SECTION 12
Number Page
1. Data form for the fathead minnow, Pimephales promelas,
embryo-larval survival and teratogenicity test. Routine
chemical and physical determinations ,. 122
2. Data form for the fathead minnow, Pimephales promelas,
embryo-larval survival and teratogenicity test.: Survival
and terata data '. . 124
3. Summary data for the fathead minnow, Pimephales promelas,
embryo-larval survival and teratogenicity test . 127
4. Flowchart for statistical analysis of fathead minnow,
Pimephales promelas, embryo-larval data 131
5. Plot of fathead minnow, Pimephales promelas, total mortality
data from the embryo-larval test 134
6. Output for USEPA Probit Analysis program, Version 1.5 140
SECTION 13
Number ' Page
1. Examples of a test board and randomizing template 156
2. Data form for the daphnid, Ceriodaphnia dubia, survival
and reproduction test. Routine chemical and physical
determinations 163
3. Data form for the daphnid, Ceriodaphnia dubia, survival
and reproduction test. Daily summary of data . : . . 166
4. Flowchart for statistical analysis of the daphnid,
Ceriodaphnia dubia, survival data 172
5. Output for USEPA Trimmed Spearman-Karber program 175
xm
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FIGURES (CONTINUED) :
SECTION 13 (Continued)
Number Page
6. Flowchart for statistical analysis of the daphnid,
Cen'odaphnia dubia, reproduction data 176
7. Plot of number of young per adult female from a daphnid,
Cen'odaphnia dubia, survival and reproduction test j. . 17.8
8. Plot of raw data, observed means, and smoothed means for
the daphnid, Cen'odaphnia dubia, reproductive data 187
9. Example of ICPIN program output for the IC25 . . 190
!
10. Example of ICPIN program output for the IC50 j. . 191
SECTION 14
Number ! Page
1. Data form for the green alga, Selenastrum capn'cornutum, I
growth test. Routine chemical and physical l
determinations . 206
2. Data form for the green alga, Selenastrum capn'cornutum,
growth test, cell density determinations . . . . L . 209
3. Flowchart for statistical analysis of the green alga,
Selenastrum capn'cornutum, growth response data :. . 213
4. Plot of Iog10 transformed cell count data from the green
alga, Selenastrum capn'cornutum, growth response test in :
Table 4 ......!.. 215
5. Plot of raw data and observed means for the green alga,
Selenastrum capn'cornutum, growth data . 224
6. ICPIN program output for the IC25 . . . ;. . 226
7. ICPIN program output for the IC50 ........ I . 227
xiv
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TABLES
I
SECTIONS 1-10
Number ' < Page
1. National interlaboratory study of chronic toxicity
test precision, 1991: Summary of responses using
a reference toxicant .; 15
2. Commercial suppliers of brine shrimp (Artemia) cysts 25
i ;,
3. Preparation of synthetic freshwater using reagent
grade chemicals ; 33
4. Preparation of synthetic freshwater using
mineral water 34
5. Percent unionized NH3 in aqueous ammonia solutions:
Temperatures 15-26ฐC and pH 6.0-8.9 41
SECTION 11
Number : Page
1. Summary of test conditions and test acceptability
criteria for fathead minnow, Pimephales promelas,
larval survival and growth toxicity tests with
effluents and receiving waters 79
, 1
2. Summary of survival and growth data for fathead minnow,
Pimephales promelas, larvae exposed to a reference
toxicant for seven days 81
3. Fathead minnow, Pimephales promelas, survival data ....... 85
4. Centered observations for Shapiro-Wilk's example 87
5. Ordered centered observations for the
Shapiro-Wilk's example ; 87
6. Coefficients and differences for Shapiro-Wilk's example .... 88
7. ANOVA table ' 90
8. ANOVA table for Dunnett's Procedure example 91
9. Calculated t values !. . 92
10. Data for Probit Analysis 94
xv
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TABLES (CONTINUED)
SECTION 11 (Continued) i
Number [ Page
11. Fathead minnow, Pimephales promelas, growth data (. . 97
12. Centered observations for Shapiro-Milk's example !. . 97
13. Ordered centered observations for Shapiro-Milk's example .... 99
14. Coefficients and differences for Shapiro-Milk's example .... 100
15. ANOVA table 102
16. ANOVA table for Dunnett's Procedure example ;. . 103
17. Calculated t values 104
18. Fathead minnow, Pimephales promelas, mean growth response .
after smoothing '. . 107
19. Precision of the fathead minnow, Pimephales promelas,
larval survival and growth test, using NaPCP as a ;
reference toxicant ; '. 108
20. Combined frequency distribution for survival NOECs
for all laboratories ; . 112
21. Combined frequency distribution for weight NOECs :
for all laboratories , . 113
SECTION 12 '
Number Page
1. Summary of test conditions and test acceptability criteria '
for fathead minnow, Pimephales promelas, embryo-larval I
survival and teratogenicity toxicity tests with effluents
and receiving waters ] . 128
2. Data from fathead minnow, Pimephales promelas, embryo-larval \
toxicity test with ground water effluent i . 132
3. Fathead minnow, Pimephales promelas, embryo-larval total
mortality data : . 133
4. Centered observations for Shapiro-Milk's example j . 135
5. Ordered centered observations for the Shapiro-Milk's example .' . 135
"""' I
I
xvi i
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TABLES (CONTINUED)
SECTION 12 (Continued) '
Number : Page
6. Coefficient and differences for the Shapiro-WiIks example ... 136
7. Assigning Ranks to the control and 3.125% effluent
concentration for the Steel's Many-One Rank Test: . . 137
8. Table of Ranks for Steel's Many-One Rank Test 138
9. Rank Sums 138
10. Data for Probit Analysis 139
11. Precision of the fathead minnow, Pimephales promelas,
embryo-larval survival and teratogenicity test, using
cadmium as a reference toxicant 141
12. Precision of the fathead minnow, Pimephales promelas,
embryo-larval, survival and teratogenicity toxicity test,
using diquat as a reference toxicant 142
13. Precision of the fathead minnow, Pimephales promelas,
embryo-larval survival and teratogenicity static-renewal
test conducted with trickling filter effluent . . 143
SECTION 13
Number : Page
i
1. Nutrient stock solutions for maintaining algal stock
cultures ........ 150
2. Final concentration of macronutrients and micronutrients
in the culture medium 151
3. Summary of test conditions and test acceptability
criteria for daphnid, Ceriodaphnia dubia, survival
and reproduction toxicity tests with effluents
and receiving waters 168
4. Summary of survival and reproduction data for the
daphnid, Ceriodaphnia dubia, exposed to an effluent
for seven days , 170
i
5. Format of the 2x2 contingency table ......... 173
6. 2x2 contingency table for control and 25% effluent 173
xv ii
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TABLES (CONTINUED)
SECTION 13 (Continued) ,
Number Page
7. Data for Trimmed Spearman-Karber Analysis '. . 174
8. The daphnid, Ceriodaphm'a dubia, reproduction data . . 177
9. Centered observations for Shapiro-Wilk's example ... . . .. L . 179
10. Ordered centered observations for Shapiro-Wilk's example . . . . 180
11. Coefficients and differences for Shapiro-Wilk's example . . i. . 181
12. ANOVA table -. .; . .... , . ...... |. . 183
13. ANOVA table for Dunnett's Procedure example ... I . 185
14. Calculated t values i. . 185
15. Daphnid, Ceriodaphm'a dubia, reproduction mean
response after smoothing [.. . 188
16. Single laboratory precision of the daphnid, Ceriodaphnia \
dubia, survival and reproduction test, using NaPCP as a ;
reference toxicant . 192
17. The daphnid, Ceriodaphnia dubia, seven-day survival and
reproduction test precision for a single laboratory using
NaPCP as the reference toxicant '. 193
18. Inter!aboratory precision of the daphnid, Ceriodaphnia
dubia, survival and reproduction test with copper \
spiked effluent !. . 194
19. Inter!aboratory precision data for the daphnid,
Ceriodaphnia dubia, summarized for eight reference
toxicants and effluents 195
SECTION 14 I
Number ; Page
1. Nutrient stock solutions for maintaining algal stock ;
cultures and test control cultures L . 200
2. Final concentration of macronutrients and micronutrients
in the culture medium I . 202
xvm
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TABLES (CONTINUED)
SECTION 14 (Continued)
Number
Page
3. Summary of test conditions and test acceptability criteria
for green alga, Selenastrum capricornutum, growth toxicity
tests with effluents and receiving waters ..... 210
4. Green alga, Selenastrum capricornutum, growth
response data 212
5. Centered observations for Shapiro-Wilk's example 214
6. Ordered centered observations for Shapiro-Wilk's example .... 216
7. Coefficients and differences for Shapiro-Wilk's example .... 217
8. ANOVA table : 219
9. ANOVA table for Dunnett's Procedure example . 220
10. Calculated t values ' 221
11. Algal mean growth response after smoothing ... 223
12. Single laboratory precision of the green alga,
Selenastrum capriccurnutum, 96-h toxicity tests,
using the reference toxicant cadmium chloride 228
xix
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ACKNOWLEDGMENTS
The principal authors of this document are Philip Lewis, Donald Klemm,
Cornelius Weber, James Lazorchak, and Florence Fulk, Environmental Systems
Laboratory-Cincinnati, OH; William Peltier, Environmental Services Division,
Region 4, Athens, GA; Teresa Norberg-King, Environmental Research Laboratory,
Duluth, MM; and Cathy Poore, Computer Sciences Corporation, Cincinnati;, OH.
Contributors to specific sections of this manual are listed below.
1. Sections 1-10; General Guidelines \
Margarete Heber, OST, Office of Water ;
Donald Klemm, EMSL-Cincinnati '
Philip Lewis, EMSL-Cincinnati :
George Morrison, ERL-Narragansett !
Teresa Norberg-King, ERL-Duluth !
William Peltier, ESD, Region 4
Cornelius Weber, EMSL-Cincinnati
2. Sections 11-13; Toxicity Test Methods j
Donald Klemm, EMSL-Cincinnati ;
James Lazorchak, EMSL-Cincinnati ;
Philip Lewis, EMSL-Cincinnati
Donald Mount, ERL-Duluth
Teresa Norberg-King, ERL-Duluth
Quentin Pickering, EMSL-Cincinnati !
1
3. Data Analysis (Sections 9, 11-13, and Appendices) j
Florence Fulk, EMSL-Cincinnati !
Laura Gast, Technology Application, Inc (TAI) |
Cathy Poore, Computer Sciences Corporation (CSC) !
Review comments from the following persons are gratefully acknowledged:
Celeste Philbrick Barr, Environmental Services Division, Biology Section,
U.S. Environmental Protection Agency, Region 1, Lexington, MA.
Michael Tucker, Bjoassay Lab. Environmental Services Division, U.S. :
Environmental Protection Agency, Region 7, Kansas City, MO. \
Michael Morton, Environmental Services Division, U.S. Environmental
Protection Agency, Region 6, Dallas, TX.
Jerry Smrchek, Environmental Effects Branch, Health and Environmental [Review
Division, U.S. Environmental Protection Agency, Washington, DC.
Robert Donaghy, Environmental Services Division, U.S. Environmental Protection
Agency, Region 3, Wheeling, WV.
Philip Crocker, Water Quality Management Branch, U.S. Environmental \.
Protection Agency, Region 6, Dallas, TX. !
Chick Steiner, Central Regional Laboratory, U.S. Environmental Protection
Agency, Region 5, Chicago, IL. \
xx
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ACKNOWLEDGMENTS (CONTINUED)
Tom Waller, Institute of Applied Sciences, University of North Texas, Denton,
TX. ,
Many useful public comments on the second edition of the freshwater
toxicity test methods "Short-Term Methods for Estimating the Chronic Toxicity
of Effluents and Receiving Waters to Freshwater Organisms", EPA/600/4-89/001
(USEPA, 1989a) were received in response to the proposed rule, published in
the Federal Register, December 4, 1989, [FR 54(231):50216-50224], regarding
the Agency's intent to include the short-term chronic toxicity tests in
Table IA, 40 CFR Part 136. These comments were considered in the preparation
of the third edition of the manual, and are included in the Public Docket for
rulemaking, located in room 2904, USEPA Headquarters, Washington, DC.
Materials in this manual w.ere taken in part from the following sources:
Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and
Amphibians, Environmental Research Laboratory, U. S. Environmental Protection
Agency, Duluth, MN, EPA-660/3-75-009 (USEPA, 1975); Handbook for Analytical
Quality Control in Water and Wastewater Laboratories, Environmental Monitoring
and Support Laboratory-Cincinnati, U. S. Environmental Protection Agency,
Cincinnati, OH, EPA-600/4-79/019 (USEPA, 1979a); Methods for chemical analysis
of water and wastes, Environmental Monitoring and Support Laboratory-
Cincinnati, U. S. Environmental Protection Agency, Cincinnati, OH, EPA-600/4-
79-020 (USEPA, 1979b); Interim NPDES Compliance Biomonitoring Inspection
Manual, Enforcement Division, Office of Water Enforcement, U. S. Environmental
Protection Agency, Washington, DC, (USEPA, 1979c); NPDES compliance inspection
manual, Office of Water Enforcement and Permits (EN-338), U. S. Environmental
Protection Agency, Washington, D. C. (USEPA, 1988a); 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, OH, EPA-600/4-85/013 (USEPA, 1985a); Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Freshwater Organisms, Environmental Monitoring Systems Laboratory-Cincinnati,
U.S. Environmental Protection. Agency, Cincinnati, OH, EPA/600/4-89/001 (USEPA
1989a); A Seven-day Life-cycle Cladoceran Test, Environ. Toxicol. Chem.
3:425-434 (Mount, D. I. and 1. J. Norberg, 1984); A New Fathead Minnow
(Pimephales promelas) Subchronic Toxicity Test, Environ. Toxicol. Chem.
4:711-718 (Norberg, T. J., arid D. I. Mount, 1985); The Selenastrum
capn'cornutum Printz Algal Assay Bottle Test, Environmental Research
Laboratory, U. S. Environmental Protection Agency, Corvallis, OR,
EPA-600/9-78-018 (USEPA, 1978a); Short-term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms,
Environmental Monitoring and Support Laboratory-Cincinnati, U. S.
Environmental Protection Agency, Cincinnati, OH, EPA-600/4-87/028 (USEPA,
1988b); Short-term Methods for Estimating the Chronic Toxicity of Effluents
and Receiving Waters to Marine and Estuarine Organisms (Second Edition),
Environmental Monitoring Systems Laboratory-Cincinnati, U.S. Environmental
Protection Agency, Cincinnati, OH, EPA/600/4-91/003 (USEPA, 1993a); Methods
for Measuring the Acute Toxicity of Effluents and Receiving Waters to
Freshwater and Marine Organisms (Fourth Edition), Environmental Monitoring
xxi '
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ACKNOWLEDGMENTS (CONTINUED)
Systems Laboratory-Cincinnati, U.S. Environmental Protection Agency,
Cincinnati, OH, EPA/600/4-90/027F (USEPA, 1993b); and Technical Support
Document for Water Quality-Based Toxics Control, Office of Water Enforcement
and Permits, Office of Water Regulations and Standards, U.S. Environmental
Protection Agency, Washington, DC, EPA/505/2-90-001 (USEPA, 1991a). ;
The fathead minnow, Pimephales promelas, larval survival and growth test
method and the daphnid, Cen'odaphnia dubia, survival and reproduction ;test
method in this manual were adapted from methods developed at the ERL-Duluth by
Donald Mount and Teresa Norberg-King. The fathead minnow, Pimephales '
promelas, embryo-larval survival and teratogenicity test method was developed
by Wesley Birge and Jeffrey Black, University of Kentucky, Lexington, junder a
cooperative agreement with the EMSL-Cincinnati. The algal growth test method
was adapted from the green alga, Selenastrum capn'cornutum, algal assay bottle
test developed at the Environmental Research Laboratory-Corvallis by William
Miller, Joseph Greene, and Tamotsu Shiroyama. \
i
Debbie Hall, Bioassessment and Ecotoxicology Branch, and Mary Sullivan,
Quality Assurance Research Division, provided valuable secretarial assistance,
and Betty Thomas, Technical Information Manager, EMSL-Cincinnati, provided an
editorial review. i
xxn
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SECTION 1
INTRODUCTION
1.1 This manual describes chronic toxicity tests for use in the National
Pollutant Discharge Elimination System (NPDES) Permits Program to identify
effluents and receiving waters containing toxic materials in chronically toxic
concentrations. The methods included in this manual are referenced in
Table IA, 40 CFR Part 136 regulations and, therefore, constitute approved
methods for chronic toxicity tests. They are also suitable for determining
the toxicity of specific compounds contained in discharges. The tests may be
conducted in a central laboratory or on-site, by the regulatory agency or the
permittee. ;
1.2 The data are used for NPDES permits development and to determine
compliance with permit toxicity limits. Data can also be:used to predict
potential acute and chronic toxicity in the receiving water, based on the
LC50, NOEC, IC50 or IC25 (see Section 9, Chronic Toxicity Endpoints and Data
Analysis) and appropriate dilution, application, and persistence factors. The
tests are performed as a part of self-monitoring permit requirements,
compliance biomonitoring inspections, toxics sampling inspections, and special
investigations. Data from chronic toxicity tests performed as part of permit
requirements are evaluated during compliance evaluation inspections and
performance audit inspections.
1.3 Modifications of these tests are also used in toxicity reduction
evaluations and toxicity identification evaluations to identify the toxic
components of an effluent, to aid in the development and implementation of
toxicity reduction plans, and to compare and control the effectiveness of
various treatment technologies for a given type of industry, irrespective of
the receiving water (USEPA, 1988c; USEPA, 1989b; USEPA 1989c; USEPA, 1989d;
USEPA, 1989e; USEPA, 1991a; USEPA, 1991b; and USEPA, 1992).
1.4 This methods manual serves as a companion to the acute toxicity test
methods for freshwater and marine organisms (USEPA, 1993b), the short-term
chronic toxicity test methods for marine and estuarine organisms (USEPA,
1993a), and the manual for evaluation of laboratories performing aquatic
toxicity tests (USEPA, 1991c).
1.5 Guidance for the implementation of toxicity tests in the NPDES program is
provided in the Technical Support Document for Water Quality-based Toxics
Control (USEPA, 1991a). ;
1.6 These freshwater short-term toxicity tests are similar to those developed
for marine and estuarine organisms to evaluate the toxicity of effluents
discharged to marine and estuarine waters under the NPDES permit program.
Methods are presented in this manual for three species of freshwater organisms
from three phylogenetic groups. The methods are all static renewal type
seven-day tests except the green alga, Selenastrum capricornutum, test which
lasts four days.
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1.7 The three species for which test methods are provided are the fathead
minnow, Pimephales promelas-, the daphnid, Ceriodaphnia dubia; and the .green
alga, Selenastrum capri'cornutum.
1.7.1 The tests included in this document are based on the following ^ethods:
1. "A new fathead minnow (Pimephales promelas) subchronic toxicity ftest,"
by Teresa J. Norberg and Donald I. Mount, 1985, Environmental Toxicology
and Chemistry (Norberg and Mount, 1985). '
2. "In-situ acute/chronic toxicological monitoring of industrial effluents
for the NPDES biomonitoring program using fish and amphibian ;
embryo/larval stages as a test organism," by Wesley J. Birge and Jeffrey
A. Black, 1981, OWEP-82-001, Office of Water Enforcement and Permits,
U.S. Environmental Protection Agency, Washington, DC (USEPA, 1981).
3. "A seven-day life-cycle cladoceran test,", by Donald I. Mount arid
Teresa Norberg, 1984, Environmental Toxicology and Chemistry (Mount and
Norberg, 1984). j
4. "The Selenastrum capricornutum Printz algal assay bottle test,";by
William E. Miller, Joseph C. Greene and Tamotsu Shiroyama, 1978,
Environmental research Laboratory, U.S. Environmental Protection
Agency, Con/all is, OR. EPA/600/9-78/018 (USEPA, 1978a). j
1.7.2 Two of the methods incorporate the chronic endpoint of growth in
addition to lethality and one incorporates reproduction. The fathead;minnow,
Pimephales promelas, embryo-larval survival and teratogenicity test
incorporates teratogenic effects in addition to lethality. The green,alga,
Selenastrum capricornutum, growth test has the advantage of a relatively short
exposure period (96 h).
1.8 The validity of the freshwater chronic methods in predicting adverse
ecological impacts of toxic discharges was demonstrated in field studies
(USEPA, 1984; USEPA, 1985b; USEPA, 1985c; USEPA, 1985d; USEPA, 1986a;:USEPA,
1986b; USEPA, 1986c; USEPA, J,986d; Birge et al., 1989; and Eagleson et al.,
1990). ;
1.9 The use of any test species or test conditions other than those described
in the methods summary tables in this manual shall be subject to application
and approval of alternate test procedures under 40 CFR 136.4 and 40 CFR 136.5.
1.10 These methods are restricted to use by, or under the supervision of,
analysts experienced in the use or conduct of aquatic toxicity tests and the
interpretation of data from aquatic toxicity testing. Each analyst must
demonstrate the ability to generate acceptable test results with these methods
using the procedures described in this methods manual. ;
1.11 This manual was prepared in the established EMSL-Cincinnati format
(USEPA, 1983). i
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SECTION 2
SHORT-TERM METHODS FOR ESTIMATING CHRONIC TOXICITY
2.1 INTRODUCTION
I
i
2.1.1 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 a<; mortality, growth,
and reproduction.
2.1.2 Acute lethality 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 (one-to-four days). '
2.1.3 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.
2.1.4 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.
2.1.5 McKim (1977) evaluated the data from 56 full life-cycle tests, 32- of
which used the fathead minnow, Pimephales promeTas, and concluded that the
embryo-larval and early juvenile life-stages were the most sensitive stages.
He proposed the use of partial life-cycle toxicity tests with the early
life-stages (ELS) of fish to establish water quality criteria.
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2.1.6 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. They
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 jfish
with embryogenic periods ranging from one-to-fourteen 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.
*
2.1.7 Because of the high cost of full life-cycle fish toxicity tests and the
emerging consensus that the ELS test data usually would be adequate for
estimating chronically safe concentrations, there was a rapid shift by aquatic
toxicologists to 30 - 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 idata
from full life-cycle tests were not available (USEPA, 1980a). i
2.1.8 Published reports of the results of ELS tests indicate that
relative sensitivity of growth and survival as endpoints may be species
dependent, toxicant dependent, or both. Ward and Parrish (1.980) examined the
literature on ELS tests that used embryos and juveniles of the sheepshead
minnow, Cypn'nodon 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.
2.1.9 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, Weltering (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, found larval
growth to be the most significant measure of effect, and survival to be
equally or less sensitive than growth in early life-stage tests with four
organic chemicals. i
i
2.1.10 Efforts to further reduce the length of partial life-cycle toxicity
tests for fish without compromising their predictive value have resulted in
the development of an eight-day, embryo-larval survival and teratogenicity
test for fish and other aquatic vertebrates (USEPA, 1981; Birge et all, 1985),
and a seven-day larval survival and growth test (Norberg and Mount, 1985).
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2.1.11 The similarity of estimates of chronically safe concentrations of
toxicants derived from short-term, embryo-larval survival and teratogenicity
tests 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).
2.1.12 Use of a seven-day, fathead minnow, Pimephales promelas, larval
survival and growth test was first proposed by Norberg and; Mount at the 1983
annual meeting of the Society for Environmental Toxicology arid Chemistry
(Norberg and Mount, 1983). This test was subsequently used by Mount and
associates in field demonstrations at Lima, OH (USEPA, 1984), and at many
other locations. Growth was frequently found to be more sensitive than
survival in determining the effects of complex effluents.
2.1.13 Norberg and Mount (1985) performed three single toxicant fathead
minnow larval growth tests with zinc, copper, and DURSBANฎ, 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.
2.1.14 Mount and Norberg (1984) developed a seven-day cladoceran partial
life-cycle test and experimented with a number of diets for use in culturing
and testing the daphnid, Ceriodaphm'a reticulata (Norberg and Mount, 1985).
As different laboratories began to use this cladoceran test, it was discovered
that apparently more than one species was involved in the tests conducted by
the same laboratory. Berner (1986) studied the problem and determined that
perhaps as many as three variant forms were involved and it was decided to
recommend the use of the more common Ceriodaphm'a dubia rather than the
originally reported Ceriodaphm'a reticulata. The method was adopted for use
in the first edition of the freshwater short-term chronic methods (USEPA,
1985e),
2.1.15 The green alga, Selenastrum capricornutum, bottle test was developed,
after extensive design, evaluation, and application, for the National
Eutrophication Research Program (USEPA, 1971). The test was later modified
for use in the assessment of receiving waters and the effects of wastes
originating from industrial, municipal, and agricultural point and non-point
sources (USEPA, 1978a).
2.1.16 The use of short-term toxicity tests including subchronic and chronic
tests in the NPDES Program is especially attractive because they provide a
more direct estimate of the safe concentrations of effluents in receiving
waters than was provided by acute toxicity tests, at an only slightly
increased level of effort, compared to the fish full life-cycle chronic and
28-day ELS tests and the 21-day daphnid, Daphm'a magna, life-cycle test.
2.2 TYPES OF TESTS
2.2.1 The selection of the test type will depend on the NPDES permit
requirements, the objectives of the test, the available resources, the
requirements of the test organisms, and effluent characteristics such as
fluctuations in effluent toxicity.
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2.2.2 Effluent chronic toxicity i's generally measured using a multi- I
concentration, or definitive test, consisting of a control and a minimum of
five effluent concentrations. The tests are designed to provide dose-response
information, expressed as the percent effluent concentration tha't affects the
hatchability, gross morphological abnormalities, survival, growth, and/or
reproduction within the prescribed period of time (four to seven days).. The
results of the tests are expressed in terms of the highest concentration that
has no statistically significant observed effect on those responses when
compared to the controls or the estimated concentration that causes a
specified percent reduction in responses versus the controls. \
2.2.3 Use of pass/fail tests consisting of a single effluent concentration
(e.g., the receiving water concentration or RWC) and a control is not
recommended. If the NPDES permit has a whole effluent toxicity limit for
acute toxicity at the RWC, it is prudent to use that permit limit as the
midpoint of a series of five effluent concentrations. This will ensur;e that
there is sufficient information on the dose-response relationship. For
example, the effluent concentrations utilized in a test may be:
(1) 100% effluent, (2) (RWC + 100)/2, (3) RWC, (4) RWC/2, and (5) RWC/14. More
specifically, if the RWC = 50%, the effluent concentrations used in the
toxicity test would be 100%, 75%, 50%, 25%, and 12.5%. ;
2.2.4 Receiving (ambient) water toxicity tests commonly employ two >
treatments, a control and the undiluted receiving water, but may also consist
of a series of receiving water dilutions.
2.2.5 A negative result from a chronic toxicity test does not preclude the
presence of toxicity. Also, because of the potential temporal variability in
the toxicity of effluents, a negative test result with a particular sample
does not preclude the possibility that samples collected at some otheir time
might exhibit chronic toxicity.
2.2.6 The frequency with which chronic toxicity tests are conducted under a
given NPDES permit is determined by the regulatory agency on the basis of
factors such as the variability and degree of toxicity of the waste,
production schedules, and process changes.
2.2.7 Tests recommended for use in this methods manual may be staticinon-
renewal or static renewal. Individual methods specify which static type of
test is to be conducted. :
2.3 STATIC TESTS
2.3.1 Static non-renewal tests - The test organisms are exposed to the same
test solution for the duration of the test. t
2.3.2 Static-renewal tests - The test organisms are exposed to a fresh
solution of the same concentration of sample every 24 h or other prescribed
interval, either by transferring the test organisms from one test chamber to
another, or by replacing all or a portion of solution in the test chambers.
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2.4 ADVANTAGES AND DISADVANTAGES OF TOXICITY TEST TYPES
2.4.1 STATIC NON-RENEWAL, SHORT-TERM TOXICITY TESTS: |
Advantages:
1. Simple and inexpensive.
2. Very cost effective in determining compliance with permit conditions.
3. Limited resources (space, manpower, equipment) required; would permit
staff to perform many more tests in the same amount of time.
4. Smaller volume of effluent required than for static renewal or flow-
through tests. i
Disadvantages:
1. Dissolved oxygen (DO) depletion may result from high chemical oxygen
demand (COD), biological oxygen demand (BOD), or metabolic wastes.
2. Possible loss of toxicants through volatilization and/or adsorption to
the exposure vessels.
3. Generally less sensitive than static renewal, because the toxic
substances may degrade or be adsorbed, thereby reducing the apparent
toxicity. Also, there is less chance of detecting slugs of toxic
wastes, or other temporal variations in waste properties.
2.4.2 STATIC RENEWAL, SHORT-TERM TOXICITY TESTS: |
Advantages:
1. Reduced possibility of DO depletion from high COD and/or BOD, or ill
effects from metabolic wastes from organisms in the test solutions.
2. Reduced possibility of loss of toxicants through volatilization and/or
adsorption to the exposure vessels.
3. Test organisms that rapidly deplete energy reserves are fed when the
test solutions are renewed, and are maintained in a healthier state.
Disadvantages: ,
1. Require greater volume of effluent than non-renewal tests.
2. Generally less chance of temporal variations in waste properties.
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SECTION 3
HEALTH AND SAFETY
3.1 GENERAL PRECAUTIONS
3.1.1 Each laboratory should develop and maintain an effective health and
safety program, requiring an ongoing commitment by the laboratory management.
This program should include (1) a safety officer with the responsibility and
authority to develop and maintain a safety program, (2) the preparation of a
formal, written, health and safety plan, which is provided to each of the
laboratory staff, (3) an ongoing training program on laboratory safety, and
(4) regularly scheduled, documented, safety inspections.
3.1.2 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. :
3.1.3 Prior to sample collection and laboratory work, personnel will
determine that all necessary safety equipment and materials have been ^obtained
and are in good condition.
3.1.4 Guidelines for the handling and disposal of hazardous materials must be
strictly followed.
3.2 SAFETY EQUIPMENT
3.2.1 PERSONAL SAFETY GEAR
3.2.1.1 Personnel should use safety equipment, as required, such as rubber
aprons, laboratory coats, respirators, gloves, safety glasses, hard hats, and
safety shoes. Plastic netting on glass beakers, flasks, and other glassware
minimizes breakage and subsequent shattering of the glass.
3.2.2 LABORATORY SAFETY EQUIPMENT
3.2.2.1 Each laboratory (including mobile laboratories) should be provided
with safety equipment such as first aid kits, fire extinguishers, fire
blankets, emergency showers, chemical spill clean up kits, and eye fountains.
3.2.2.2 Mobile laboratories should be equipped with a telephone or other
means to enable personnel to summon help in case of emergency.
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
8
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listed in Section 3, Health and Safety, Subsection 3.5). It is recommended
that personnel collecting samples and performing toxicity tests 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. Fume and canopy hoods over the toxicity test areas
must be used whenever possible. ;
3.3.3 It is advisable to cleanse exposed parts of the body immediately after
collecting effluent samples. ' , '
3.3.4 All containers are to be adequately labeled to indicate their contents.
3.3.5 Staff should be familiar with safety guidelines on Material Safety Data
Sheets for reagents and other chemicals purchased from suppliers.
Incompatible materials should not be stored together. Good housekeeping
contributes to safety and reliable results.
3.3.6 Strong acids and volatile organic solvents employed in glassware
cleaning must be used in a fume hood or under an exhaust canopy over the work
area.
3.3.7 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.
3.3.8 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, polio, and
hepatitis B.
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
safety manuals, including USEPA (1986e) and Walters and Jameson (1984).
3.6 WASTE DISPOSAL
3.6.1 Wastes generated during toxicity testing must be properly handled and
disposed of in an appropriate manner. Each testing facility will have its own
waste disposal requirements based on local, state, and Federal rules and
regulations. It is extremely important that these rules and regulations be
known, understood, and complied with by all persons responsible for, or
otherwise involved in performing the toxicity testing activities. Local fire
officials should be notified of any potentially hazardous conditions.
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SECTION 4
QUALITY ASSURANCE
4.1 INTRODUCTION
4.1.1 Development and maintenance of a toxicity test laboratory quality
assurance (QA) program (USEPA, 1991a) requires an ongoing commitment by
laboratory management. Each toxicity test laboratory should (1) appoint a
quality assurance officer with the responsibility and authority to deyelop and
maintain a QA program; (2) prepare a quality assurance plan with stated data
quality objectives (DQOs); (3) prepare a written description of laboratory
standard operating procedures (SOPs) for culturing, toxicity-testing,
instrument calibration, sample chain-of-custody procedures, laboratory sample
tracking system, glassware cleaning, etc.; and (4) provide an adequate,
qualified technical staff for culturing and testing the organisms, and
suitable space and equipment to assure reliable data. :
4.1.2 QA practices for toxicity testing laboratories must address all
activities that affect the quality of the final effluent toxicity test data,
such as: (1) effluent sampling and handling; (2) the source and condition of
the test organisms; (3) condition of equipment; (4) test conditions; (5)
instrument calibration; (6) replication; (7) use of reference toxicants;
(8) record keeping; and (9) data evaluation. !
4.1.3 Quality control practices, on the other hand, consist of the more
focused, routine, day-to-day activities carried out within the scope of the
overall QA program. For more detailed discussion of quality assurance and
general guidance on good laboratory practices and laboratory evaluation
related to toxicity testing, see FDA, (1978); USEPA, (1979d), USEPA (1980b),
USEPA (1980c), and USEPA (1991c); DeWoskin (1984); and Taylor (1987).;
4.1.4 Guidance for the evaluation of laboratories performing toxicity tests
and laboratory evaluation criteria may be found in USEPA (1991c).
4.2 FACILITIES, EQUIPMENT, AND TEST CHAMBERS
4.2.1 Separate test organism culturing and toxicity testing areas shbuld be
provided to avoid possible loss of cultures due to cross-contamination.
Ventilation systems should be designed and operated to prevent recirculation
or leakage of air from chemical analysis laboratories or sample storage and
preparation areas into organism culturing or testing areas, and from testing
and sample preparation areas into culture rooms.
4.2.2 Laboratory and toxicity test 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, Equipment and Supplies;
and specific toxicity test method). ;
10
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4.3 TEST ORGANISMS
4.3.1 The test organisms used in the procedures described in this manual are
the fathead minnow, PimephaTes promelas, the daphnid, Cen'odaphnia dubia, and
the green alga, Selenastrum capn'cornutum. The fish and invertebrates should
appear healthy, behave normally, feed well, and have low mortality in the
cultures, during holding, and in test controls. Test organisms should be
positively identified to species (see Section 6, Test Organisms).
4.4 LABORATORY WATER USED FOR CULTURING AND TEST DILUTION MATER
4.4.1 The quality of water used for test organism culturing and for dilution
water used in toxicity tests is extremely important. Water for these two uses
should come from the same source. The dilution water used in effluent
toxicity tests will depend in part on the objectives of the study and
logistical constraints, as discussed in detail in Section 7, Dilution Water.
For tests performed to meet MPDES objectives, synthetic, moderately hard water
should be used. The dilution water used for internal quality assurance tests
with organisms, food, and reference toxicants should be the water routinely
used with success in the laboratory. Types of water are discussed in
Section 5, Facilities, Equipment and Supplies. Water used for culturing and
test dilution should be analyzed for toxic metals and organics at least
annually or whenever difficulty is encountered in meeting minimum
acceptability criteria for control survival and reproduction or growth. The
concentration of the metals Al, As, Cr, Co, Cu, Fe, Pb, Ni, and Zn, expressed
as total metal, should not exceed 1 /^g/L each, and Cd, Hg, and Ag, expressed
as total metal, should not exceed 100 ng/L each. Total organochlorine
pesticides plus PCBs should be less than 50 ng/L (APHA, 1992). Pesticide
concentrations should not exceed USEPA's Ambient Water Quality chronic
criteria values where available.
4.5 EFFLUENT AND RECEIVING MATER SAMPLING AND HANDLING
4.5.1 Sample holding times and temperatures of effluent samples collected for
on-site and off-site testing must conform to conditions described in
Section 8, Effluent and Receiving Water Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests.
4.6 TEST CONDITIONS ',
i
4.6.1 Water temperature must be maintained within the limits specified for
each test. The temperature of test solutions must be measured by placing the
thermometer or probe directly into the test solutions, or by placing the
thermometer in equivalent volumes of water in surrogate vessels positioned at
appropriate locations among the test vessels. Temperature should be recorded
continuously in at least one test vessel for the duration of each test.' Test
solution temperatures must be maintained within the limits specified for each
test. DO concentration and pH should be checked at the beginning of each test
and daily throughout the test period.
11
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4.7 QUALITY OF TEST ORGANISMS
I
4.7.1 If the laboratory performs short-term chronic toxicity tests routinely
but does not have an ongoing test organism culturing program and must obtain
the test organisms from an outside source, the sensitivity of a batch of test
organisms must be determined with a reference toxicant in a short-term chronic
toxicity test performed monthly (see Subsections 4.14, 4.15, 4.16, and, 4.17).
Where acute or short-term chronic toxicity tests are performed with effluents
or receiving waters using test organisms obtained from outside the test
laboratory, concurrent toxicity tests of the same type must be performed with
a reference toxicant, unless the test organism supplier provides control chart
data from at least the last five monthly short-term chronic toxicity tests
using the same reference toxicity and control conditions (see Section 6, Test
Organism).
4.7.2 The supplier should certify the species identification of the test
organisms, and provide the taxonomic reference (citation and page) or !name(s)
of the taxonomic expert(s) consulted. j
4.7.3 If the laboratory maintains breeding cultures, the sensitivity !of the
offspring should be determined in a short-term chronic toxicity test performed
with a reference toxicant at least once each month (see Subsections 4.|l4,
4.15, 4.16, and 4.17). If preferred, this reference toxicant test may be
performed concurrently with an effluent toxicity test. However, if a given
species of test organism produced by inhouse cultures is used only monthly, or
less frequently in toxicity tests, a reference toxicant test must be performed
concurrently with each short-term chronic effluent and/or receiving water
toxicity test.
4.7.4 If routine reference toxicant tests fail to meet acceptability
criteria, the test must be immediately repeated. If the failed reference
toxicant test was being performed concurrently with an effluent or receiving
water toxicity test, both tests must be repeated (For exception, see i
Section 4, Quality Assurance, Subsection 4.16.5). |
4.8 FOOD QUALITY
4.8.1 The nutritional quality of the food used in culturing and testing fish
and invertebrates is an important factor in the quality of the toxicity test
data. This is especially true for the unsaturated fatty acid content of brine
shrimp nauplii, Artemia. Problems with the nutritional suitability of the
food will be reflected in the survival, growth, and reproduction of the test
organisms in cultures and toxicity tests. Artemia cysts, and other foods must
be obtained as described in Section 5, Facilities, Equipment, and Supplies.
4.8.2 Problems with the nutritional suitability of food will be reflected in
the survival, growth, and reproduction of the test organisms in cultures and
toxicity tests. If a batch of food is suspected to be defective, the
performance of organisms fed with the new food can be compared with the
performance of organisms fed with a food of known quality in side-by-side
tests. If the food is used for culturing, its suitability should be
determined using a short-term chronic test which will determine the affect of
12
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food quality on growth or reproduction of each of the relevant test species in
culture, using four replicates with each food source. Where applicable, foods
used only in chronic toxicity tests can be compared with a food of known
quality in side-by-side, multi-concentration chronic tests, using the
reference toxicant regularly employed in the laboratory QA program. For list
of commercial sources of Artemia cysts, see Table 2 of Section 5, Facilities,
Equipment, and Supplies.
s
4.8.3 New batches of food used in culturing and testing s;hould be analyzed
for toxic organics and metals or whenever difficulty is encountered in meeting
minimum acceptability criteria for control survival and reproduction or
growth. If the concentration of total organochlorine pesticides exceeds 0.15
ng/g wet weight, or the concentration of total organochlorine pesticides plus
PCBs exceeds 0.30 /j,g/g wet weight, or toxic metals (Al, As, Cr, Cd, Cu, Pb,
Ni, Zn, expressed as total metal) exceed 20 ng/g wet weight, the food should
not be used (for analytical methods see AOAC, 1990 and USDA, 1989). For foods
(e.g., such as YCT) which are used to culture and test organisms, the quality
of the food should meet the requirements for the laboratory water used for
culturing and test dilution water as described in Section 4.4 above.
4.9 ACCEPTABILITY OF SHORT-TERM CHRONIC TOXICITY TESTS
4.9.1 For the tests to be acceptable, control survival in fathead minnow,
Pimephales promeTas, and the daphnid, Cen'odaphm'a dubia, tests must be 80% or
greater. At the end of the test, the average dry weight of surviving
seven-day-old fathead minnows in control chambers must equal or exceed
0.25 mg. In Cen'odaphm'a dubia controls, at least 60% of the surviving adults
should have produced their third brood in 7 ฑ 1 days, and the number of young
per surviving adult must be 15 or greater. In algal toxicity tests, the mean
cell density in the controls after 96 h must equal or exceed 2 x 10 cells/mL
and not vary more than 20% among replicates. If these criteria are not met,
the test must be repeated.
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 experience and
professional judgment of the laboratory investigator and the reviewing staff
of the regulatory authority. Any deviation from test specifications must be
noted when reporting data from the test.
4.10 ANALYTICAL METHODS
4.10.1 Routine chemical and physical analyses for culture and dilution water,
food, and test solutions must include established quality assurance practices
outlined in USEPA methods manuals (USEPA, 1979a and USEPA, 1979b).
4.10.2 Reagent containers should be dated and catalogued when received from
the supplier, and the shelf life should not be exceeded. Also, working
solutions should be dated when prepared, and the recommended shelf life should
be observed.
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4.11 CALIBRATION AND STANDARDIZATION ;
4.11.1 Instruments used for routine measurements of chemical and physical
parameters such as pH, DO, temperature, and conductivity, must be calibrated
and standardized according to instrument manufacturer's procedures asj
indicated in the general section on quality assurance (see USEPA Methods
150.1, 360.1, 170.1, and 120.1 in USEPA, 1979b). Calibration data are
recorded in a permanent log book. ;
4.11.2 Wet chemical methods used to measure hardness, alkalinity and total
residual chlorine must be standardized prior to use each day according to the
procedures for those specific USEPA methods (see USEPA Methods 130.2 and 310.1
in USEPA, 1979b).
4.12 REPLICATION AND TEST SENSITIVITY
4.12.1 The sensitivity of the tests will depend in part on the number of
replicates per concentration, the significance level selected, and the type of
statistical analysis. If the variability remains constant, the sensitivity of
the test will increase as the number of replicates is increased. Thelminimum
recommended number of replicates varies with the objectives of the test and
the statistical method used for analysis of the data.
4.13 VARIABILITY IN TOXICITY TEST RESULTS
4.13.1 Factors which can affect test success and precision include (1) the
experience and skill of the laboratory analyst; (2) test organism age*
condition, and sensitivity; (3) dilution water quality; (4) temperature
control; and (5) the quality and quantity of food provided. The results will
depend upon the species used and the strain or source of the test organisms,
and test conditions, such as temperature, DO, food, and water quality. The
repeatability or precision of toxicity tests is also a function of the number
of test organisms used at each toxicant concentration. Jensen (1972)
discussed the relationship between sample size (number of fish) and the
standard error of the test, and considered 20 fish per concentration as
optimum for Probit Analysis.
4.14 TEST PRECISION
4.14.1 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 at least
five tests with a reference toxicant.
4.14.2 Test precision can be estimated by using the same strain of organisms
under the same test conditions and employing a known toxicant, such as a
reference toxicant.
4.14.3 Inter!aboratory precision data from chronic toxicity tests with two
species using the reference toxicants potassium chloride and copper sulfate
are shown in Table 1. Additional precision data for each of the tests
14 '
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described in this manual are presented in the sections describing the
individual test methods.
TABLE 1. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST PRECISION,
1991: SUMMARY OF RESPONSES USING A REFERENCE TOXICANT1
Organism
Endpoint
No. Labs % Effluent'
SD
CV(%)
Pimephales
promelas
Cen'odaphnia
dubia
Survival, NOEC
Growth, IC25
Growth, IC50
Growth, NOEC
Survival, NOEC
Reproduction, IC25
Reproduction, IC50
Reproduction, NOEC
146
124
117
142
162
155
150
156
NA
4.67
6.36
NA
NA
2.69
3.99
NA
NA
1.87
2.04
NA
i NA
1.96
i 2.35
NA
NA
40.0
32.1
NA
NA
72.9
58.9
NA
From a national study of interlaboratory precision of toxicity test data
performed in 1991 by the Environmental Monitoring Systems Laboratory-
Cincinnati ,, U.S. Environmental Protection Agency, Cincinnati, OH 45268.
Participants included Federal, state, and private laboratories engaged in
NPDES permit compliance monitoring. ;
Expressed as % effluent; in reality it was a reference toxicant (KC1)
but was not known by the persons conducting the tests.
4.14.4 Additional information on toxicity test precision!is provided in the
Technical Support Document for Water Quality-based Control (see pp. 2-4, and
11-15 in USEPA, 1991a). ;
4.14.5 In cases where the test data are used in Probit Analysis or other
point estimation techniques (see Section 9, Chronic Toxicity Test Endpoints
and Data Analysis), precision can be described by the mean, standard
deviation, and relative standard deviation (percent coefficient of variation,
or CV) of the calculated endpoints from the replicated tests. In cases where
the test data are used in the Linear Interpolation Method, precision can be
estimated by empirical confidence intervals derived by using the ICPIN Method
(see Section 9, Chronic Toxicity Test Endpoints and Data Analysis). However,
in cases where the results are reported in terms of the No-Observed-Effect
Concentration (NOEC) and Lowest-Observed-Effect Concentration (LOEC) (see
Section 9, Chronic Toxicity Test Endpoints and Data Analysis) precision can
only be described by listing the NOEC-LOEC interval for each test. It is not
possible to express precision in terms of a commonly used statistic. However,
when all tests of the same toxicant yield the same NOEC-LOEC interval, maximum
15
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precision has been attained. The "true" no effect concentration could fall
anywhere within the interval, NOEC ฑ (NOEC minus LOEC).
4.14.6 It should be noted here that the dilution factor selected for;a test
determines the width of the NOEC-LOEC interval and the inherent maximum
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
ฑ 300%. With a dilution factor of 0.5, the NOEC could be considered to have a
relative variability of ฑ 100%. As a result of the variability of different
dilution factors, USEPA recommends the use of the dilution factor of 0.5 or
greater. Other factors which can affect test precision include: test
organism age, condition, and sensitivity; temperature control; and feeding.
4.15 DEMONSTRATING ACCEPTABLE LABORATORY PERFORMANCE
4.15.1 It is a laboratory's responsibility to demonstrate its ability to
obtain consistent, precise results with reference toxicants before it [performs
toxicity tests with effluents for permit compliance purposes. To meet this
requirement, the intralaboratory precision, expressed as percent coefficient
of variation (CV%), of each type of test to be used in the laboratory should
be determined by performing five or more tests with different batches !of test
organisms, using the same reference toxicant, at the same concentrations, with
the same test conditions (i.e., the same test duration, type of dilution
water, age of test organisms, feeding, etc.), and the same data analysis
methods. A reference toxicant concentration series (0.5 or higher) should be
selected that will consistently provide partial mortalities at two or more
concentrations.
4.16 DOCUMENTING ONGOING LABORATORY PERFORMANCE
4.16.1 Satisfactory laboratory performance is demonstrated by performing at
least one acceptable test per month with a reference toxicant for each|
toxicity test method commonly used in the laboratory. For a given test
method, successive tests must be performed with the same reference toxicant,
at the same concentrations, in the same dilution water, using the same data
analysis methods. Precision may vary with the test species, reference
toxicant, and type of test.
4.16.2 A control chart should be prepared for each combination of reference
toxicant, test species, test conditions, and endpoints. Toxicity endpoints
from five or six tests are adequate for establishing the control charts.
Successive toxicity endpoints (NOECs, IC25s, LC50s, etc.) should be plotted
and examined to determine if the results (X.) are within prescribed limits
(Figure 1). The types of control charts illustrated (see USEPA, 1979a) are
used to evaluate the cum'ulative trend of results from a series of samples.
For endpoints that are point estimates (LC50s and IC25s), the cumulative
mean (X) and upper and lower control limits (ฑ 2S) are re-calculated with each
successive test result. Endpoints from hypothesis tests (NOEC, NOAEC); from
each test are plotted directly on the control chart. The control limits would
consist of one concentration interval above and below the concentration
16
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representing the central tendency. After two years of data collection, or a
minimum of 20 data points, the control (cusum) chart should be maintained
using only the 20 most recent data points.
4.16.3 The outliers, which are values falling outside the upper and lower
control limits, and trends of increasing or decreasing sensitivity, are
readily identified. In the case of endpoints that are point estimates (LCBOs
and IC25s), at the Pp 05 probability level, one in 20 tests would be expected
to fall outside of the control limits by chance alone. If more than one out
of 20 reference toxicant tests fall outside the control limits, the effluent
toxicity tests conducted during the month in which the second reference
toxicant test failed are suspect, and should be considered as provisional and
subject to careful review. Control limits for the NOECs will also be exceeded
occasionally, regardless of how well a laboratory performs.
4.16.4 If the toxicity value from a given test with a reference toxicant
falls well outside the expected range for the other test organisms when using
the standard dilution water and other test conditions, 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.
4.16.5 Performance-should improve with experience, and the control limits for
endpoints that are point estimates should gradually narrow. However, control
limits of ฑ 2S will be exceeded 5% of the time by chance alone, regardless of
how well a laboratory performs. Highly proficient laboratories which develop
very narrow control limits may be unfairly penalized if a test result which
falls just outside the control limits is rejected de facto. For this reason,
the width of the control limits should be considered by the permitting
authority in determining whether the outliers should be rejected.
4.17 REFERENCE TOXICANTS
4.17.1 Reference toxicants such as sodium chloride (NaC'l), potassium chloride
(KC1), cadmium chloride (CdCU), copper sulfate (CuSOJ, sodium dodecyl
sulfate (SDS), and potassium dichromate (K2Cr207), are suitable for use in the
NPDES Program and other Agency programs requiring aquatic toxicity tests.
EMSL-Cincinnati hopes to -release USEPA-certified solutions of cadmium and
copper for use as reference toxicants through cooperative research and
development agreements with commercial suppliers, and will, continue to develop
additional reference toxicants for future release. Interested parties can
determine the availability of "EPA Certified" reference toxicants by checking
the EMSL-Cincinnati electronic bulletin board, using a modem to access the
following telephone number: 513-569-7610. Standard reference materials also
can be obtained from commercial supply houses, or can be prepared inhouse
using reagent grade chemicals. The regulatory agency should be consulted
before reference toxicant(s) are selected and used.
17
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Where:
a
o
UPPER CONTROL LIMIT
CENTRAL TENDENCY
LOWER CONTROL LIMIT
0
10
15
20
O
O
O"
UPPER CONTROL LIMIT (X + 2 S)
CENTRAL TENDENCY
LOWER^ONTROL LIMIT (X - 2 S)
0 5 10 15 20 ""
TOXICITYTEST WITH REFERENCE TOXICANTS
X =
n
S =
\
Ey- 2 _ ji=l
^
n
n-l
X
S
= Successive toxicity values from toxicity tests.
= Number of tests.
= Mean toxicity value.
- Standard deviation.
18
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4.18 RECORD KEEPING :
4.18.1 Proper record keeping is important. A complete file should be
maintained for each individual toxicity test or group of tests on closely
related samples. This file should contain a record of the sample chain-of-
custody; a copy of the sample log sheet; the original bench sheets for the
test organism responses during the toxicity test(s); chemical analysis data on
the sample(s); detailed records of the test organisms used in the test(s),
such as species, source, age, date of receipt, and other pertinent information
relating to their history and health; information on the calibration of
equipment and instruments; test conditions employed; and results of reference
toxicant tests. Laboratory data should be recorded on a real-time basis to
prevent the loss of information or inadvertent introduction of errors into the
record. Original data sheets should be signed and dated by the laboratory
personnel performing the tests. :
4.18.2 The regulatory authority should retain records pertaining to discharge
permits. Permittees are required to retain records pertaining to permit
applications and compliance for a minimum of 3 years [40 CFR 122.41(j)(2)].
4.19 VIDEO TAPES OF USEPA CULTURE AND TOXICITY TEST METHODS
4.19.1 Three video-based training packages are available :from the National
Technical Information Service i(NTIS), Department of Commerce, 5285 Port Royal
Road, Springfield, VA 22161. Credit card orders can be placed by calling
toll-free (800) 788-6282, or by FAX at 703-321-8547, or by mail at the above
address. .For other information call 703-487-4650.
1. Order # ELA18254: "U.S. EPA Freshwater Culturing Methods for
Ceriodaphnia dubia and the Fathead Minnow (Pimephales promelas),"
consisting of a 24-minute video and 33-page supplemental report on
culturing Ceriodaphnia, and an 18 minute video and 22-page report on
culturing fathead minnows, and a copy of Short-term Methods for
Estimating the chronic Toxicity of Effluents and Receiving Waters to
Freshwater Organisms (EPA-600/4-89/001). Price $60..00..
2. Order # ELA18036: "U.S. EPA Test Methods for Freshwater Toxicity
Tests," consisting of a 23-minute video and 26-page ,supplemental report
on Ceriodaphnia survival and reproduction toxicity tests, and a 15-
minute video and 18-page report on fathead minnow survival and growth
toxicity tests, and a copy of Short-term Methods for Estimating the
chronic Toxicity of Effluents and Receiving Waters to Freshwater
Organisms (EPA-600/4-89/001). Price $45.00. ;
3. Order # ELA18301: U.S. EPA Culturing and Test Methods for Freshwater
Effluent Toxicity Tests using Ceriodaphnia dubia and Fathead Minnows
(Pimephales promelas), consisting of all four videos and supplemental
reports, and a copy of the short-term toxicity test manual. Price
$90.00.
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4.20 SUPPLEMENTAL REPORTS FOR TRAINING VIDEO TAPES
4.20.1 Supplemental Reports for Training Video Tapes are included in draining
packages above.
20
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SECTION 5
FACILITIES, EQUIPMENT, AND SUPPLIES
5.1 GENERAL REQUIREMENTS
1
5.1.1 Effluent toxicity tests may be performed in a fixed or mobile
laboratory. Facilities must include equipment for rearing and/or holding
organisms. Culturing facilities for test organisms may be desirable in fixed
laboratories which perform large numbers of tests. 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, receiving water, dechlorinated tap water, or
reconstituted synthetic water. Dechlorination can be accomplished by carbon
filtration, or the use of sodium thiosulfate. Use of 3.6 mg (anhydrous)
sodium thiosulfate/L will reduce 1.0 mg chlorine/L. After dechlorination,
total residual chlorine should be non-detectable. Air used for aeration must
be free of oil and toxic vapors. Oil-free air pumps should be used where
possible. Particulates can be removed from the air using BALSTONฎ Grade BX or
equivalent filters (Balston, Inc., Lexington, Massachusetts), and oil and
other organic vapors can be removed using activated carbon filters (BALSTONฎ,
C-l filter, or equivalent).
5.1.2 The facilities must be well ventilated and free from fumes. Laboratory
ventilation systems should be checked to ensure that return air from chemistry
laboratories and/or sample holding areas is not circulated to test organism
culture rooms or toxicity test rooms, or that air from toxicity test rooms
does not contaminate culture areas. Sample preparation, culturing, and
toxicity test areas should be separated to avoid cross cointamination of
cultures or toxicity test solutions with toxic fumes. Air pressure
differentials between such rooms should not result in a net flow of
potentially contaminated air to sensitive areas through open or loosely-
fitting doors. Organisms should be shielded from external disturbances.
5.1.3 Materials used for exposure chambers, tubing, etc., that come in
contact with the effluent and dilution water should be carefully chpsen.
Tempered glass and perfluorocarbon plastics (TEFLONฎ) should be used whenever
possible to minimize sorption and leaching of toxic substances. These
materials may be reused following decontamination. Containers made of
plastics, such as polyethylene, polypropylene, polyvinyl chloride, TYGONฎ,
etc., may be used as test chambers or to ship, store and transfer effluents
and receiving waters, but they should not be reused unless absolutely
necessary, because they could carry over adsorbed toxicants from one test to
another, if reused. However, these containers may be repeatedly reused for
storing uncontaminated waters, such as deionized or laboratory-prepared
dilution waters and receiving waters. Glass or disposable polystyrene
containers can be used for test chambers. The use of large (> 20 L) glass
carboys is discouraged for safety reasons.
5.1.4 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
21
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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 (see Section 5, Facilities, Equipment and
Supplies, Subsection 5.3.2). Fiberglass and stainless steel, in addition to
the previously mentioned materials, can be used for holding, acclimating, and
dilution water storage tanks, and in the water delivery system, but once
contaminated with pollutants the fiberglass should not be reused. All
material should be flushed or rinsed thoroughly with the test media before
using in the test.
5.1.5 Copper, galvanized material, rubber, brass, and lead must not come in
contact with culturing, holding, acclimation, or dilution water, or wi|th
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.
5.1.6 Silicone adhesive used to construct glass test chambers absorbs some
organochlorine and organophosphorus pesticides, which are difficult to remove.
Therefore, as little of the adhesive as possible should be in contact with
water. Extra beads of adhesive inside the containers should be removed.
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 toxicity test method. ;
5.3 CLEANING TEST CHAMBERS AND LABORATORY APPARATUS
5.3.1 New plasticware used for sample collection or organism exposure vessels
does not require thorough cleaning before use. It is sufficient to ri;nse new
sample containers once with dilution water before use. New glassware ;must be
soaked overnight in 10% acid (see below) and rinsed well in deionized water
and dilution water.
5.3.2 All non-disposable sample containers, test vessels, tanks, and [other
equipment that have come in contact with effluent must be washed after use to
remove contaminants as described below. ;
1. Soak 15 min in tap water and scrub with detergent, or clean in an
automatic dishwasher.
2. Rinse twice with tap water. ;
3. Carefully rinse once with fresh, dilute (10%, V:V) hydrochloric jor
nitric acid to remove scale, metals and bases. To prepare a 10%
solution of acid, add 10 ml of concentrated acid to 90 mL of deiionized
water. ;
4. Rinse twice with deionized water.
5. Rinse once with full-strength, pesticide-grade acetone to remove
organic compounds (use a fume hood or canopy).
6. Rinse three times with deionized water.
5.3.3 Special requirements for cleaning glassware used in the green alga,
Selenastrum capricornutum, toxicity tests (Method 1003.0 Section 14). Prepare
22
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2,
3,
4,
5.
all graduated cylinders, test flasks, bottles, volumetric flasks, centrifuge
tubes and vials used in algal assays as follows:
1. Wash with non-phosphate detergent solution, preferably heated to > 50ฐC
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.
Rinse with tap water.
Test flasks should be thoroughly rinsed with acetone and a 10% solution
(by volume) of reagent grade hydrochloric acid (HC1). It may be
advantageous to soak the flasks in 10% HC1 for several days. 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
Rinse twice with MILLIPOREฎ MILLI-Qฎ OR QPAK2, or equivalent, water.
New test flasks, and all flasks which through use may become
contaminated with toxic organic substances, must be Tinsed with
pesticide-grade acetone or heat-treated before use. To thermally
degrade organics, place glassware in a high temperature oven at 400ฐC
for 30 min. After cooling, go to 7. If acetone is used, go to 6.
6. Rinse thoroughly with MILLIPOREฎ MILLI-Qฎ or QPAK,, or equivalent
water, and dry in an 105ฐC oven. All glassware should be autoclaved
before use and between uses. ;
7. Cover the mouth of each chamber with aluminum foil or other closure, as
appropriate, before storing. :
5.3.4 The use of sterile, disposable pipets will eliminate the need for pipet
washing and minimize the possibility of contaminating the cultures with toxic
substances.
i
5.3.5 All test chambers and equipment must be thoroughly rinsed with the
dilution water immediately prior to use in each test.
5.4 APPARATUS AND EQUIPMENT FOR CULTURIN6 AND TOXICITY TESTS
5.4.1 Apparatus and equipment requirements for culturing and testing are
specified in each toxicity test method. Also, see USEPA, 1993b.
5.4.2 WATER PURIFICATION SYSTEM \
i
5.4.2.1 A good quality deionized water, providing 18 mega-ohm, laboratory
grade water, should be available in the laboratory and in sufficient capacity
tor laboratory needs. Deionized water may be obtained from MILLIPOREฎ Milli-
Qฎ, MILLIPOREฎ QPAK or equivalent system. If large quantities of high
quality deionized water are needed, it may be advisable to supply the
laboratory grade deionizer with preconditioned water from a Culliqanฎ
Continentalฎ, or equivalent.
23
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MO
5.5 REAGENTS AND CONSUMABLE MATERIALS
5.5.1 SOURCES OF FOOD FOR CULTURE AND TOXICITY TESTS
1. Brine shrimp, Artemia sp., cysts -- A list of commercial sources is
listed in Table 2.
2. Frozen adult brine shrimp, Artemia -- Available from most pet supply
shops or from San Francisco Bay Brand, 8239 Enterprise Dr., Newark, CA
94560 (415-792-7200). ,
3. Flake fish food -- TETRAMINฎ and BIORILฎ are available from most pet
4 Trout'chow -- Available from Zeigler Bros., P.O. Box 95, Gardners, PA
17324 (717-677-6181 or 800-841-6800); Glencoe Mills, 1011 Elliott St.,
Glencoe, MN 55336 (612-864-3181); or Murray Elevators, 118 West,4800
South, Murray, UT 84107 (800-521-9092). :
5. CEROPHYLLฎ -- Available from Ward's Natural Science Establishment,
Inc., P.O. Box 92912, Rochester, NY 14692-9012 (716-359-2502) or as
cereal leaves from Sigma Chemical Company, P.O. Box 14508, St. Louis,
63178 (800-325-3010).
6. Yeast -- Packaged dry yeast, such as Fleischmann's, or equivalent, can
be purchased at the local grocery store or is available from Lake States
Yeast, Rhine!and, WI. .
7. Alfalfa Rabbit Pellets -- Available from feed stores as Purina rabbit
chow '
8. Algae - Available from (1) the American Type Culture Collection; 12301
Parklawn Drive, Rockville, MD 10852; or (2) the Culture Collection of
Algae, Botany Department, University of Texas, Austin, TX 78712.
5.5.1.1 All food should be tested for nutritional suitability and chemically
analyzed for organochlorine pesticides, PCBs, and toxic metals (see Section 4,
Quality Assurance).
5.5.2 Reagents and consumable materials are specified in each toxicity test
method section. Also, see Section 4, Quality Assurance.
5.6 TEST ORGANISMS '
5.6.1 Test organisms should be obtained from inhouse cultures or from
commercial suppliers (see specific test method; Section 4, Quality Assurance;
and Section 6, Test Organisms). '
I
5.7 SUPPLIES ;
5.7.1 See test methods (see Sections 11-14) for specific supplies.
24
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TABLE 2. COMMERCIAL SUPPLIERS OF BRINE SHRIMP (ARTEMIA) CYSTS1'2
Aquafauna Biomarine
P.O. Box 5
Hawthorne, CA 90250
Tel. (310) 973-5275
Fax. (310) 676-9387
(Great Salt Lake North Arm,
San Francisco Bay) i
Argent Chemical
8702 152nd Ave. NE
Redmond, WA 98052
Tel. (800) 426-6258
Tel. (206) 855-3777
Fax. (206) 885-2112
(Platinum Label - San Francisco
Bay; Gold Label - San Francisco
Bay, Brazil; Silver Label -
Great Salt Lake, Australia;
Bronze Label - China, Canada,
other)
Bonneville Artemia Intl., Inc.
P.O. Box 511113
Salt Lake City, UT 84151-1113
Tel. (801) 972-4704
Fax. (801) 972-7495
Ocean Star International
P.O. Box 643
Snowville, UT 84336
Tel. (801) 872-8217
Fax. (801) 872-8272
(Great Salt Lake)
Sanders Brine Shrimp Co.
3850 South 540 West
Ogden, UT 84405
Tel. (801) 393-5027
(Great Salt Lake)
Sea Critters Inc.
P.O. Box 1508
Tavernier, FL 33070
Tel. (305) 367-2672
Aquarium Products !
180L Penrod Court
Glen Burnie, MD 21061
Tel. (800) 368-2507
Tel. (301) 761-2100
Fax. (410) 761-6458
(Columbia)
INVE Artemia Systems
Oeverstraat 7
B-9200 Baasrode, Belgium
Tel. 011-32-52-331320
Fax. 011-32-52-341205
(For marine species - AF grade)
[small nauplii], UL grade [large
nauplii], for freshwater species-
Hi grade [small nauplii], EG grade
[large nauplii]
Golden West Artemia
411 East 100 South
Salt Lake City, UT 84111
Tel. (801) 975-1222
Fax. (801) 975-1444:
San Francisco Bay Brand
8239 Enterprise Drive
Newark, CA 94560 ;
Tel. (510) 792-7200
Fax. (510) 792-5360
(Great Salt Lake,
San Francisco Bay)
Western Brine Shrimp
957 West South Temple
Salt Lake City, UT 84104
Tel. (801) 364-3642
Fax. (801) 534-0211:
(Great Salt Lake)
2 List from David A. Bengston, University of Rhode Island, Narragansett, RI
The geographic sources from which the vendors obtain the brine shrimp
cysts are shown in parentheses. i
25
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SECTION 6
TEST ORGANISMS
6.1 TEST SPECIES ;
6.1.1 The species used in characterizing the chronic toxicity of effluents
and/or receiving waters will depend on the requirements of the regulatory
authority and the objectives of the test. It is essential that good quality
test organisms be readily available throughout the year from inhouse or
commercial sources to meet NPDES monitoring requirements. The organisms used
in the toxicity tests must be identified to species. If there is any doubt as
to the identity of the test organism, representative specimens should be sent
to a taxonomic expert to confirm the identification.
6.1.2 Toxicity test conditions and culture methods for the species listed in
Subsection 6.1.3 are provided in this manual also, see USEPA, 1993b. ;
6.1.3 The organisms used in the short-term chronic toxicity tests described
in this manual are the fathead minnow, Pimephales promeTas, the daphnid,
Ceriodaphnia dubia (Berner, 1986), and the green alga, Selenastrum
capricornutum.
6.1.4 Some states have developed culturing and testing methods for indigenous
species that may be as sensitive, or more sensitive, than the species
recommended in Subsection 6.1.3. However, USEPA allows the use of indigenous
species only where state regulations require their use or prohibit importation
of the recommended species in Subsection 6.2.6. Where state regulations
prohibit importation of non-native fishes or the use of recommended test
species, permission must be requested from the appropriate state agency prior
to their use.
6.1.5 Where states have developed culturing and testing methods, for
indigenous species other than those recommended in this manual, data comparing
the sensitivity of the substitute species and the one or more recommended
species must be obtained in side-by-side toxicity tests with reference
toxicants and/or effluents, to ensure that the species selected are at least
as sensitive as the recommended species. These data must be submitted to the
permitting authority (State or Region) if required. USEPA acknowledges that
reference toxicants prepared from pure chemicals may not always be j
representative of effluents. However, because of the observed and/or
potential variability in the quality and toxicity of effluents, it is not
possible to specify a representative effluent.
6.1.6 Guidance for the selection of test organisms where the salinity of the
effluent and/or receiving water requires special consideration is provided in
the Technical Support Document for Water Quality-based Toxics Control (USEPA,
1991a).
1. Where the salinity of the receiving water is < l%o, freshwater lorganisms
are used regardless of the salinity of the effluent.
26
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2. Where the salinity of the receiving water is > l%o, :the choice of
organisms depends on state water quality standards and/or permit
requirements. ,
6.2 SOURCES OF TEST ORGANISMS
6.2.1 The test organisms recommended in this manual can be cultured in the
laboratory using culturing and handling methods for each organism described in
the respective test method sections. The fathead minnow, Pimephales promelas,
culture method is given in Section 11 and not repeated in Section 12. Also,
see USEPA (1993b). ;'
6.2.2 Inhouse cultures should be established wherever it is cost effective.
If inhouse cultures cannot be maintained or it is not. cost effective, test
organisms or starter cultures should be purchased from experienced commercial
suppliers (see USEPA, 1993b).
6.2.3 Starter cultures of the green algae, Selenastrum cupricornutum,
5. minutum, and Chlamydomonas reinhardti are available from the following
sources:
1. American Type Culture Collection (Culture No. ATCC 22662), 12301
Parklawn Drive, Rockville, MD 10852.
2. Culture Collection of Algae, Botany Department, University of Texas,
Austin, TX 78712.
6.2.4 Because the daphnid, Cen'odaphnia dubia, must be cultured individually
in the laboratory for at least seven days before the test begins, it will be
necessary to obtain a starter culture from a commercial source at least three
weeks before the test is to begin if they are not being cultured inhouse.
6.2.5 If, because of their source, there is any uncertainty concerning the
identity of the organisms, it is advisable to have them examined by a
taxonomic specialist to confirm their identification. For detailed guidance
on identification, see the individual test methods.
6:2.6 FERAL (NATURAL OCCURRING, WILD CAUGHT) ORGANISMS
6.2.6.1 The use of test organisms taken from the receiving water has strong
appeal, and would seem to be a logical approach. However, it is generally
impractical and not recommended for the following reasons:
1. Sensitive organisms may not be present in the receiving water because of
previous exposure to the effluent or other pollutants.
2. It is often difficult to collect organisms of the required age and
quality from the receiving water.
3. Most states require collecting permits, which may be difficult to
obtain. Therefore, it is usually more cost effective to culture the
organisms in the laboratory or obtain them from private, state, or
Federal sources. The fathead minnow, Pimephales promelas, the daphnid,
Cen'odaphnia dubia, and the green alga, Selenastrum capricornutum, are
easily cultured in the laboratory or readily available commercially.
27
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4. The required QA/QC records, such as the single laboratory precision
data, would not be available. ;
5. Since it is mandatory that the identity of the test organism be!
known to species level, it would be necessary to examine each organism
caught in the wild to confirm its identity. This would usually be
impractical or, at the least, very stressful to the organisms.
6. Test organisms obtained from the wild must be observed in the laboratory
for a minimum of one week prior to use, to assure that they arelfree of
signs of parasitic or bacterial infections and other adverse effects.
Fish captured by electroshocking must not be used in toxicity testing.
6.2.6.2 Guidelines for collecting natural occurring organisms are provided in
USEPA (1973), USEPA (1990) and USEPA (1993c). '
6.2.7 Regardless of their source, test organisms should be carefully'observed
to ensure that they are free of signs of stress and disease, and in good
physical condition. Some species of test organisms can be obtained from
commercial stock certified as "disease-free".
6.3 LIFE STAGE
6.3.1 Young organisms are often more sensitive to toxicants than are|adults.
For this reason, the use of early life stages, such as larval fish, is
required for all tests. There may be special cases, however, where the
limited availability of organisms will require some deviation from the
recommended life stage. In a given test, all ^organisms should be ;
approximately the same age and should be taken from the same source. (Since
age may affect the results of the tests, it would enhance the value and
comparability of the data if the same species in the same life stages iwere
used throughout a monitoring program at a given facility.
6.4 LABORATORY CULTURIN6
I
6.4.1 Instructions for culturing and/or holding the recommended test |
organisms are included in the respective test methods (also, see USEPA,
1993b).
6.5 HOLDING AND HANDLING TEST ORGANISMS
6.5.1 Test organisms should not be subjected to changes of more than |3ฐC in
water temperature in any 12 h period or 2 units of pH in any 24-h period.
6.5.2 Organisms should be handled as little as possible. When handling is
necessary, it should be done as gently, carefully, and quickly as possible to
minimize stress. Organisms that are dropped or touch a dry surface or are
injured during handling must be discarded. Dipnets are best for handling
larger organisms. These nets are commercially available or can be made from
small-mesh nylon netting, silk batting cloth, plankton netting, or similar
material. Wide-bore, smooth glass tubes (4 to 8 mm ID) with rubber bulbs or
pipettors (such as PROPIPETTEฎ) should be used for transferring smaller
organisms such as larval fish. I
28
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6.5.3 Holding tanks for fish are supplied with good quality water (see
Section 5, Facilities, Equipment, and Supplies) with flow-through rate of at
least two tank volumes per day. Otherwise use a recirculation system where
water flows through an activated carbon or undergravel filter to remove
dissolved metabolites. Culture water can also be piped through high intensity
ultraviolet light sources for disinfection, and to photodegrade dissolved
organics.
6.5.4 Crowding must be avoided because it will stress the organisms and lower
the DO concentrations to unacceptable levels. The solution of oxygen depends
on temperature and altitude. The DO must be maintained at a minimum of 4.0
mg/L. Aerate gently if necessary.
6.5.5 The organisms should be observed carefully each day for signs of
disease, stress, physical damage, or mortality. Dead and abnormal organisms
should be removed as soon as observed. It is not uncommon for some fish
mortality (5-10%) to occur during the first 48 h in a holding tank because of
individuals that refuse to feed on artificial food and die of starvation.
Organisms in the holding tanks should generally be fed as in the cultures (see
culturing methods in the respective methods).
6.5.6 Fish should be fed as much as they will eat at least once a day with
live brine shrimp nauplii, Artemia, or frozen adult brine shrimp, or dry food
(frozen food should be completely thawed before use). Adult brine shrimp can
be supplemented with commercially prepared food such as TETRAMINฎ.or BIORILฎ
flake food, or equivalent. Excess food and fecal material should be removed
from the bottom of the tanks at least twice a week by siphoning.
6.5.7 Fish should be observed carefully each day for signs of disease,
stress, physical damage, and mortality. Dead and abnormal specimens should be
removed as soon as observed. It is not uncommon to have some fish (5-10%)
mortality during the first 48 h in a holding tank because of individuals that
refuse to feed on artificial food and die of starvation. :Fish in the holding
tanks should generally be fed as in the cultures (see culturing methods in the
respective methods).
6.5.8 A daily record of feeding, behavioral observations^ and mortality
should be maintained.
6.6 TRANSPORTATION TO THE TEST SITE ;
6.6.1 Organisms are transported from the base or supply laboratory to a
remote test site in culture water or standard dilution water in plastic bags
or large-mouth screw-cap (500 ml) plastic bottles in styrofoam coolers.
Adequate DO is maintained by replacing the air above the water in the bags
with oxygen from a compressed gas cylinder, and sealing tine bags or by use of
an airstone supplied by a portable pump, the DO concentration must not fall
below 4.0 mg/L.
6.6.2 Upon arrival at the test site, the organisms are transferred to
receiving water if receiving water is to be used as the test dilution water.
All but a small volume of the holding water (approximately 5%) is removed by
29
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siphoning and replaced slowly over a 10 to 15 minute period with dilution
water. If receiving water is to be used as the dilution water, caution must
be exercised in exposing the test organisms to it, because of the possibility
that it might be toxic. For this reason, it is recommended that only
approximately 10% of the test organisms be exposed initially to the dilution
water. If this group does not show excessive mortality or obvious signs of
stress in a few hours, the remainder of the test organisms may be transferred
to the dilution water.
*
6.6.3 A group of organisms must not be used for a test if they appear:to be
unhealthy, discolored, or otherwise stressed, or if mortality appears to
exceed 10% preceding the test. If the organisms fail to meet these criteria,
the entire group must be discarded and a new group obtained. The mortality
may be due to the presence of toxicity, if the receiving water is used; as
dilution water, rather than a diseased condition of the test organisms. If
the acclimation process is repeated with a new group of test organisms and
excessive mortality occurs, it is recommended that an alternative source of
dilution water be used. ;
6.7 TEST ORGANISM DISPOSAL
6.7.1 When the toxicity test(s) is concluded, all test organisms (including
controls) should be humanely destroyed and disposed of in an appropriate
manner.
30
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SECTION 7
DILUTION WATER !
7.1 TYPES OF DILUTION WATER
7.1.1 The type of dilution water used in effluent toxicity tests will depend
largely on the objectives of the study.
7.1.1.1 If the objective of the test is to estimate the chronic toxicity of
the effluent, which is the primary objective of NPDES permit-related toxicity
testing, a synthetic (standard) dilution water (moderately hard water) is
used. If the test organisms have been cultured in water which is different
from the test dilution water, a second set of controls, using culture water,
should be included in the test. ' ;
7.1.1.2 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 receiving water (if
non-toxic), collected either upstream and outside the influence of the
outfall, or with other uncontaminated natural water (ground or surface water)
or standard dilution water having approximately the same characteristics
(hardness, alkalinity, and conductivity) as the receiving^water. Seasonal
variations in the quality of receiving waters may affect effluent toxicity.
Therefore, the pH, alkalinity, hardness, and conductivity1of receiving water
samples should be determined before each use. If the test organisms have been
cultured in water which is different from the test dilution water, a second
set of controls, using culture water, should be included in the test.
7.1.1.3. If the objective of the test is to determine the1additive or
mitigating effects of the discharge on already contaminated receiving water,
the test is performed using dilution water consisting of receiving water
collected immediately upstream or outside the influence of the outfall.
A second set of controls, using culture water, should be included in the test.
7.2 STANDARD, SYNTHETIC DILUTION WATER
7.2.1 Standard, synthetic dilution water is prepared with deionized water and
reagent grade chemicals or mineral water (Tables 3 and 4). The source water
for the deionizer can be ground water or tap water.
7.2.2 DEIONIZED WATER USED TO PREPARE STANDARD, SYNTHETIC, DILUTION WATER
7.2.2.1 Deionized water is obtained from a MILLIPOREฎ MlLLI-Qฎ, MILLIPOREฎ
QPAK2 or equivalent system. It is advisable to provide a preconditioned
(deionized) feed water by using a Culliganฎ, Continentalฎ;, or equivalent
system in front of the MILLIPOREฎ System to extend the life of the MILLIPOREฎ
cartridges (see Section 5, Facilities, Equipment, and Supplies).
7.2.2.2 The recommended order of the cartridges in a four-cartridge deionizer
(i.e., MILLI-Qฎ System or equivalent) is (1) ion exchange, (2) ion exchange,
31
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(3) carbon, and (4) organic cleanup (such as ORGANEX-Qฎ, or equivalent)
followed by a final bacteria filter. The QPAK2 water system is a sealed
system which does not allow for the rearranging of the cartridges. However,
the final cartridge is an ORGANEX-Qฎ filter, followed by a final bacteria
filter. Commercial laboratories using this system have not experience^1 any
difficulty in using the water for culturing or testing. Reference to jthe
MILLI-Qฎ systems throughout the remainder of the manual includes all
HILLIPOREฎ or equivalent systems.
7.2.3 STANDARD, SYNTHETIC FRESHWATER j
7.2.3.1 To prepare 20 L of synthetic, moderately hard, reconstituted water,
use the reagent grade chemicals in Table 3 as follows:
1. Place 19 L of MILLI-Qฎ, or equivalent, water in a properly cleaned
plastic carboy.
2. Add 1.20 g of MgS04, 1.92 g NaHC03, and O.OSOg KC1 to the carboy.
3. Aerate overnight.
4. Add 1.20 g of CaS04ซ2H20 to 1 L of MILLI-Qฎ or equivalent deionized
water in a separate flask. Stir on magnetic stirrer until calcium
sulfate is dissolved, add to the 19 L above, and mix well. '
5. For Cen'odaphnia dubia culturing and testing, add sufficient sodium
selenate (Na2Se04) to provide 2 /ig selenium per liter of final dilution
water. '
6. Aerate the combined solution vigorously for an additional 24 h tb
dissolve the added chemicals and stabilize the medium.
7. The measured pH, hardness, etc., should be as listed in Table 3.
7.2.3.2 If large volumes of synthetic reconstituted water will be needed, it
may be advisable to mix 1 L portions of concentrated stock solutions of
NaHC03, MgS04, and KC1 for use in preparation of the reconstituted waters.
7.2.3.3 To prepare 20 L of standard, synthetic, moderately hard,
reconstituted water, using mineral water such as PERRIERฎ Water, or equivalent
(Table 4), follow the instructions below. !
1. Place 16 L of MILLI-Qฎ or equivalent water in a properly cleaned plastic
carboy.
2. Add 4 L of PERRIERฎ Water, or equivalent. ;
3. Aerate vigorously for 24 h to stabilize the medium. i
4. The measured pH, hardness and alkalinity of the aerated water will be as
indicated in Table 4. .[
5. This synthetic water is referred to as diluted mineral water (DMW) in
the toxicity test methods. !
7.3 USE OF RECEIVING WATER AS DILUTION WATER ;
7.3.1 If the objectives of the test require the use of uncontaminated
receiving water as dilution water, and the receiving water is uncontaminated,
it may be possible to collect a sample of the receiving water upstream of, or
close to, but outside of the zone influenced by the effluent. However, if the
receiving water is contaminated, it may be necessary to collect the sample in
32 ;
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TABLE 3. PREPARATION OF SYNTHETIC FRESHWATER USING REAGENT GRADE
CHEMICALS1
Water
Type
Reagent
Added (mg/L)2
NaHC03 CaS04.2H20
Very soft
Soft
Moderately
Hard
Hard
Very hard
12.
48.
96.
192.
384.
0
0
0
0
0
7.
30.
60.
120.
240.
5
0
0
0
0
MgS04
7
30
60
120
240
.5
.0
.0
.0
.0
KC1
0.5
2.0
4.0
8.0
16.0
Final Water
z /,
pH3
6.4-6
7.2-7
7.4-7
7.6-8
8.0-8
.8
.6
.8
.0
.4
Hardness
: 10-13
40-48
: 80-100
1 160-180
280-320
Quality
Alka-
linity4
10-13
30-35
60-70
110-120
225-245
Taken in part from Marking and Dawson (1973).
Add reagent grade chemicals to deionized water. i
Approximate equilibrium pH after 24 h of aeration.
Expressed as mg CaC03/L. ;
an area "remote" from the discharge site, matching as closely as possible the
physical and chemical characteristics of the receiving water near the outfall.
7.3.2 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,
or in the case where large volumes are required for flow through tests, the
sample should be chilled to 4ฐC during or immediately following collection,
and maintained at that temperature prior to use in the test.
7.3.3 Receiving water containing debris or indigenous organisms that may be
confused with or attack the test organisms should be filtered through a sieve
having 60 /m mesh openings prior to use.
7.3.4 Where toxicity-free dilution water is required in ;a test, the water is
considered acceptable if test organisms show the required survival, growth,
and reproduction in the controls during the test. ;
7.3.5 The regulatory authority may require that the hardness of the dilution
water be comparable to the receiving water at the discharge site. This
requirement can be satisfied by collecting an uncontaminetted receiving water
with a suitable hardness, or adjusting the hardness of an otherwise suitable
receiving water by addition of reagents as indicated in Table 3.
33
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TABLE 4. PREPARATION OF SYNTHETIC FRESHWATER USING MINERAL WATER1
Final Water Quality
Volume of
Water
Type
Mineral Watef
Added
(mi/ir
Proportion
of Mineral
Water
(%)
PH3
Hardness4
Al ka-
linity4
Very soft 50
Soft 100
Moderately Hard 200
Hard 400
Very hard
2.5
10.0
20.0
40.0
7.2-8.1
7.9-8.3
7.9-8.3
7.9-8.3
10-13
40-48
80-100
160-180
10-13
30-35
60-70
110-120
From Mount et al. (1987), and data provided by Philip Lewis,
EMSL-Cincinnati, OH. ;
Add mineral water to Milli-Qฎ water, or equivalent, to prepare Diluted
Mineral Water (DMW).
Approximate equilibrium pH after 24 h of aeration.
Expressed as mg CaCO,/L.
Dilutions of PERRIERฎ Water form a precipitate when concentrations
equivalent to "very hard water" are aerated. :
7.4 USE OF TAP WATER AS DILUTION WATER
7.4.1 The use of tap water as dilution water is discouraged unless it Jis
dechlorinated and passed through a deionizer and carbon filter. Tap water can
be dechlorinated by deionization, carbon filtration, or the use of sodium
thiosulfate. Use of 3.6 mg/L (anhydrous) sodium thiosulfate will reduce
1.0 mg chlorine/L (APHA, 1992). Following dechlbrination, total residual
chlorine should not exceed 0.01 mg/L. Because of the possible toxicity of
thiosulfate to test organisms, a control lacking thiosulfate should be
included in toxicity tests utilizing thiosulfate-dechlorinated water.
7.4.2 To be adequate for general laboratory use following dechlorinatiion, the
tap water is passed through a deionizer and carbon filter to remove toxic
metals and organics, and to control hardness and alkalinity.
7.5 DILUTION WATER HOLDING
7.5.1 A given batch of dilution water should not be used for more than
14 days following preparation because of the possible build-up of bacterial,
fungal, or algal slime growth and the problems associated with it. The
container should be kept covered and the contents should be protected from
light. . !
34
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SECTION 8
EFFLUENT AND RECEIVING WATER SAMPLING, SAMPLE HANDLING,
AND SAMPLE PREPARATION FOR TOXICITY TESTS
8.1 EFFLUENT SAMPLING
8.1.1 The effluent sampling point should be the same as that specified in the
NPDES discharge permit (USEPA, 1988a). Conditions for exception would be:
(1) better access to a sampling point between the final treatment and the
discharge outfall; (2) if tne processed waste is chlorinated prior to
discharge, 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.
8.1.2 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 time, 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 prohibitively large number of
separate samples and tests. Collection of a 24-h composite .sample, however,
may dilute toxicity spikes, and average the quality of the effluent over the
sampling period. Sampling recommendations are provided below (also see USEPA,
1993b).
8.1.3 Aeration during collection and transfer of effluents should be
minimized to reduce the loss of volatile chemicals.
8.1.4 Details of date, time, location, duration, and procedures used for
effluent sample and dilution water collection should be recorded.
8.2 EFFLUENT SAMPLE TYPES
8.2.1 The advantages and disadvantages of effluent grab and composite samples
are listed below:
8.2.1.1 GRAB SAMPLES \
Advantages:
1. Easy to collect; require a minimum of equipment and on-site time.
2. Provide a measure of instantaneous toxicity. Toxicity spikes are not
masked by dilution.
35
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Disadvantages:
1. Samples are collected over a very short period of time and on a
relatively infrequent basis. The chances of detecting a spike ip
toxicity would depend on the frequency of sampling and the probability
of missing a spike is high.
8.2.1.2 COMPOSITE SAMPLES
Advantages:
1.
2,
A single effluent sample is collected over a 24-h period.
The sample is collected over a much longer period of time than a single
grab sample and contains all toxicity spikes.
Disadvantages:
1. Sampling equipment is more sophisticated and expensive, and must:be
placed on-site for at least 24 h.
2. Toxicity spikes may not be detected because they are masked by dilution
with less toxic wastes.
8.3 EFFLUENT SAMPLING RECOMMENDATIONS
8.3.1 When tests are conducted on-site, test solutions can be renewed daily
with freshly collected samples, except for the green alga, Selenastrum
capricornutum, test which is not renewed.
8.3.2 When tests are conducted off-site, a minimum of three samples are
collected. If these samples are collected on Test Days 1, 3, and 5, the first
sample would be used for test initiation, and for test solution renewal on Day
2. The second sample would be used for test solution renewal on Days 3 and 4.
The third sample would be used for test solution renewal on Days 5, 6, and 7.
-,
8.3.3 Sufficient sample volume must be collected to perform the required
toxicity and chemical tests. A 4-L (1-gal) CUBITAINERฎ will provide
sufficient sample volume for most tests. '
8.3.4 THE FOLLOWING EFFLUENT SAMPLING METHODS ARE RECOMMENDED: !
8.3.4.1 Continuous Discharges
1. If the facility discharge is continuous, but the calculated retention
time of a continuously discharged effluent is less than 14 days and the
variability of the waste is unknown, at a minimum, four grab samples or
four composite samples are collected over a 24-h period. For example, a
grab sample is taken every.6 h (total of four samples) and each sample
is used for a separate toxicity test, or four successive 6-h composite
samples are taken and each is used in a separate test.
2. If the calculated retention time of a continuously discharged effluent
is greater than 14 days, or if it can be demonstrated that the :
36
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wastewater does not vary more than 10% in toxicity over a 24-h period,
regardless of retention time, a single grab sample is collected for a
single toxicity test.
3. The retention time of the effluent in the wastewater treatment facility
may be estimated from calculations based on the volume of the retention
basin and rate of wastewater inflow. However, the calculated retention
time may be much greater than the actual time because of
short-circuiting in the holding basin. Where short-circuiting is
suspected, or sedimentation may have reduced holding basin capacity, a
more accurate estimate of the retention time can be obtained by carrying
out a dye study. !
8.3.4.2. Intermittent discharges ;
8.3.4.2.1 If the facility discharge is intermittent, a single grab sample is
collected midway during each discharge period. Examples of intermittent
discharges are: ;
1. When the effluent is continuously discharged during a single 8-h work
shift (one sample is collected) or two successive 8-h work shifts (two
samples are collected).
2. When the facility retains the wastewater during an B-h work shift, and
then treats and releases the wastewater as a batch discharge (one sample
is collected).
3. When, at the end the shift, clean up activities result, in the discharge
of a slug of toxic wastes (one sample is collected).
8.4 RECEIVING WATER SAMPLING
8.4.1 Logistical problems and difficulty in securing sampling equipment
generally preclude the collection of composite receiving water samples for
toxicity tests. Therefore, based on the requirements of the test, a single
grab sample or daily grab sample of receiving water is collected for use in
the test.
8.4.2 The sampling point is determined by the objectives, of the test. In
rivers, samples should be collected from mid-stream and at mid-depth, if
accessible. In lakes the samples are collected at mid-depth.
8.4.3 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, and the rate and degree of mixing, may be difficult to ascertain.
Therefore, 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.
37 ;
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8.5 EFFLUENT AND RECEIVING WATER SAMPLE HANDLING, PRESERVATION, AND SHIPPING
8.5.1 Unless the samples are used in an on-site toxicity test the day of
collection, they should be chilled and maintained at 4ฐC until used to inhibit
microbial degradation, chemical transformations, and loss of highly volatile
toxic substances. :
8.5.2 Composite samples should be chilled as they are collected.
samples should be chilled immediately following collection.
Grab
8.5.3 If the effluent has been chlorinated, total residual chlorine must be
measured immediately following sample collection.
8.5.4 Sample holding time begins when the last grab sample in a series is
taken (i.e., when a series of four grab samples are taken over a 24-h period),
or when a 24-h composite sampling period is completed. If the data from the
samples are to be acceptable for use in the NPDES Program, the lapsed ^time
(holding time) from sample collection to first use of the sample in test
initiation must not exceed 36 h. EPA believes that 36 h is adequate time to
deliver the samples to the laboratories performing the test in most cases. In
the isolated cases, where the permittee can document that this delivery time
cannot be met, the permitting authority can allow an option for on-site
testing or a variance for an extension of shipped sample holding time. The
request for a variance in sample holding time, directed to the USEPA Regional
Administrator under 40 CFR 136.3(e) must include supportive data which show
that the toxicity of the effluent sample is not reduced (e.g., because of
volatilization and/or sorption of toxics on the sample container surfaces) by
extending the holding time beyond more than 36 h. However in no case should
more than 72 h elapse between collection and first use of the sample. In
static-renewal tests, the original sample may also be used to prepare test
solutions for renewal at 24 h and 48 h after test initiation, if stored at
4ฐC, with minimum head space, as described in Subsection 8.5. Guidance for
determining the persistence of the sample is provided in Subsection 8.7.
8.5.5 To minimize the loss of toxicity due to volatilization of toxic
constituents, all sample containers should be "completely" filled, leaving no
air space between the contents and the lid. ;
8.5.6 SAMPLES USED IN ON-SITE TESTS
8.5.6.1 Samples collected for on-site tests should be used within 24 h.
8.5.7 SAMPLES SHIPPED TO OFF-SITE FACILITIES !
8.5.7.1 Samples collected for off-site toxicity testing are to be chilled to
4ฐC during or immediately after collection, and shipped iced to the performing
laboratory. Sufficient ice should be placed with the sample in the shipping
container to ensure that ice will still be present when the sample arrives at
the laboratory and is unpacked. Insulating material must not be placed
between the ice and the sample in the shipping container. ;
38
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8 5.7.2 Samples may be shipped in one or more 4-L (1-gal) CUBITAINERSฎ or new
plastic "milk" jugs. All sample containers should be rinsed with source water
before being filled with sample. After use with receiving water or effluents,
CUBITAINERSฎ and plastic jugs are punctured to prevent reuse.
8573 Several sample shipping options are available, including Express
Mail, air express, bus, and courier service. Express Mail is delivered seven
days a week. Saturday and Sunday shipping and receiving schedules of private
.carriers vary with the carrier.
8.6 SAMPLE RECEIVING
8.6.1 Upon arrival at the laboratory, samples are logged in and the
temperature is measured and recorded. If the samples are not immediately
prepared for testing, they are stored at 4ฐC until used.
8.6.2 Every effort must be made to initiate the test with an effluent sample
on the day of arrival in the laboratory, and the sample holding time should
not exceed 36 h unless a variance has been granted by the NPDES permitting
authority.
8.7 PERSISTENCE OF EFFLUENT TOXICITY DURING SAMPLE SHIPMENT AND HOLDING
8.7.1 The persistence of the toxicity of an effluent prior to its use in a
toxicity test is of interest in assessing the validity of toxicity test data,
and in determining the possible effects of allowing an extension of the
holding time. Where a variance in holding time (> 36 h, but < 72 h) is
requested by a permittee, (see Subsection 8.5.4 above), information on the
effects of the extension in holding time on the toxicity of samples must be
obtained by comparing the results of multi-concentration chronic toxicity
tests performed on effluent samples held 36 h with toxicity test results using
the same samples after they were held for the requested,:longer period. The
portion of the sample set aside for the second test should be held under the
same conditions as during shipment and holding.
8.8 PREPARATION OF EFFLUENT AND RECEIVING WATER SAMPLES FOR TOXICITY TESTS
8.8.1 When aliquots are removed from the sample container, the head space
above the remaining sample should be held to a minimum. Air which enters a
container upon removal of sample should be expelled by compressing the
container before reclosing, if possible (i.e., where a CUBITAINERฎ is used),
or by using an appropriate discharge valve (spigot).
8.8.2 With the daphnid, Cen'odaphm'a dubia, and fathead minnow, Pimephales
promelas, tests, effluents and receiving waters must be filtered through a
60-Aim plankton net to remove indigenous organisms that may attack or be
confused with the test organisms (see the daphnid, Cen'odaphm'a dubia, test
method for details). Receiving waters used in green alga, Selenastrum
capn'cornutum, toxicity tests must be filtered through a;0.45-Mm 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-mm holes to remove
debris and/or break up large floating or suspended solids. Because filtration
39
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may increase the DO in the effluent, the DO should be checked both before and
after filtering. Caution: filtration may remove some toxicity.
8.8.3 If the samples must be warmed to bring them to the prescribed test
temperature, supersaturation of the dissolved oxygen and nitrogen may'become a
problem. To avoid this problem, the effluent-an.d dilution water are checked
with a DO probe after reaching test temperature and, if the DO is greater than
100% saturation or lower than 4.0 mg/L, the solutions are aerated moderately
(approximately 500 mL/min) for a few minutes, using an airstone, until the DO
is within the prescribed range (> 4.0 mg/L). Caution: avoid excessive
aeration.
8.8.4 The DO concentration in the samples should be near saturation prior to
use. Aeration will bring the DO and other gases into equilibrium with air,
minimize oxygen demand, and stabilize the pH. However, aeration" during
collection, transfer, and preparation of samples should be minimized to reduce
the loss of volatile chemicals.
8.8.4.1 Aeration during the test may alter the results and should be iused
only as a last resort to maintain the required DO. Aeration can reduce the
apparent toxicity of the test solutions by stripping them of highly volatile
toxic substances, or increase their toxicity by altering pH. However, the DO
in the test solutions must not be allowed to fall below 4.0 mg/L.
8.8.4.2 In static tests (renewal or non-renewal), low DOs may commonly occur
in the higher concentrations of wastewater. Aeration is accomplished by
bubbling air through a pipet at a rate of 100 bubbles/min. If aeration is
necessary, all test solutions must be aerated. It is advisable to monjitor the
DO closely during the first few hours of the test. Samples with a potential
DO problem generally show a downward trend in DO within 4 to 8 h after the
test is started. Unless aeration is initiated during the first 8 h of the
test, the DO may be exhausted during an. unattended period, thereby .
invalidating the test.
8.8.5 At a minimum, pH, conductivity, and total residual chlorine are
measured in the undiluted effluent or receiving water, and pH and conductivity
are measured in the dilution water. :
8.8.5.1 It is recommended that total alkalinity and total hardness also be
measured in the undiluted effluent test water, receiving water, and the
dilution water.
8.8.6 Total ammonia is measured in effluent and receiving water samples where
toxicity may be contributed by un-ionized ammonia (i.e., where total ammonia
ฃ 5 mg/L). The concentration (mg/L) of un-ionized (free) ammonia in a-sample
is a function of temperature and pH, and is calculated using the percentage
value obtained from Table 5, under the appropriate pH and temperature, and
multiplying it by the concentration (mg/L) of total ammonia in the sample.
8.8.7 Effluents and receiving waters can be dechlorinated using 6.7 mg/L
anhydrous sodium thiosulfate to reduce 1 mg/L chlorine (APHA, 1992). Note
that the amount of thiosulfate required to dechlorinate effluents is greater
40 !
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TABLE 5. PERCENT UNIONIZED NH3 IN AQUEOUS AMMONIA SOLUTIONS:
15-26ฐC AND pH 6.0-8.91
TEMPERATURES
pH
TEMPERATURE (ฐC)
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9
15
0.0274
0.0345
0.0434
0.0546
0.0687
0.0865
0.109
0.137
0.172
0.217
0.273
0.343
0.432
0.543
0.683
0.858
1.08
1.35
1.70
2.13
2.66
3.33
4.16
5.18
6.43
7.97
9.83
12.07
14.7
17.9
16
0.0295
0.0372
0.0468
0.0589
0.0741
0.0933
0.117
0.148
0.186
0.234
0.294
0.370
0.466
0.586
0.736
0.925
1.16
1.46
1.83
2.29
2.87
3.58
4.47
5.56
6.90
8.54
10.5
12.9
15.7
19.0
17
0.0318
0.0400
0.0504
0.0634
0.0799
0.1005
0.127
0.159
0.200
0.252
0.317
0.399
0.502
0.631
0.793
0.996
1.25
1.57
1.97
2.46
3.08
3.85
4.80
5.97
7.40
9.14
11.2
13.8
16.7
20.2
18
0.0343
0.0431
0.0543
0.0683
0.0860
0.1083
0.136
0.171
0.216
0.271
0.342
0.430
0.540
0.679
0.854
1.07
1.35
1.69
2.12
2.65
3.31
4.14
5.15
6.40
7.93
9.78
12.0
14.7
17.8
21.4
19
0.0369
0.0464
0.0584
0.0736
0.0926
0.1166
0.147
0.185
0.232
0.292
0.368
0.462
0.581
0.731
0.918
1.15
1.45
1.82
2.28
2.85
3.56
4.44
5.52
6.86
8.48
10.45
12.8
15.6
18.9
22.7
20
0.0397
0.0500
0.0629
0.0792
0.0996
0.1254
0.158
0.199
0.250
0.314
0.396
0.497
0.625
0.786
0.988
1.24
1.56
1.95
2.44
3.06
3.82
4.76
5.92
7.34
9.07
11.16
13.6
16.6
20.0
24.0
21
0.0427
0.0537
0.0676
0.0851
0.107
0.135
0.170
0.214
0.269
0.338
0.425
0.535
0.672
0.845
1.061
1.33
1.67
2.10
2.62
3.28
4.10
5.10
6.34
7.85
9.69
11.90
14.5
17.6
21.2
25.3
22
0.0459
0.0578
0.0727
0.0915
0.115
0.145
0.182
0.230
0.289
0.363
0.457
0.575
0.722
0.908
1.140
1.43
1.80
2.25
2.82
3.52
4.39
5.46
6.78
8.39
10.3
12.7
15.5
18.7
22.5
26.7
23
0.0493
0.0621
0.0781
0.0983
0.124 ,
0.156
0.196
0.247
0.310
0.390
0.491 :
0.617
0.776
0.975
1.224
1.54
1.93
2.41
3.02
3.77
4.70
5.85
7.25
8.96 ;
11.0
13.5
16.4
19.8
23.7
28.2 .
24
0.0530
0.0667
0.0901
0.1134
0.133
0.167
0.210
0.265
0.333
0.419
0.527
0.663
0.833
1.05
1.31
1.65
2.07
2.59
3.24
4.04
5.03
6.25
7.75
9.56
11.7
14.4
17.4
21.0
25.1
29.6
25
0.0568
0.0716
0.0901
0.1134
0.143
0.180
0.226
0.284
0.358
0.450
0.566
0.711
0.893
1.12
1.41
1.77
2.21
2.77
3.46
4.32
5.38
6.68
8.27
10.2
12,5
15.2
18.5
22.2
26.4
31.1
26
0.0610
0.0768
0.0966
0.1216
0.153
0.193
0.242
0.305
0.384
0.482
0.607
0.762
0.958
1.20
1.51
1.89
2.37
2.97
3.71
4.62
5.75
7.14
8.82
10.9
13.3
16.2
19.5
23.4
27.8
32.6
Table provided by Teresa Norberg-King, ERL, Duluth, ;Minnesota. Also
see Emerson et al. (1975), Thurston et al. (1974), and USEPA (1985a).
than the amount needed to dechlorinate tap water (see Section 7, Dilution
Water, Subsection 7.4.1). Since thiosulfate may contribute to sample
toxicity, a thiosulfate control should be used in the test in addition to the
normal dilution water control. !
8.8.8 Mortality or impairment of growth or reproduction due to pH alone may
occur if the pH of the sample falls outside the range of 6.0 - 9.0. Thus, the
presence of other forms of toxicity (metals and organics) in the sample may be
masked by the toxic effects of low or high pH. The question about the
presence of other toxicants can be answered only by performing two parallel
tests, one with an adjusted pH, and one without an adjusted pH. Freshwater
samples are adjusted to pH 7.0 by adding IN NaOH or IN HC1 dropwise, as
required, being careful to avoid overadjustment.
41
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8.9 PRELIMINARY TOXICITY RANGE-FINDING TESTS
8.9.1 USEPA Regional and State personnel generally have observed that it is
not necessary to conduct a toxicity range-finding test prior to initiating a
static, chronic, definitive toxicity test. However, when preparing to perform
a static test with a sample of completely unknown quality, or before
initiating a flow-through test, it is advisable to conduct a preliminary
toxicity range-finding test.
8.9.2 A toxicity range-finding test ordinarily consists of a down-scaled,
abbreviated static acute test in which groups of five organisms are exposed to
several widely-spaced sample dilutions in a logarithmic series, such as 100%,
10.0%, 1.00%, and 0.100%, and a control, for 8-24 h. Caution: if the sample
must also be used for the full-scale definitive test, the 36-h limit on
holding time (see Subsection 8.5.4) must not be exceeded before the definitive
test is initiated. :
8.9.3 It should be noted that the toxicity (LC50) of a sample observed in a
range-finding test may be significantly different from the toxicity observed
.in the follow-up chronic definitive test because: (1) the definitive test is
longer; and (2) the test may be performed with a sample collected at a
different time, and possibly differing significantly in the level of toxicity.
8.10 HULTI-CONCENTRATION (DEFINITIVE) EFFLUENT TOXICITY TESTS
i
8.10.1 The tests recommended for use in determining discharge permit >
compliance in the NPDES program are multi-concentration, or definitive, tests
which provide (1) a point estimate of effluent toxicity in terms of an IC25,
IC50, or LC50, or (2) a no-observed-effect-concentration (NOEC) defined in
terms of mortality, growth, reproduction, and/or teratogenicity and obtained
by hypothesis testing. The tests may be static renewal or static non-renewal.
8.10.2 The tests consist of a control and a minimum of five effluent
concentrations commonly selected to approximate a geometric series, such as
100%, 50%, 25%, 12.5%, and 6.25%, using a > 0.5 dilution series.
8.10.3 These tests are also to be used in determining compliance with permit
limits on the mortality of the receiving water concentration (RWC) of
effluents by bracketing the RWC with effluent concentrations in the following
manner: (1) 100% effluent, (2) [RWC + 100]/2, (3) RWC, (4) RWC/2, andi (5)
RWC/4. For example, where the RWC = 50%, the effluent concentrations used in
the test would be 100%, 75%, 50%, 25%, and 12.5%.
8.10.4 If acute/chronic ratios are to be determined by simultaneous acute and
short-term chronic tests with a single species, using the same sample,'both
types of tests must use the same test conditions, i.e., pH, temperature, water
hardness, salinity, etc.
42
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8.11 RECEIVING WATER TESTS
8.11.1 Receiving water toxicity tests generally consist of 100% receiving
water and a control. The total hardness of the control should be comparable
to the receiving water.
8.11.2 The data from the two treatments are analyzed by hypothesis testing to
determine if test organism survival in the receiving water differs
significantly from the control. Four replicates and 10 organisms per
replicate are required for each treatment (see Summary of Test Conditions and
Test Acceptability Criteria in the specific test method).
8.11.3 In cases where the objective of the test is to estimate the degree of
toxicity of the receiving water, a multi-concentration test is performed by
preparing dilutions of the receiving water, using a > 0.5 dilution series,
with a suitable control water.
43
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SECTION 9
CHRONIC TOXICITY TEST ENDPOINTS AND DATA ANALYSIS
9.1 ENDPOINTS
9.1.1 The objective of chronic aquatic toxicity tests with effluents
and pure
compounds is to estimate the highest "safe" or "no-effect concentration" of
these substances. For practical reasons, the responses observed in these
tests are usually limited to hatchability, gross morphological abnormalities,
survival, growth, and reproduction, and the results of the tests are usually
expressed in terms of the highest toxicant concentration that has no
statistically significant observed effect on these responses, when compared to
the controls. The terms currently used to define the endpoints employed in
the rapid, chronic and sub-chronic toxicity tests have been derived from the
terms previously used for full life-cycle tests. As shorter chronic tests
were developed, it became common practice to apply the same terminology to the
endpoints. The terms used in this manual are as follows:
9.1.1.1 Safe Concentration - The highest concentration of toxicant that will
permit normal propagation of fish and other aquatic life in receiving waters.
The concept of a "safe concentration" is a biological concept, whereas the
"no-observed-effect concentration" (below) is a statistically defined
concentration.
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 (short-term) test, that causes no observable adverse effects on the
test organisms (i.e., the highest concentration of toxicant in which the
values for the observed responses are not statistically significantly
different from the controls). This value is used, along with other factors,
to determine toxicity limits in permits.
9.1.1.3 Lowest-Observed-Effect-Concentration (LOEC) - The lowest
concentration of toxicant to which organisms are exposed in a life-cycle or
partial life-cycle (short-term) test, which causes adverse effects on the test
organisms (i.e., where the values for the observed responses are statistically
significantly different from the controls).
9.1.1.4 Effective Concentration (EC) - A point estimate of the toxicant
concentration that would cause an observable adverse affect on a quantal, "all
or nothing," response (such as death, immobilization, or serious
incapacitation) in a given percent of the organisms, calculated by point
estimation techniques. If the observable effect is death or immobility, the
term, Lethal Concentration (LC), should be used (see Subsection 9.1.1.5). A
certain EC or LC value might be judged from a biological standpoint to
represent a threshold concentration, or lowest concentration that would cause
an adverse effect on the observed response.
9.1.1.5 Lethal Concentration (LC) - The toxicant concentration that would
cause death in a given percent of the test population. Identical to EC when
44
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the observed adverse effect is death. For example, the LC50 is the
concentration of toxicant that would cause death in 50% of the test
population.
9.1.1.6 Inhibition Concentration (1C) - The toxicant concentration that would
cause a given percent reduction in a non-quanta! biological measurement for
the test population. For example, the IC25 is the concentration of toxicant
that would cause a 25% reduction in mean young per female or in growth for the
test population, and the IC50 is the concentration of toxicant that would
cause a 50% reduction.
9.2 RELATIONSHIP BETWEEN ENDPOINTS DETERMINED BY HYPOTHESIS TESTING AND POINT
ESTIMATION TECHNIQUES
9.2.1 If the objective of chronic aquatic toxicity tests with effluents and
pure compounds is to estimate the highest "safe or no-effect concentration" of
these substances, it is imperative to understand how the statistical endpoints
of these tests are related to the "safe" or "no-effect" concentration. NOECs
and LOECs are determined by hypothesis testing (Dunnett's Test, a t test with
the Bonferroni adjustment, Steel's Many-one Rank Test, or the Wilcoxon Rank
Sum Test with the Bonferroni adjustment), whereas LCs, ICs, and ECs are
determined by point estimation techniques (Probit Analysis, Spearman-Karber
Method, Trimmed Spearman-Karber Method, Graphical Method or Linear
Interpolation Method). There are inherent differences between the use of a
NOEC or LOEC derived from hypothesis testing to estimate a "safe"
concentration, and the use of a LC, EC, 1C, or other point estimates derived
from curve fitting, interpolation, etc.
9.2.2 Most point estimates, such as the LC, 1C, or EC, are derived from a
mathematical model that assumes a continuous dose-response relationship. By
definition, any LC, 1C, or EC value is an estimate of some amount of adverse
effect. Thus the assessment of a "safe" concentration must be made from a
biological standpoint rather than with a statistical test,. In this instance,
the^biologist must determine some amount of adverse effect that is deemed to
be "safe", in the sense that from a practical biological viewpoint it will not
affect the normal propagation of fish and other aquatic life in receiving
waters.
9.2.3 The use of NOECs and LOECs, on the other hand, assumes either (1) a
continuous dose-response relationship, or (2) a non-continuous (threshold)
model of the dose-response relationship.
9.2.3.1 In the case of a continuous dose-response relationship, it is also
assumed that adverse effects that are not "statistically observable" are also
not important from a biological standpoint, since they are not pronounced
enough to test as statistically significant against some measure of the
natural variability of the responses.
9.2.3.2 In the case of non-continuous dose-response relationships, it is
assumed that there exists a true threshold, or concentration below which there
is no adverse effect on aquatic life, and above which there is an adverse
45
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effect. The purpose of the statistical analysis in this case is to estimate
as closely as possible where that threshold lies. i
9.2.3.3 In either case, it is important to realize that the amount of adverse
effect that is statistically observable (LOEC) or not observable (NOEC) is
highly dependent on all aspects of the experimental design, such as the number
of concentrations of toxicant, number of replicates per concentration; number
of organisms per replicate, and use of randomization. Other factors that
affect the sensitivity of the test include the choice of statistical analysis,
the choice of an alpha level, and the amount of variability between responses
at a given concentration. i
9.2.3.4 Where the assumption of a continuous dose-response relationship is
made, by definition some amount of adverse effect might be present at the
NOEC, but is not great enough to be detected by hypothesis testing.
9.2.3.5 Where the assumption of a non-continuous dose-response relationship
is made, the NOEC would indeed be an estimate of a "safe" or "no-effect"
concentration if the amount of adverse effect that appears at the threshold is
great enough to test as statistically significantly different from the
'controls in the face of all aspects of the experimental design mentioned
above. If, however, the amount of adverse effect at the threshold were not
great enough to test as statistically different, some amount of adverse effect
might be present at the NOEC. In any case, the estimate of the NOEC with
hypothesis testing is always dependent on the aspects of the experimental
design mentioned above. For this reason, the reporting and examination of
some measure of the sensitivity of the test (either the minimum significant
difference or the percent change from the control that this minimum difference
represents) is extremely important. ;
9.2.4 In summary, the assessment of a "safe" or "no-effect" concentration
cannot be made from the results of statistical analysis alone, unless;(1) the
assumptions of a strict threshold model are accepted, and (2) it is assumed
that the amount of adverse effect present at the threshold is statistically
detectable by hypothesis testing. In this case, estimates obtained from a
statistical analysis are indeed estimates of a "no-effect" concentration.
If the assumptions are not deemed tenable, then estimates from a statistical
analysis can only be used in conjunction with an assessment from a biological
standpoint of what magnitude of adverse effect constitutes a "safe"
concentration. In this instance, a "safe" concentration is not necessarily a
truly "no-effect" concentration, but rather a concentration at which the
effects are judged to be of no biological significance. ;
9.2.5 A better understanding of the relationship between endpoints derived by
hypothesis testing (NOECs) and point estimation techniques (LCs, ICs,j and ECs)
would be very helpful in choosing methods of data analysis. Norberg-King
(1991) reported that the IC25s were comparable to the NOECs for 23 effluent
and reference toxicant data sets analyzed. The data sets included short-term
chronic toxicity tests for the fathead minnow, Pimephales promelas, and the
daphnid, Ceriodaphm'a dubia. Birge et al. (1985) reported that LCls derived
from Probit Analysis of data from short-term embryo-larval tests with
reference toxicants were comparable to NOECs for several organisms.
46
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Similarly, USEPA (1988d) reported that the IC25s were comparable to the NOECs
for a set of daphnid, Ceriodaphnia dubia, chronic tests with a single
reference toxicant. However, the scope of these comparisons was very limited,
and sufficient information is not yet available to establish an overall
relationship between these two types of endpoints, especially when derived
from effluent toxicity test data.
9.3 PRECISION
9.3.1 HYPOTHESIS TESTS !
9.3.1.1 When hypothesis tests are used to analyze toxicity test data, it is
not possible to express precision in terms of a commonly used statistic.
The results of the test are given in terms of two endpoints, the No-Observed-
Effect Concentration (NOEC) and the Lowest-Observed-Effect Concentration
(LOEC). The NOEC and LOEC are limited to the concentrations selected for the
test. The width of the NOEC-LOEC interval is a function of the dilution
series, and differs greatly depending on whether a dilution factor of 0.3 or
0.5 is used in the test design. Therefore, USEPA recommends the use of the
> 0.5 dilution factor (see Section 4, Quality Assurance). It is not possible
to place confidence limits on the NOEC and LOEC derived from a given test, and
it is difficult to quantify the precision of the NOEC-LOEC endpoints between
tests. If the data from a series of tests performed with the same toxicant,
toxicant concentrations, and test species, were analyzed with hypothesis
tests, precision could only be assessed by a qualitative comparison of the
NOEC-LOEC intervals, with the understanding that maximum precision would be
attained if all tests yielded the same NOEC-LOEC interval; In practice, the
precision of results of repetitive chronic tests is considered acceptable if
the NOECs vary by no more than one concentration intervaliabove or below a
central tendency. Using these guidelines, the "normal" range of NOECs from
toxicity tests using a 0.5 dilution factor (two-fold difference between
adjacent concentrations), would be four-fold.
9.3.2 POINT ESTIMATION TECHNIQUES :
9.3.2.1 Point estimation techniques have the advantage of providing a point
estimate of the toxicant concentration causing a given amount of adverse
(inhibiting) effect, the precision of which can be quantitatively assessed
(1) within tests by calculation of 95% confidence limits, and (2) across tests
by calculating a standard deviation and coefficient of variation.
9.4 DATA ANALYSIS
9.4.1 ROLE OF THE STATISTICIAN \
9.4.1.1 The use of the statistical methods described in this manual for
routine data analysis does not require the assistance of a statistician.
However, the interpretation of the results of the analysis of the data from
any of the toxicity tests described in this manual can become problematic
because of the inherent variability and sometimes unavoidable anomalies in
biological data. If the data appear unusual in any way, or fail to meet the
necessary assumptions, a statistician should be consulted. Analysts who are
47
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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.
9.4.1.2 The statistical methods recommended in this manual are not the only
possible methods of statistical analysis. Many other methods have been
proposed and considered. Certainly there are other reasonable and defensible
methods of statistical analysis for this kind of toxicity data. Among
alternative hypothesis tests some, like Williams' Test, require additional
assumptions, while others, like the bootstrap methods, require computer-
intensive computations. Alternative point estimation approaches most probably
would require the services of a statistician to determine the appropriateness
of the model (goodness of fit), higher order linear or nonlinear models,
confidence intervals for estimates generated by inverse regression, etc.
In addition, point estimation or regression approaches would require the
'specification by biologists or toxicologists of some low level of adverse
effect that would be deemed acceptable or safe. The statistical methods
contained in this manual have been chosen because they are (1) applicable to
most of the different toxicity test data sets for which they are recommended,
(2) powerful statistical tests, (3) hopefully "easily" understood by
nonstatisticians, and (4) amenable to use without a computer, if necessary.
9.4.2 PLOTTING THE DATA :
9.4.2.1 The data should be plotted, both as a preliminary step to help detect
problems and unsuspected trends or patterns in the responses, and as an aid in
interpretation of the results. Further discussion and plotted sets of data
are included in the methods and the Appendices. ;
9.4.3 DATA TRANSFORMATIONS ;
9.4.3.1 Transformations of the data, (e.g., arc sine square root and logs),
are used where necessary to meet assumptions of the proposed analyses, such as
the requirement for normally distributed data.
9.4.4 INDEPENDENCE, RANDOMIZATION, AND OUTLIERS ,
9.4.4.1 Statistical independence among observations is a critical assumption
in all statistical analysis of toxicity data. One of the best ways to insure
independence is to properly follow rigorous randomization procedures.
Randomization techniques should be employed at the start of the test, ;
including the randomization of the placement of test organisms in the test
chambers and randomization of the test chamber location within the array of
chambers. Discussions of statistical independence, outliers and
randomization, and a sample randomization scheme, are included in Appendix A.
9.4.5 REPLICATION AND SENSITIVITY !
9.4.5.1 The number of replicates employed for each toxicant concentration is
an important factor in determining the sensitivity of chronic toxicity tests.
Test sensitivity generally increases as the number of replicates is increased,
but the point of diminishing returns in sensitivity may be reached rather
48
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quickly. The level of sensitivity required by a hypothesis test or the
confidence interval for a point estimate will determine the number of
replicates, and should be based on the objectives for obtaining the toxicity
UclLctป
9.4.5.2 In a statistical analysis of toxicity data, the choice of a
particular analysis and the ability to detect departures from the assumptions
of the analysis, such as the normal distribution of the data and homogeneity
of variance, is also dependent on the number of replicates. More than the
minimum number of replicates may be required in situations where it is
imperative to obtain optimal statistical results, such as with tests used in
enforcement cases or when it is not possible to repeat the tests. For
example, when the data are analyzed by hypothesis testing,: the nonparametric
alternatives cannot be used unless there are at least four replicates at each
toxicant concentration.
i
9.4.6 RECOMMENDED ALPHA LEVELS !
9.4.6.1 The data analysis examples included in the manual specify an alpha
level of 0.01 for testing the assumptions of hypothesis tests and an alpha
level of 0.05 for the hypothesis tests themselves. These levels are common
and well accepted levels for this type of analysis and are presented as a
recommended minimum significance level for toxicity test data analysis.
9.5 CHOICE OF ANALYSIS
9.5.1 The recommended statistical analysis of most data from chronic toxicity
tests with aquatic organisms follows a decision process illustrated in the
flowchart in Figure 2. An initial decision is made to use point estimation
techniques (the Probit Analysis, the Spearman-Karber Method, the Trimmed
Spearman-Karber Method, the Graphical Method, or Linear Interpolation Method)
and/or to use hypothesis testing (Dunnett's Test, the t test with the
Bonferroni adjustment, Steel's Many-one Rank Test, or the Wilcoxon Rank Sum
Test with the Bonferroni adjustment). NOTE: For the NPDES Permit Program, the
point estimation techniques are the preferred statistical methods in
calculating end points for effluent toxicity tests. If hypothesis testing is
chosen, subsequent decisions are made on the appropriate procedure for a given
set of data, depending on the results of the tests of assumptions as
illustrated in the flowchart. A specific flow chart is included in the
analysis section for each test.
95.2 Since a single chronic toxicity test might yield information on more
than one parameter (such as survival, growth, and reproduction), the lowest
estimate of a "no-observed-effect concentration" for any of the responses
would be used as the "no-observed-effect concentration" for each test It
follows logically that in the statistical analysis of the data, concentrations
that had a significant toxic effect on one of the observed responses would not
be subsequently tested for an effect on some other response.'. This is one
reason for excluding concentrations that have shown a statistically
significant reduction in survival from a subsequent hypothesis test for
effects on another parameter such as reproduction. A second reason is that
the exclusion of such concentrations usually results in a more powerful and
49
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DATA (SURVIVAL, GROWTH, REPRODUCTION, ETC.)
POINT
ESTIMATION
HYPOTHESIS TESTING
TRANSFORMATION?
ENDPOINT ESTIMATE
LC, EC, 1C
SHAPIRO-WIUCS TEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETT'STEST
HETEROGENEOUS
VARIANCE
NO STATISTICAL ANALYSIS
RECOMMENDED
NO
ซ*-
4 OR MORE
REPLICATES?
YES
EQUAL NUMBER OF
REPLICATES?
T-TESTWITH
BONFERRONI
ADJUSTMENT
EQUAL NUMBER OF
REPLICATES?
YES
NO
STEEL'S MANY-ONE
RANKTEST
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 2. Flowchart for statistical analysis of test data.
50
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appropriate statistical analysis. In performing the point estimation
techniques recommended in this manual, an all-data approach is used. For
example, data from concentrations above the NOEC for survival are included in
determining ICp estimates using the Linear Interpolation Method.
9.5.3 ANALYSIS OF GROWTH AND REPRODUCTION DATA ;
9.5.3.1 Growth data from the fathead minnow, Pimephales promelas, larval
survival and growth test are analyzed using hypothesis testing or point
estimation techniques according to the flowchart in Figure 2. The above
mentioned growth data may also be analyzed by generating a point estimate with
the Linear Interpolation Method. Data from effluent conceintrations that have
tested significantly different from the control for survival are excluded from
further hypothesis tests concerning growth effects. Growth is defined as the
dry weight per original number of test organisms when group weights are
obtained. When analyzing the data using point estimation techniques, data
from all concentrations are included in the analysis.
9.5.3.2 Reproduction data from the daphnid, Ceriodaphnia dubia, survival and
reproduction test are analyzed using hypothesis testing or point estimation
techniques according to the flowchart in Figure 2. In hypothesis testing,
data from effluent concentrations that have significantly lower survival than
the control, as determined by Fisher's Exact test, are not!included in the
hypothesis tests for reproductive effects. Data from all concentrations are
included when using point estimation techniques.
9.5.4 ANALYSIS OF ALGAL GROWTH RESPONSE DATA '
9.5.4.1 The growth response data from the green alga, Selenastrum
capn'cornutum, toxicity test, after an appropriate transformation, if
necessary, to meet the assumptions of normality and homogeneity of variance,
may be analyzed by hypothesis testing according to the flowchart in Figure 2.
Point estimates, such as the IC25 and IC50, would also be appropriate in
analyzing algal growth data.
9.5.5 ANALYSIS OF MORTALITY DATA
9.5.5.1 Mortality data are analyzed by Probit Analysis, if appropriate, or
other point estimation techniques (i.e., the Spearman-Karber Method, the
Trimmed Spearman-Karber Method, or the Graphical Method) (see Appendices I-L
and the discussion below). The mortality data can also be analyzed by
hypothesis testing, after an arc sine square root transformation (see Appendix
B-F), according to the flowchart in Figure 2. i '
}
9.5.5.2 Mortality data from the daphnid, Ceriodaphnia dubia, survival and
reproduction test are analyzed by Fisher's Exact Test (Appendix G) prior to
the analysis of the reproduction data. The mortality data may also be
analyzed by Probit Analysis, if appropriate or other methods (see
Subsection 9.5.5.1).
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9.6 HYPOTHESIS TESTS
9.6.1 DUNNETT'S PROCEDURE
9.6.1.1 Dunnett's Procedure is used to determine the NOEC. The procedure
consists of an analysis of variance (ANOVA) to determine the error term, which
is then used in a multiple comparison procedure for comparing each of;the
treatment means with the control mean, in a series of paired tests (see
Appendix C). Use of Dunnett's Procedure requires at least three replicates
per treatment to check the assumptions of the test. In cases where the
numbers of data points (replicates) for each concentration are not equal, a
t test may be performed with Bonferroni's adjustment for multiple comparisons
(see Appendix D), instead of using Dunnett's Procedure. ;
9.6.1.2 The assumptions upon which the use of Dunnett's Procedure is
contingent are that the observations within treatments are normally
distributed, with homogeneity of variance. Before analyzing the data, these
assumptions must be tested using the procedures provided in Appendix B.
9.6.1.3 If, after suitable transformations have been carried out, the
normality assumptions have not been met, Steel's Many-one Rank Test should be
used if there are four or more data points (replicates) per toxicant
concentration. If the numbers of data points for each toxicant concentration
are not equal, the Wilcoxon Rank Sum Test with Bonferroni's adjustment should
be used (see Appendix F).
9.6.1.4 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.
9.6.1.5 A step-by-step example of the use of Dunnett's Procedure is provided
in Appendix C.
9.6.2 T TEST WITH THE BONFERRONI ADJUSTMENT , '
9.6.2.1 At test with Bonferroni's adjustment is used as an alternative to
Dunnett's Procedure when the number of replicates is not the same for all
concentrations. This test sets an upper bound of alpha on the overall error
rate, in contrast to Dunnett's Procedure, for which the overall error rate is
fixed at alpha. Thus Dunnett's Procedure is a more powerful test.
9.6.2.2 The assumptions upon which the use of the t test with Bonferroni's
adjustment is contingent are that the observations within treatments are
normally distributed, with homogeneity of variance. These assumptions must be
tested using the procedures provided in Appendix B.
9.6.2.3 The estimate of the safe' concentration derived from this tes't is
reported in terms of the NOEC. A step-by-step example of the use of the
t test with Bonferroni's adjustment is provided in Appendix D.
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9.6.3 STEEL'S MANY-ONE RANK TEST ;
9.6.3.1 Steel's Many-one Rank Test is a multiple comparison procedure for
comparing several treatments with a control. This method is similar to
Dunnett's Procedure, except that it is not necessary to meet the assumption of
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).
9.6.3.2 It is necessary to have at least four replicates per toxicant
concentration to use Steel's test. Unlike Dunnett's procedure, the
sensitivity of this test cannot be stated in terms of the minimum difference
between treatment means and the control mean that can be detected as
statistically significant. .
9.6.3.3 The estimate of the safe concentration is reported as the NOEC.
A step-by-step example of the use of Steel's Many-one Rank Test is provided in
Appendix E. ;
9.6.4 WILCOXON RANK SUM TEST WITH THE BONFERRONI ADJUSTMENT
9.6.4.1 The Wilcoxon Rank Sum Test with the Bonferroni Adjustment is a
nonparametric test for comparing treatments with a control. The data are
ranked and the analysis proceeds exactly as in Steel's Test except that
Bonferroni's adjustment for multiple comparisons is used instead of Steel's
tables. When Steel's test can be used ('i.e., when there are equal numbers of
data points per toxicant concentration), it will be more powerful (able to
detect smaller differences as statistically significant) than the Wilcoxon
Rank Sum Test with Bonferroni's adjustment.
9.6.4.2 The estimate of the safe concentration is reported as the NOEC.
A step-by-step example of the use of the Wilcoxon Rank Sum Test with
Bonferroni Adjustment is provided in Appendix F.
9.6.5 A CAUTION IN THE USE OF HYPOTHESIS TESTING '. '
9.6.5.1 If in the calculation of an NOEC by hypothesis testing, two tested
concentrations cause statistically significant adverse effects, but an
intermediate concentration did not cause statistically significant effects,
the results should be used with extreme caution.
9.7 POINT ESTIMATION TECHNIQUES
9.7.1 PROBIT ANALYSIS '
9.7.1.1 Probit Analysis is used to estimate the LCI, LC50, EC1, or EC50 and
the associated 95% confidence interval. The analysis consists of adjusting
the data for mortality in the control, and then using a maximum likelihood
technique to estimate the parameters of the underlying log tolerance
distribution, which is assumed to have a particular shape.'
53
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9.7.1.2 The assumption upon which the use of Probit Analysis is contingent
is a normal distribution of log tolerances. If the normality assumption is
not met, and at least two partial mortalities are not obtained, Probit
Analysis should not be used. It is important to check the results of IProbit?
Analysis to determine if use of the analysis is appropriate. The chi-square
test for heterogeneity provides one good test of appropriateness of tfje
analysis. The computer program (see Appendix I) checks the chi-square
statistic calculated for the data set against the tabular value, and provides
an error message if the calculated value exceeds the tabular value.
9.7.1.3 A discussion of Probit Analysis, and examples of computer program
input and output, are found in Appendix I. ,
9.7.1.4 In cases where Probit Analysis is not appropriate, the LC50 and
associated confidence interval may be estimated by the Spearman-Karber Method
(Appendix J) or the Trimmed Spearman-Karber Method (Appendix K). If the test
results in 100% survival and 100% mortality in adjacent treatments (all or
nothing effect), the LC50 may be estimated using the Graphical Method
(Appendix L).
9.7.2 LINEAR INTERPOLATION METHOD ;
9.7.2.1 The Linear Interpolation Method (see Appendix M) is a procedure to
calculate a point estimate of the effluent or other toxicant concentration
[Inhibition Concentration, (1C)] that causes a given percent reduction (e.g.,
25%, 50%, etc.) in the reproduction or growth of the test organisms. The
procedure was designed for general applicability in the analysis of data from
short-term chronic toxicity tests.
9.7.2.2 Use of the Linear Interpolation Method is based on the assumptions
that the responses (1) are monotonically non-increasing (the mean response for
each higher concentration is less than or equal to the mean response for the
previous concentration), (2) follow a piecewise linear response function, and
(3) are from a random, independent, and representative sample of test data.
The assumption for piecewise linear response cannot be tested statistically,
and no defined statistical procedure is provided to test the assumption for
monotonicity. Where the observed means are not strictly monotonic by
examination, they are adjusted by smoothing. In cases where the responses at
the low toxicant concentrations are much higher than in the controls,jthe
smoothing process may result in a large upward adjustment in the control mean.
L
9.7.2.3 The inability to test the monotonicity and piecewise linear response
assumptions for this method makes it difficult to assess when the method is,
or is not, producing reliable results. Therefore, the^ method should be used
with caution when the results of a toxicity test approach an "all or nothing"
response from one concentration to the next in the concentration series, and
when it appears that there is a large deviation from monotonicity. See
Appendix M for a more detailed discussion of the use of this method and a
computer program available for performing calculations.
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SECTION 10 I
REPORT PREPARATION
The toxicity data are reported, together with other appropriate data. The
following general format and content are recommended for the report:
10.1 INTRODUCTION
1. Permit number
2. Toxicity testing requirements of permit I
3. Plant location
4. Name of receiving water body
'5. Contract Laboratory (if the tests are performed under contract)
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)
8. Design flow of treatment facility at time of sampling
10.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION WATER
1. Effluent Samples I
a. Sampling point
b. Collection dates and times I
c. Sample collection method
d. Physical and chemical data
e. Mean daily discharge on sample collection date
f. Lapsed time from sample collection to delivery
g. Sample temperature when received at the laboratory
2. Receiving Water 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)
f. Sample temperature when received at the laboratory
g. Lapsed time from sample collection to delivery
55
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3. Dilution Water Samples ;
a. Source
b. Collection date(s) and time(s)
c. Pretreatment ;
d. Physical and chemical characteristics :
10.4 TEST METHODS
1. Toxicity test method used (title, number, source)
2. Endpoint(s) of test
3. Deviation(s) from reference method, if any, and the reason(s)
4. Date and time test started
5. Date and time test terminated
6. Type and volume of test chambers
7. Volume of solution used per chamber ;
8. Number of organisms per test chamber ;
9. Number of replicate test chambers per treatment
10. Acclimation of test organisms (temperature mean and range) '
11. Test temperature (mean and range)
12. Specify if aeration was needed
13. Feeding frequency, arid amount and type of food ,
10.5 TEST ORGANISMS ]
1. Scientific name and how determined
2. Age
3. Life stage
4. Mean length and weight (where applicable)
5. Source . :
6. Diseases and treatment (where applicable)
7. Taxonomic key used for species identification ;
10.6 QUALITY ASSURANCE ;
1. Reference toxicant used routinely; source ;
2. Date and time of most recent reference toxicant test, test results,
and current control (cusum) chart
3. Dilution water used in reference toxicant test
4. Results (NOEC or, where applicable, LOEC, LC50, EC50, IC25 !
and/or IC50) !
5. Physical and chemical methods used
10.7 RESULTS
1. Provide raw toxicity data in tabular form, including daily records of
affected organisms in each concentration (including controls), and
plots of toxicity data
2. Provide table of LCSOs, NOECs, IC25, IC50, etc.
3. Indicate statistical methods used to calculate endpoints
4. Provide summary table of physical and chemical data
5. Tabulate QA data
56
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10.8 CONCLUSIONS AND RECOMMENDATIONS i
1. Relationship between test endpoints and permit limits
2. Actions to be taken i
57
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SECTION 11
TEST METHOD
FATHEAD MINNOW, PIMEPHALES PROMELAS,
LARVAL SURVIVAL AND GROWTH TEST
METHOD 1000.0
11.1 SCOPE AND APPLICATION
11.1.1 This method estimates the chronic toxicity of 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.
11.1.2 Daily observations on mortality make it possible to also calculate
acute toxicity for desired exposure periods (i.e., 24-h, 48-h, 96-h LC50s).
11.1.3 Detection limits of the toxicity of an effluent or pure substance are
organism dependent.
11.1.4 Brief excursions in 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
degradable or highly volatile toxicants present in the source may not be
detected in the test.
11.1.5 This test method is commonly used in one of two forms: (1) a '
definitive test, consisting of a minimum of five effluent concentrations and a
control, and (2) a receiving water test(s), consisting of one or more
receiving water concentrations and a control.
11.2 SUMMARY OF METHOD
11.2.1 Fathead minnow, Pimephales promelas, larvae 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 weight of the
larvae.
11.3. INTERFERENCES
11.3.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment and Supplies).
11.3.2 Adverse effects of low dissolved oxygen (DO) concentrations, high
concentrations of suspended and/or dissolved solids, and extremes of pH,
alkalinity, or hardness, may mask the presence of toxic substances.
58
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11.3.3 Improper effluent sampling and sample handling may adversely affect
test results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).
11.3.4 Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival and confound test results.
11.3.5 Food added during the test may sequester metals and other toxic
substances and confound test results. Daily renewal of solutions, however,
will reduce the probability of reduction of toxicity caused by feeding.
11.4 SAFETY
11.4.1 See Section 3, Health and Safety. !
11.5 APPARATUS AND EQUIPMENT
11.5.1 Fathead minnow and brine shrimp culture units -- see USEPA, 1985a and
USEPA, 1993b. This test requires 180-360 larvae. It is preferable to obtain
larvae from an in-house fathead minnow culture unit. If it is not feasible to
culture fish in-house, embryos or newly hatched larvae can be shipped in well
oxygenated water in insulated containers.
11.5.2 Samplers -- automatic sampler, preferably with sample cooling
capability, that can collect a 24-h composite sample of 5 L.
11.5.3 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
11.5.4 Environmental chamber or equivalent facility with temperature control
(25 ฑ 16C).
11.5.5 Water purification system -- MILLIPORE MILLI-Qฎ, deionized water or
equivalent (see Section 5, Facilities, Equipment, and Supplies).
11.5.6 Balance -- analytical, capable of accurately weighing to 0.00001 g.
11.5.7 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. ;
i
11.5.8 Test chambers -- four (minimum of three) borosilicate glass or
non-toxic disposable plastic test chambers are required for each concentration
and control. Test chambers.may be 1 L, 500 ml or 250 ml beakers, 500 ml
plastic cups, or fabricated rectangular (0.3 cm thick) glass chambers,' 15 cm x
7.5 cm x 7.5 cm. To avoid potential contamination from the air and excessive
evaporation of test solutions during the test, the chambers should be covered
with safety glass plates or sheet plastic (6 mm thick).
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11.5.9 Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware, 10-1000 ml for making test solutions. 5.10
11.5.10 Volumetric pipets -- Class A, 1-100 ml.
11.5.11 Serological pipets -- 1-10 ml, graduated. ;
11.5.12 Pipet bulbs and fillers -- PROPIPETฎ, or equivalent. j
11.5.13 Droppers, and glass tubing with fire polished edges, 4 mm ID -- for
transferring larvae.
11.5.14 Wash bottles -- for rinsing small glassware and instrument electrodes
and probes.
11.5.15 Thermometers, glass or electronic, laboratory grade -- for measuring
water temperatures. i
11.5.16 Bulb-thermograph or electronic-chart type thermometers -- for
continuously recording temperature. ;
11.5.17 Thermometers, National Bureau of Standards Certified (see USEPA
Method 170.1, USEPA, 1979b) --to calabrate laboratory themometers.
11.5.18 Meters, pH, DO, and specific conductivity -- for routine physical and
chemical measurements. \
11.5.19 Drying oven -- 50-105ฐC range for drying larvae.
11.6 REAGENTS AND CONSUMABLE MATERIALS
11.6.1 Sample containers -- for sample shipment and storage (see Sectjion 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
11.6.2 Data sheets (one set per test) -- for recording data.
11.6.3 Vials, marked -- 18-24 per test, containing 4% formalin or 70% ethanol
to preserve larvae (optional).
11.6.4 Weighing boats, aluminum -- 18-24 per test.
11.6.5 Tape, colored -- for labelling test chambers.
11.6.6 Markers, waterproof -- for marking containers, etc.
11.6.7 Reagents for hardness and alkalinity tests -- see USEPA Methods 130.2
and 310.1, USEPA, 1979b.
11.6.8 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument
manufacturer) -- for instrument calibration (see USEPA Method 150.1, USEPA,
1979b).
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11.6.9 Specific conductivity standards -- see USEPA Method 120.1, USEPA,
1979b.
11.6.10 Membranes and filling solutions for DO probe (see USEPA Method 360.1,
USEPA, 1979b), or reagents -- for modified Winkler analysis.
11.6.11 Laboratory quality control samples and standards .-- for calibration
of the above methods.
11.6.12 Reference toxicant solutions (see Section 4, Quality Assurance).
11.6.13 Ethanol (70%) or formalin (4%) -- for use as a preservative for the
fish larvae. . . '
11.6.14 Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms (see Section 5,
Facilities, Equipment, and Supplies).
11.6.15 Effluent, receiving water, and dilution water -- ;see Section 7,
Dilution Water; and Section 8,, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.
11.6.16 Brine Shrimp, Artemia, Nauplii -- for feeding cultures and test
organisms ;
11.6.16.1 Newly-hatched Artemia nauplii are used as food (see USEPA, 1993b)
for fathead minnow, Pimephales promelas, larvae in toxicity tests and frozen
brine shrimp and flake food are used in the maintenance of continuous stock
cultures. Although there are many commercial sources of brine shrimp cysts,
the Brazilian or Colombian strains are currently preferred because the
supplies examined have had low concentrations of chemical residues and produce
nauplii of suitably small size. For commercial sources of brine shrimp,
Artemia, cysts, see Table 2 of Section 5,* Facilities, Equipment, and Supplies
and Section 4, Quality Assurance.
11.6.16.2 Each new batch of brine shrimp, Artemia, cysts must be evaluated
for size (Vanhaecke and Sorgelloos, 1980, and Vanhaecke et al., 1980) and
nutritional suitability (see Leger et al., 1985; Leger et al., 1986) against
known suitable reference cysts by performing a side by side larval growth test
using the "new" and "reference" cysts. The "reference" cysts used in the
suitability test may be a previously tested and acceptable batch of cysts, or
may be obtained from the Quality Assurance Branch, Environmental Monitoring
Systems Laboratory, Cincinnati, OH 45268; 513-569-7325. A sample of
newly-hatched Artemia nauplii from each new batch of cysts should be
chemically analyzed. The Artemia cysts should not be used if the
concentration of total organochlorine exceeds 0.15 ng/g wet weight or the
total concentration of organochlorine pesticides plus PCBs exceeds 0.30 //g/g
wet weight. (For analytical methods see USEPA, 1982).
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11.6.16.3 Artemia nauplii are obtained as follows:
1. Add 1 L of seawater, or a solution prepared by adding 35.0 g uniodized
salt (NaCl) or artificial sea salts to 1 L deionized water, to a 2-1
separatory funnel, or equivalent.
2. Add 10 ml Artemia cysts to the separatory funnel and aerate for 24-h at
27ฐC. (Hatching time varies with incubation temperature and the
geographic strain of Artemia used) (see USEPA, 1991b; USEPA, 1993b and
ASTM, 1993).
3. After 24 h, cut off the air supply in the separatory funnel. Artemia
nauplii are phototactic, and will concentrate at the bottom of the
funnel if it is covered for 5-10 min. To prevent mortality, do not
leave the concentrated nauplii at the bottom of the funnel more than 10
min without aeration.
4. Drain the nauplii into a beaker or funnel fitted with a < 150 //tn Nitexฎ
or stainless steel screen, and rinse with seawater, or equivalent,
before use.
11.6.16.4 Testing Artemia nauplii as food for toxicity test organisms.
11.6.16.4.1 The primary criterion for acceptability of each new supply of
brine shrimp cysts is the ability of the nauplii to support good survival and
growth of the fathead minnow larvae (see Subsection 11.12). The larvae used
to evaluate the suitability of the brine shrimp nauplii must be of the same
geographical origin, species, and stage of development as those used routinely
in the toxicity tests. Sufficient data to detect differences in survival and
growth should be obtained by using three replicate test vessels, each
containing a minimum of 15 larvae, for each type of food.
11.6.16.4.2 The feeding rate and frequency, test vessels, volume of control
water, duration of the test, and age of the nauplii at the start of the test,
should be the same as used for the routine toxicity tests. |
11.6.16.4.3 Results of the brine shrimp nutrition assay, where there are only
two treatments, can be evaluated statistically by use of a t test. The "new"
food is acceptable if there are no statistically significant differences in
the survival and growth of the larvae fed the two sources of nauplii.
11.6.17 TEST ORGANISMS, FATHEAD MINNOWS, PIMEPHALES PROMELAS <
11.6.17.1 Newly hatched fish less than 24 h old should be used for the test.
If organisms must be shipped to the testing site, fish up to 48 h old'may be
used, all hatched within a 24-h window.
11.6.17.2 If the fish are kept in a holding tank or container, most of the
water should be siphoned off to concentrate the fish. The fish are th'en
transferred one at a time randomly to the test chambers until each chamber
contains ten fish. Alternately, fish may be placed one or two at a time into
small beakers or plastic containers until they each contain five fish. Three
(minimum of two) of these beakers/plastic containers are then assigned to
randomly-arranged control and exposure chambers.
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11.6.17.2.1 The fish are transferred directly to the test vessels or
intermediate beakers/plastic containers, using a large-bore, fire-polished
glass tube (6 mm to 9 mm I.D. X 30 cm long) equipped with a rubber bulb, or a
large volumetric pipet with tip removed and fitted with a safety type bulb
filler. The glass or plastic containers should only contain a small volume of
dilution water.
11.6.17.2.2 It is important to note that larvae should not be handled with a
dip net. Dipping small fish with a net may result in damage to the fish and
cause mortality. ;
11.6.17.3 The test is conducted with four (minimum of three) test chambers at
each toxicant concentration and control. Fifteen (minimum of ten) embryos are
placed in each replicate test chamber. Thus 60 (minimum of 30) fish are
exposed at each test concentration.
11.6.17.4 Sources of organisms
11.6.17.4.1 Fathead minnows, Pimephales promelas, may be obtained from
commercial biological supply houses. Fish obtained from outside sources for
use as brood stock or in toxicity tests may not always be of suitable age and
quality. Fish provided by supply houses should be guaranteed to be of (1) the
correct species, (2) disease free, (3) in the requested age range, and (4) in
good condition. This can be done by providing the record of the date on which
the eggs were laid and hatched, and information on the sensitivity of
contemporary fish to reference toxicants.
11.6.17.5 Inhouse Sources of Fathead Minnows, Pimephales promelas
11.6.17.5.1 Problems in obtaining suitable fish from outside laboratories can
be avoided by developing an inhouse laboratory culture facility. Fathead
minnows, Pimephales promelas., can be easily cultured in the laboratory from
eggs to adults in static, recirculating, or flow-through 'systems. The larvae,
juveniles, and adult fish should be kept in 60 L (15 gal) or 76 L (20 gal)
rearing tanks supplied with reconstituted water, dechlorinated tap water, or
natural water. The water should be analyzed for toxic metals and organics
quarterly (see Section 4, Quality Assurance). ',
11.6.17.5.1.1 If a static or recirculating system is used, it is necessary to
equip each tank with an outside activated carbon filter system, similar to
those sold for tropical fish hobbyists (or one large activated carbon filter
system for a series of tanks) to prevent the accumulation of toxic metabolic
wastes (principally nitrite and ammonia) in the water.
11.6.17.5.2 Flow-through systems require large volumes of water and may not
be feasible in some laboratories. The culture tanks should be shielded from
extraneous disturbances using opaque curtains, and should be isolated from
toxicity testing activities to prevent contamination.
11.6.17.5.3 To avoid the possibility of inbreeding of the inhouse brood
stock, fish from an outside source should be introduced yearly into the
culture unit.
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11.6.17.5.4 Dissolved oxygen -- The DO concentration in the culture tanks
should be maintained near saturation, using gentle aeration with 15 cm air
stones if necessary. Brungs (1971), in a carefully controlled long-term
study, found that the growth of fathead minnows was reduced significantly at
all dissolved oxygen concentrations below 7.9 mg/L. Soderberg (1982)
presented an analytical approach to the re-aeration of flowing water for
culture systems.
11.6.17.5.5 Culture Maintenance
11.6.17.5.5.1 Adequate procedures for culture maintenance must be followed to
..avoid poor water quality in the culture system. The spawning and brood stock
culture tanks should be kept free of debris (excess food, detritus, waste,
etc.) by siphoning the accumulated materials (such as dead brine shrimp
nauplii or cysts) from the bottom of the tanks daily with a glass siphon tube
attached to a plastic hose leading to the floor drain. The tanks are more
thoroughly cleaned as required. Algae, mostly diatoms and green algae,
growing on the glass of the spawning tanks are left in place, except for the
front of the tank, which is kept clean for observation. To avoid excessive
build-up of algal growth, the walls of the tanks are periodically scraped.
The larval culture tanks are cleaned once or twice a week to reduce the mass
of fungus growing on the bottom of the tank. ;
11,6.17.5.5.2 Activated.charcoal and floss in the tank filtration systems
should be changed weekly, or more often if needed. Culture water may be
maintained by preparation of reconstituted water or use of dechlorinated tap
water. Distilled or deionized water is added as needed to compensate for
evaporation.
11.6.17.5.5.3 Before new fish are placed in tanks, salt deposits are removed
by scraping or with 5% acid solution, the tanks are washed with detergent,
sterilized with a hypochlorite solution, and rinsed well with hot tap water
and then with laboratory water. :
11.6.17.5.6 Obtaining Embryos for Toxicity Tests ;
11.6.17.5.6.1 Embryos can be shipped to the laboratory from an outside source
or obtained from adults held in the laboratory as described below. ;
11.6.17.5.6.2 For breeding tanks, it is convenient to use 60 L (15 gal) or
76 L (20 gal) aquaria. The spawning unit is designed to simulate conditions
in nature conducive to spawning, such as water temperature and photoperiod.
Spawning tanks must be held at a temperature of 25 ฑ 2ฐC. Each aquarium is
equipped with a heater, if necessary, a continuous filtering unit, and
spawning substrates. The photoperiod for the culture system should be1
maintained at 16 h light and 8 h darkness. For the spawning tanks, this
photoperiod must be rigidly controlled. A convenient photoperiod is 5:00 AM
to 9:00 PM. Fluorescent lights should be suspended about 60 cm above the
surface of the water in the brood and larval tanks. Both DURATESTฎ and cool-
white fluorescent lamps have been used, and produce similar results. A"
illumination level of 50 to 100 ft-c is adequate.
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11.6.17.5.6.3 To simulate the natural spawning environment, it is necessary
to provide substrates (nesting territories) upon which the eggs can be
deposited and fertilized, and which are defended and cared for by the males.
The recommended spawning substrates consist of inverted half-cylinders,
7.6 cm x 7.6 cm (3 in x 3 in) of Schedule 40 PVC pipe. The substrates should
be placed equi-distant from each other on the bottom of the tanks.
11.6.17.5.6.4 To establish a breeding unit, 15-20 pre-spawning adults six to
eight months old are taken from a "holding" or culture tank and placed in a
76-1 spawning tank. At this point, it is not possible to distinguish the
sexes. However, after less than a week in the spawning tank, the breeding
males will develop their distinct coloration and territorial behavior, and
spawning will begin. As the breeding males are identified, all but two are
removed, providing a final ratio of 5-6 females per male. The excess spawning
substrates are used as shelter by the females. i
11.6.17.5.6.5 Sexing of the fish to ensure a correct female/male ratio in
each tank can be a problem. However, the task usually becomes easier as
experience is gained (Flickinger, 1966). Sexually mature females usually have
large bellies and a tapered snout. The sexually mature males are usually
distinguished by their larger overall size, dark vertical color bands, and the
spongy nuptial tubercles on the snout. Unless the males exhibit these
secondary breeding characteristics, no reliable method has been found to
distinguish them from females. However, using the coloration of the males and
the presence of enlarged urogenital structures and other characteristics of
the females, the correct selection of the sexes can usually be achieved by
trial and error.
11.6.17.5.6.6 Sexually immature males are usually recognized by their
aggressive behavior and partial banding. These undeveloped males must be
removed from the spawning tanks because they will eat the eggs and constantly
harass the mature males, tiring them and reducing the fecundity of the
breeding unit. Therefore, the fish in the spawning tanks must be carefully
checked periodically for extra males.
11.6.17.5.6.7 A breeding unit should remain in their spawning tank about four
months. Thus, each brood tank or unit is stocked with new spawners about
three times a year. However, the restocking process is rotated so that at any
one time the spawning tanks contain different age groups of brood fish.
11.6.17.5.6.8 Fathead minnows spawn mostly in the early morning hours. They
should not be disturbed except for a morning feeding (8:00 AM) and daily
examination of substrates for eggs in late morning or early afternoon. In
nature, the male protects, cleans, and aerates the eggs until they hatch.
In, the laboratory, however, it is necessary to remove the eggs from the tanks
to prevent them from being eaten by the adults, for ease of handling, for
purposes of recording embryo count and hatchability, and for the use of the
newly hatched young fish for toxicity tests.
11.6.17.5.6.9 Daily, beginning six to eight hours after 'the lights are turned
on (11:00 AM - 1:00 PM), the substrates in the spawning tanks are each lifted
carefully and inspected for embryos. Substrates without embryos are
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immediately returned to the spawning tank. Those with embryos are immersed in
clean water in a collecting tray, and replaced with a clean substrate. A
daily record is maintained of each spawning site and the estimated number of
embryos on the substrate.
11.6.17.5.6.10 Three different methods are described for embryo incubation.
1. Incubation of Embryos on the Substrates: Several (2-4) substrates
are placed on end in a circular pattern (with the embryos on the innerside) in
10 cm of water in a tray. The tray is then placed in a constant temperature
water bath, and the embryos are aerated with a 2.5 cm airstone placed in the
center of the circle. The embryos are examined daily, and the dead and
fungused embryos are counted, recorded, and removed with forceps. At an
incubation temperature of 25ฐC, 50% hatch occurs in five days. At 22ฐC
embryos incubated on aerated tiles require 7 days for 50% hatch.
2. Incubation of Embryos in a Separatorv Funnel: The embryos are removed
from the substrates with a rolling action of the index finger ("rolled off")
(Cast and Brungs, 1973), their total volume is measured, and the number of
embryos is calculated using a conversion factor of approximately 430
embryos/mL. The embryos are incubated in about 1.5 L of water in a 2 L
separatory funnel maintained in a water bath. The embryos are stirred in the
separatory funnel by bubbling air from the tip of a plastic micro-pipette
placed at the bottom, inside the separatory funnel. During the first two
days, the embryos are taken from the funnel daily, those that are dead and
fungused are removed, and those that are alive are returned to the separatory
funnel in clean water. The embryos hatch in four days at a temperature of
25ฐC. However, usually on day three the eyed embryos are removed from:the
separatory funnel and placed in water in a plastic tray and gently aerated
with an air stone. Using this method, the embryos hatch in five days.;
Hatching time is greatly influenced by the amount of agitation of the embryos
and the incubation temperature. If on day three the embryos are transferred
from the separatory funnel to a static, unaerated container, a 50% hatch will
occur in six days (instead of five) and a 100% hatch will occur in seven days.
If the culture system is operated at 22ฐC, embryos incubated on aerated tiles
require seven days for 50% hatch.
3. Incubation in Embryo Incubation Cups: The embryos are "rolled off" the
substrates, and the total number is estimated by determining the volume. The
embryos are then placed in incubation cups attached to a rocker arm assembly
(Mount, 1968). Both flow-through and static renewal incubation have been
used. On day one, the embryos are removed from the cups and those that are
dead and fungused are removed. After day one only dead embryos are removed
from the cups. During the incubation period, the eggs are examined daily for
viability and fungal growth, until they hatch. Unfertilized eggs, and eggs
that have become infected by fungus, should be removed with forceps using a
table top magnifier-illuminator. Non-viable eggs become milky and opaque, and
are easily recognized. The non-viable eggs are very susceptible to fungal
infection, which may then spread throughout the egg mass. Removal of fungus
should be done quickly, and the substrates should be returned to the
incubation tanks as rapidly as possible so that the good eggs are not damaged
by desiccation. Hatching takes four to five days at an optimal temperature of
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25ฐC. Hatching can be delayed several (two to four) days by incubating at
lower temperatures. A large plastic tank receiving recirculating water from a
temperature control unit, can be used as a water bath for incubation of
embryos. [
11.6.17.5.6.11 Newly-hatched larvae are transferred daily from the egg
incubation apparatus to small rearing tanks, using a large bore pipette, until
the hatch is complete. New rearing tanks are set up on a daily basis to
separate fish by age group. Approximately 1500 newly hatched larvae are
placed in a 60-L (15 gal) or 76-L (20 gal) all-glass aquarium for 30 days.
A density of 150 fry per liter is suitable for the first four weeks. The
water temperature in the rearing tanks is allowed to follow ambient laboratory
temperatures of 20-25ฐC, but sudden, extreme variations in temperature must be
avoided.
11.6.17.5.7 Food and Feeding \
11.6.17.5.7.1 The amount of food and feeding schedule affects both growth and
egg production. The spawning fish and pre-spawners in holding tanks usually
are fed all the adult frozen brine shrimp and tropical fish flake food or dry
commercial fish food (No. 1 or No. 2 granules) that they can eat (ad libitum)
at the beginning of the work day and in the late afternoon (8:00 AM and 4:00
PM). The fish are fed twice a day (twice a day with dry food and once a day
with adult shrimp) during the week and once a day on weekends.
11.6.17.5.7.2 Fathead minnow larvae are fed freshly-hatched brine shrimp
(Artemia) nauplii twice daily until they are four weeks old. Utilization of
older (larger) brine shrimp nauplii may result in starvation of the young fish
because they are unable to ingest the larger food organisms (see
Subsection 11.6.16 or USEPA, 1993b for instructions on the preparation of
brine shrimp nauplii).
11.6.17.5.7.3 Fish older than four weeks are fed frozen brine shrimp and
commercial fish starter (#1 and #2), which is ground fish meal enriched with
vitamins. As the fish grow, larger pellet sizes are used, as appropriate.
(Starter, No. 1 and N. 2 granules, U.S. Fish and Wildlife Service Formulation
Specification Diet SD9-30, can be obtained from Zeigler E!ros., Inc., P.O. Box
90, Gardners, PA 17324). Newly hatched brine shrimp nauplii, and frozen adult
brine shrimp (San Francisco Bay Brand) are fed to the fish cultures in volumes
based on age, size, and number of fish in the tanks.
11.6.17.5.7.4 Fish in the larval tanks (from hatch to 30 days old) are fed
commercial starter fish food at the beginning and end of the work day, and
newly hatched brine shrimp nauplii (from the brine shrimp culture unit) once a
day, usually mid-morning and mid-afternoon. ;
11.6.17.5.7.5' Attempts should be made to avoid introducing Artemia cysts and
empty shells when the brine shrimp nauplii are fed to the fish larvae. Some
of the mortality of the larval fish observed in cultures could be caused from
the ingestion of these materials.
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11.6.17.5.8 Disease Control
11.6.17.5.8.1 Fish are observed daily for abnormal appearance or behavior.
Bacterial or fungal infections are the most common diseases encountered.
However, if normal precautions are taken, disease outbreaks will rarely, if
ever, occur. Hoffman and Mitchell (1980) have put together a list of isome
chemicals that have been used commonly for fish diseases and pests.
11.6.17.5.8.2 In aquatic culture systems where filtration is utilized, the
application of certain antibacterial agents should be used with caution.
A treatment with a single dose of antibacterial drugs can interrupt nitrate
reduction and stop nitrification for various periods of time, resulting in
changes in pH, and in ammonia, nitrite and nitrate concentrations (Collins et
al., 1976). These changes could cause the death of the culture organisms.
11.6.17.5.8.3 Do not transfer equipment from one tank to another without
first disinfecting tanks and nets. If an outbreak of disease occurs, any
equipment, such as nets, airlines, tanks, etc., which has been exposed to
diseased fish should be disinfected with sodium hypochlorite. Also to avoid
the contamination of cultures or spread of disease, each time nets are used to
remove live or dead fish from tanks, they are first sterilized with sodium
hypochlorite or formalin, and rinsed in hot tap water. Before a new lot of
fish is transferred to culture tanks, the tanks are cleaned and sterilized as
described above.
11.6.17.5.8.4 It is recommended that chronic toxicity tests be performed
monthly with a reference toxicant. Newly hatched fathead minnow larvae less
than 24 h old are used to monitor the chronic toxicity of the reference
toxicant to the test fish produced by the culture unit (see Section 4, Quality
Assurance).
11.6.J7.5.9 Record Keeping
11.6.17.5.9.1 Records, kept in a bound notebook, include: (1) type of food
and time of feeding for all fish tanks; (2) time of examination of the tiles
for embryos, the estimated number of embryos on the tile, and the tile:
position number; (3) estimated number of dead embryos and embryos with fungus
observed during the embryonic development stages; (4) source of all fish; (5)
daily observation of the condition and behavior of the fish; and (6J~ dates and
results of reference toxicant tests performed (see Section 4, Quality
Assurance). ;
i
11.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE
11.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.
11.8 CALIBRATION AND STANDARDIZATION
11.8.1 See Section 4, Quality Assurance.
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11.9 QUALITY CONTROL
11.9.1 See Section 4, Quality Assurance.
11.10 TEST PROCEDURES
11.10.1 TEST SOLUTIONS
11.10.1.1 Receiving Waters
11.10.1.1.1 The sampling point is determined by the objectives of the test.
Receiving water toxicity is determined with samples used directly as collected
or after samples are passed through a 60 pm NITEXฎ filter and compared without
dilution, against a control. Using four replicate chambers per test, each
containing 250 mL, and 400 mL for chemical analyses, would require
approximately 1.5 L or more of sample per test per day.
11.10.1.2 Effluents
11.10.1.2.1 The selection of the effluent test concentrations should be based
on the objectives of the study. A dilution factor of 0.5 is commonly used.
A dilution factor of 0.5 provides precision of ฑ 100%, and testing of
concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%). Test precision shows
little improvement as the dilution factor is increased beyond 0.5, and
declines rapidly if a smaller dilution factor is used. Therefore, USEPA
recommends the use of the > 0.5 dilution factor.
11.10.1.2.2 If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%). If a high rate of mortality is observed during the first
1 to 2 h of the test, additional dilutions should be added at the lower range
of effluent concentrations.
11.10.1.2.3 The volume of effluent required for daily renewal of four
replicates per concentration, each containing 250 ml of test solution, is
approximately 2.5 L. Sufficient test solution (approximately 1500 ml) is
prepared at each effluent concentration to provide 400 ml additional volume
for chemical analyses at the high, medium, and low test concentrations.
If the sample is used for more than one daily renewal of test solutions, the
volume must be increased proportionately.
11.10.1.2.4 Tests should begin as soon as possible, preferably within 24 h of
sample collection. The maximum holding time following retrieval of the sample
from the sampling device should not exceed 36 h for off-site toxicity tests
unless permission is granted by the permitting authority. In no case should
the sample be used for the first time in a test more than 72 h after sample
collection (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).
11.10.1.2.5 Just prior to test initiation (approximately 1 h) the temperature
of sufficient quantity of the sample to make the test solutions should be
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adjusted to the test temperature and maintained at that temperature during the
addition of dilution water.
11.10.1.2.6 The DO of the test solutions should be checked prior to the test
initiation. If any of the solutions are supersaturated with oxygen or any
solution has a DO concentration below 4.0 mg/L, all of the solutions and the
control must be gently aerated.
11.10.1.3 Dilution Water
11.10.1.3.1 Dilution water may be uncontaminated receiving water, a standard
synthetic (reconstituted) water, or some other uncontaminated natural water
(see Section 7, Dilution Water).
11.10.2 START OF THE TEST '
11.10.2.1 Label the test chambers with a marking pen. Use of color-coded
tape to identify each treatment and replicate is helpful. A minimum of five
effluent concentrations and a control are used for each effluent test. : Each
treatment (including the control) should have four (minimum of three)
replicates.
11.10.2.2 Tests performed in laboratories that have in-house fathead minnow
breeding cultures should use larvae less than 24 h old. When eggs or larvae
must be shipped to the test site from a remote location, it may be necessary
to use larvae older than 24 h because of the difficulty in coordinating.test
organism shipments with field operations. However, in the latter case, the
larvae should not be more than 48 h old at the start of the test and should
all be within 24 h of the same age.
11.10.2.3 Randomize the position of test chambers at the beginning of the
test (see Appendix A). Maintain the chambers in this configuration throughout
the test. Preparation of a position chart may be helpful.
11.10.2.4 The larvae are pooled and placed one or two at a time into each
randomly arranged test chamber or intermediate container in sequential order,
until each chamber contains 15 (minimum of 10) larvae, for a total of
60 larvae (minimum of 30) for each concentration (see Appendix A). 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 should be kept to a minimum to avoid unnecessary
dilution of the test concentrations.
11.10.2.4.1 The chambers may be placed on a light table to facilitate
counting the larvae.
11.10.3 LIGHT, PHOTOPERIOD, AND TEMPERATURE
11.10.3.1 The light quality and intensity should be at ambient laboratory
levels, which is approximately 10-20 nE/nr/s, or 50 to 100 foot 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 ฑ 1ฐC.
70 ;
-------
11.10.4 DISSOLVED OXYGEN (DO) CONCENTRATION
11.10.4.1 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 be measured in the new solutions at the start of the
test (Day 0) and before daily renewal of the test solutions on subsequent
days. The DO concentrations should not fall below 4.0 mg/L (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests). 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, KIMAXฎ 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. ',
11.10.5 FEEDING
11.10.5.1 The fish in each test chamber are fed 0.1 g (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 or, as a minimum, 0.15 g are
fed twice daily at an interval of 6 h. Equal amounts of riauplii must be added
to each replicate chamber to reduce variability in larval weight. Sufficient
numbers of nauplii should be provided to assure that some remain alive in the
test chambers at the next feeding, but not in excessive amounts which will
result in depletion of DO below acceptable levels (below 4.0 mg/L).
11.10.5.2 The feeding schedule will depend on when the test solutions are
renewed. If the test is initiated after 12:00 PM, the larvae may be fed only
once the first day. On following days, the larvae normally would be fed at
the beginning of the work day, at least 2 h before test solution renewal, and
at the end of the work day, after test solution renewal. However, if the test
solutions are changed at the beginning of the work day, the first feeding
would be after test solution renewal in the morning, and the remaining
feeding(s) would be at the appropriate intervals. The larvae are not fed
during the final 12 h of the test. ,
11.10.5.3 The nauplii should be rinsed with freshwater to remove salinity
before use (see USEPA, 1993b). At feeding time pipette about 5 mL (5 g) of
concentrated newly hatched brine shrimp nauplii into a 120 mesh nylon net or
plastic cup with nylon mesh bottom. Slowly run freshwater through the net or
rinse by immersing the cup in a container of fresh water several times.
Resuspend the brine shrimp in 10 mL of fresh water in a 30 ml beaker or simply
set the cup of washed brine shrimp in % inch of fresh water so that the cup
contains about 10 mL of water. Allow the container to set for a minute or two
to allow dead nauplii and empty cysts to settle or float to the surface before
collecting the brine shrimp from just below the surface in a pipette for
feeding. Distribute 2 drops (0.1 g) of the brine shrimp to each test chamber.
If the survival rate in any test chamber falls below 50%, reduce the feeding
in that chamber to 1 drop of brine shrimp at each subsequent feeding.
71
-------
11.10.6 OBSERVATIONS DURING THE TEST
11.10.6.1 Routine Chemical and Physical Determinations
11.10.6.1.1 DO is measured at the beginning and end of each 24-h exposure
period in at least one test chamber at each test concentration and in the
control. I
11.10.6.1.2 Temperature and pH are measured at the end of each 24-h exposure
period in at least one test chamber at each test concentration and in the
control. Temperature should also be monitored continuously or observed and
recorded daily for at least two locations in the environmental control system
or the samples. Temperature should be measured in a sufficient number of test
vessels at least at the end of the test to determine the temperature variation
in the environmental chamber.
11.10.6.1.3 The pH is measured in the effluent sample each day before new
test solutions are made.
11.10.6.1.4 Conductivity, alkalinity and hardness are measured in each new
sample (100% effluent or receiving water) and in the control.
11.10.6.1.5 Record all the measurements on the data sheet (Figure 1).
11.10.6.2 Routine Biological Observations
11.10.6.2.1 The number of live larvae in each test chamber are recorded daily
(Figure 2), and the dead larvae are discarded.
11.10.6.2.2 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 these operations.
11.10.7 DAILY CLEANING OF TEST CHAMBERS :
11.10.7.1 Before the daily renewal of test solutions, uneaten and dead
Artemia, dead fish larvae, 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 or pipet
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. Any incidence of removal of live larvae from the
test chambers during cleaning, and subsequent return to the chambers, should
be noted in the records.
72
-------
Discharger:
Location: _
Analyst:
Dates:
Day i
Cone:
Temp.
D.O. Initial
Final
pH Initial
Final
Al kal initv
Hardness
Conductivity
Chi orine
1
2
3
4
5
6
7
.
Remarks
Day
Cone:
Temp.
D.O. Initial
Final
pH Initial
Final
Al kal initv
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
Remarks
Day
Cone:
Temp.
D.O. Initial
Final
pH Initial
Final
Al kal initv
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
i
!
Remarks
Figure 1. Data form for the fathead minnow, Pimephales promelas, larval
survival and growth test. Routine chemical and physical
determinations.
73
-------
Discharger:
Location:
Analyst:
Dates:
Day
Cone:
Terno.
D.O. Initial
Final
oH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
Remarks
Day
Cone:
Terno.
D.O. Initial
Final
oH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
Remarks
Day
Cone:
Terno.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
Remarks
Figure 1. Data form for the fathead minnow, Pimephales promelas, larval
survival and growth test. Routine chemical and physical
determinations (CONTINUED). :
74
-------
Discharger:
Location:
Dates:
Analyst:
No. Surviving Organisms
Cone: Rep.
Day
No.
Control :
Cone:
Cone:
Cone:
Cone:
Cone:
Cone:
Cone:
1
2
3
4
5
6
7
Remarks
Comments
Figure 2. Mortality data for the fathead minnow, Pimephales promelas,
larval survival and growth test.
75
-------
11.10.8 TEST SOLUTION RENEWAL
11.10.8.1 Freshly prepared solutions are used to renew the tests daily
immediately after cleaning the test chambers. For on-site toxicity studies,
fresh effluent or receiving water samples should be collected daily, and no
more than 24 h should elapse between collection of the samples and their use
in the tests (see Section 8, Effluent and Receiving Water Sampling, Sample
Holding, and Sample Preparation for Toxicity Tests). For off-site tejsts, a
minimum of three samples are collected, preferably on days one, three, and
five. Maintain the samples in the refrigerator at 4ฐC until used.
11.10.8.2 For test solution renewal, the water 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 (250 mL) should be added slowly by pouring down the side of
the test chamber to avoid excessive turbulence and possible injury to the
larvae.
11.10.9 TERMINATION OF THE TEST !
11.10.9.1 The test is terminated after seven days of exposure. At test
termination, dead larvae are removed and discarded. The surviving larvae in
each test chamber (replicate) are counted and immediately prepared as a group
for dry weight determination, or are preserved as a group in 70% ethanol or
4% formalin. Preserved organisms are dried and weighed within 7 days. For
safety, formalin should be used under a hood.
11.10.9.2 For immediate drying and weighing, place live larvae onto !a 500 /j,m
mesh screen in a large beaker to wash away debris that might contribute to the
dry weight. Each group of larvae is rinsed with deionized water to remove
food particles, transferred to a tared weighing boat that has been properly
labeled, and dried at 60ฐC, for 24 h or at 100ฐC for a minimum of 6 h.
Immediately upon removal from the drying oven, the weighing boats are placed
in a dessicator until weighed, to prevent the absorption of moisture from the
air. All weights should be measured to the nearest 0.01 mg and recorded on
data sheets (Figure 3). Subtract tare weight to determine the dry weight of
the larvae in each replicate. For each test chamber, divide the final dry
weight by the number of original larvae in the test chamber to determine the
average individual dry weight and record on the data sheet (Figure 3)j. For
the controls, also calculate the mean weight per surviving fish in the test
chamber to evaluate if weights met test acceptability criteria (See
Section 11.11). Average weights should be expressed to the nearest 0.001 mg.
11.10.9.3 Prepare a summary table as illustrated in Figure 4. .
11.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA i
11.11.1 A summary of test conditions and test acceptability criteria! is
presented in Table 1.
76
-------
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77
-------
Discharger:
Location:
Test Dates:
Analyst:
TREATMENT
NO. LIVE LARVAE
SURVIVAL
101 "\
(A)
MEAN DRY WGT OF
LARVAE (MG)
ฑ SD
TEMPERATURE
RANGE (ฐC)
DISSOLVED
OXYGEN RANGE
(MG/L)
HARDNESS
CONDUCTIVITY
CONTROL
i
'
1
COMMENTS:
Figure 4. Summary data for the fathead minnow, Pimephales promelas,
larval survival and growth test.
78
-------
TABLE 1. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
FOR FATHEAD MINNOW, PIMEPHALES PROMELAS, LARVAL SURVIVAL AND
GROWTH TOXICITY TESTS WITH EFFLUENTS AND RECEIVING WATERS
1. Test type: Static renewal :
2. Temperature (ฐC): 25 ฑ 1ฐC
3. Light quality: Ambient laboratory illumination
4. Light intensity: 10-20 ME/m2/s (50-100 ft-c)(ambient
laboratory levels)
5. Photoperiod: 16 h light, 8 h darkness
6. Test chamber size: 500 mL (minimum)
!
7. Test solution volume: 250 mL (minimum)
8. Renewal of test ;
solutions: Daily I
9. Age of test organisms: Newly hatched larvae less than 24 h old.
If shipped, not more than 48 h old, 24 h
range in age
10. No. larvae per test chamber: 15 (minimum of 10) '
11. No. replicate chambers
per concentration: 4 (minimum of 3)
12. No. larvae per
concentration: 60 (minimum of 30) ;
13. Source of food: Newly hatched Artemia nauplii (less than
24 h old)
14. Feeding regime: Feed 0.1 g newly hatched (less than 24-h
old) brine shrimp nauplii three times
daily at 4-h intervals or, as a minimum,
0.15 g twice daily, 6 h between feedings
(at the beginning of the work day prior to
renewal, and at the end of the work day
following renewal). Sufficient nauplii
are added to provide an excess. Larvae
fish are not fed during the final 12 h of
the test
79
-------
TABLE 1. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
FOR FATHEAD MINNOW, PIMEPHALES PROMELAS, LARVAL SURVIVAL AND
GROWTH TOXICITY TESTS WITH EFFLUENTS AND RECEIVING WATERS
(CONTINUED) '
15. Cleaning:
16. Aeration:
17. Dilution water:
18. Test concentrations:
19. Dilution factor
20. Test duration:
21. Endpoints:
22. Test acceptability
criteria:
23. Sampling
requirements:
Siphon daily, immediately before test
solution renewal
None, unless DO concentration falls below
4.0 mg/L. Rate should not exceed
100 bubbles/min
Uncontaminated source of receiving or
other natural water, synthetic water
prepared using MILLIPORE MILLI-Qฎ or
equivalent deionized water and reagent
grade chemicals, or DMW (see Section 7,
Dilution Water)
Effluents: Minimum of 5 and a.control
Receiving Water: 100% receiving water or
minimum of 5 and a control
Effluents: > 0.5 ;
Receiving waters: None or > 0.5 !
7 days
Survival and growth (weight)
80% or greater survival in controls; ;
average dry weight per surviving organism
in control chambers equals or exceeds:0.25 mg
For on-site tests, samples collected
daily, and used within 24 h of the time
they are removed from the sampling device;
For off-site tests, a minimum of
three samples collected on days one,
three and five with a maximum holding time
of 36 h before first use (see Section 8,
24. Sample volume required: 2.5 L/day
80
-------
11.12 ACCEPTABILITY OF TEST RESULTS
11.12.1 For the test results to be acceptable, survival in the controls must
be at least 80%. The average dry weight per surviving control larvae at the
end of the test should equal or exceed 0.25 mg.
11.13 DATA ANALYSIS
11.13.1 GENERAL
11.13.1.1 Tabulate and summarize the data.
growth response data is shown in Table 2.
A sample set of survival and
TABLE 2. SUMMARY OF SURVIVAL AND GROWTH DATA FOR FATHEAD MINNOW,
PIMEPHALES PROMELAS, LARVAE EXPOSED TO A REFERENCE TOXICANT
FOR SEVEN DAYS
NaPCP
Cone.
(M9/L)
0
32
64
128
256
512
Proportion of
Survival in Replicate
Chambers
A B C D
1.0
0.8
0.9
0.9
0.7
0.4
1.0
0.8
1.0
0.9
0.9
0.3
0,9
1.0
1.0
0.8
1.0
0.4
0.9
0.8
1.0
1.0
0.5
0.2
Mean
Prop.
Surv
0
0
0
0
0
0
.95
.85
.975
.90
.775
.325
Avg
Rep!
A
0.711
0.517
0.602
0.566
0.455
0.143
Dry Wgt (mg) In Mean
icate Chambers Dry Wgt
B C D (mg)
0.662
0.501
0.669
0.612
0.502
0.163
0.646
0,723
0.694
0,410
0.606
0.195
0.690
0.560
0.676
0.672
0.508
0.099
0.677
0.575
0.660
0.565
0.454
0.150
Four replicates of 10 larvae each.
11.13.1.2 The endpoints of toxicity tests using the fathead minnow,
Pimephales promelas, larvae are based on the adverse effects on survival and
growth. The LC50, the IC25, and the IC50 are calculated using point
estimation techniques (see Section 9, Chronic Toxicity Test Endpoints and Data
Analysis). LOEC and NOEC values for survival and growth are obtained using a
hypothesis testing approach such as Dunnett's Procedure (Dunnett, 1955) or
Steel's Many-one Rank Test (Steel, 1959; Miller, 1981) (see Section 9).
Separate analyses are performed for the estimation of the LOEC and NOEC
endpoints and for the estimation of the LC50, IC25 and IC50. Concentrations
81 i
-------
at which there is no survival in any of the test chambers are excluded from
the statistical analysis of the NOEC and LOEC for survival and growth, but
included in the estimation of the LC50, IC25, and IC50. See the Appendices
for examples of the manual computations, and examples of data input and
program output.
11.13.1.3 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 Appendix B. The assistance of a
statistician is recommended for analysts who are not proficient in statistics.
11.13.2 EXAMPLE OF ANALYSIS OF FATHEAD MINNOW, PIMEPHALES PROMELAS,
SURVIVAL DATA
11.13.2.1 Formal statistical analysis of the survival data is outlined in
Figures 5 and 6. The response used in the analysis is the proportion of
animals surviving in each test or control chamber. Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and for the
estimation of the LC50, EC50, and 1C endpoints. Concentrations at which there
is no survival in any of the test chambers are excluded from statistical
analysis of the NOEC and LOEC, but included in the estimation of the 1C, EC,
and LC endpoints.
11.13.2.2 For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
Underlying assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality is the Shapiro-Wilk's
Test, and Bartlett's Test is used to test for homogeneity of variance. If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test, is used to determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure. ',
11.13.2.3 If unequal numbers of replicates occur among the concentration
levels tested, there are parametric and nonparametric alternative analyses.
The parametric analysis is a t test with the Bonferroni adjustment (see
Appendix D). The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
nonparametric alternative (see Appendix F).
11.13.2.4 Probit Analysis (Finney, 1971; see Appendix I) is used to estimate
the concentration that causes a specified percent decrease in survival from
the control. In this analysis, the total mortality data from all test
replicates at a given concentration are combined. If the data do not fit the
Probit analysis, the Spearman-Karber Method, the Trimmed Spearman-Karber
Method, or the Graphical Method may be used (see Appendices I-L).
82
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STATISTICAL ANALYSIS OF FATHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
SURVIVAL HYPOTHESIS TESTING
SURVIVAL DATA
PROPORTION SURVIVING
i
r
ARC SINE
TRANSFORMATION
SHAPIRO-WIUCSTEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
HETEROGENEOUS
VARIANCE
J
1
EQUAL NUME
REHLICA!
NO
^
IER OF
ES?
YES
T TP^T WITH
"^ 1 YVI 1 [I r\t IklKIL 1 1 IQ
BONFERRONI uu TptT &
ADJUSTMENT lt&l
i
EQ
YES
J
STEEL'S MANY-ONE
RANK TEST
t
ENDPOINT ESTIMATES
NOEC, LOEC
JAL NUMBER OF
REPLICATE-S?
1 NO
WILOOXON RANK SUM
TESiTWITH
BONFEHROIMI ADJUSTMENT
'
Figure 5. Flowchart for statistical analysis of the fathead minnow, Pimephales
promelas, larval survival data by hypothesis testing.
83
-------
STATISTICAL ANALYSIS OF FATHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
SURVIVAL POINT ESTIMATION
MORTALITY DATA
#DEAD
TWO OR MORE
PARTIAL MORTALITIES?
NO
YES
IS PROSIT MODEL
APPROPRIATE?
(SIGNIFICANT X2 TEST)
NO
ONE OR MORE
PARTIAL MORTALITIES?
NO
^
YES
IYES
GRAPHICAL METHOD
LC50
PROBIT METHOD
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONG.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONG.?
NO
YES
SPEARMAN-KARBER
METHOD
TRIMMED SPEARMAN
KARBER METHOD
LC50 AND 95%
CONFIDENCE
INTERVAL
Figure 6. Flowchart for statistical analysis of the fathead minnow, Pimephales
promelas, larval survival data by point estimation. '(
84
-------
11.13.2.5 Example of Analysis of Survival Data
11.13.2.5.1 This example uses the survival data from the Fathead Minnow
Larval Survival and Growth Test (Table 2). The proportion surviving in each
replicate must first be transformed by the arc sine square root transformation
procedure described in Appendix B. The raw and transformed data, means and
variances of the transformed observations at each toxicant concentration and
control are listed in Table 3. A plot of the survival proportions is provided
in Figure 7.
TABLE 3. FATHEAD MINNOW, PIMEPHALES PROMELAS, SURVIVAL DATA
Replicate
Control
NaPCP Concentration (ttq/L)
32
64
128
256
512
RAW
ARC SINE
TRANS-
FORMED
Mean(Y,)
s?
^
A
B
C
D
A
B
C
D
1
- 1
0
0
1
1
1
1
1
0
1
.0
.0
.9
.9
.412
.412
.249
.249
.330
.0088
0
0
1
0
1
1
1
1
1
0
2
.8
.8
.0
.8
.107
.107
.412
.107
.183
.0232
0.9
1.0
1.0
1.0
1.249
1.412
1.412
1,412
1.371
0.0066
3
0
0
0
1
1
1
1
1
1
0
4
.9
.9
.8
.0
.249
.249
.107
.412
.254
.0155
0.
0.
1.
0.
0.
1.
1.
0.
1.
0.
5
7
9
0
5
991
249
412
785
109
0768
0.4
0.3
0.4
0.2
0.685
0.580
0.685
0.464
0.604
0.0111
6
11.13.2.6 Test for Normality
11.13.2.6.1 The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration. The centered
observations are summarized in Table 4.
11.13.2.6.2 Calculate the denominator, D, of the statistic:
D =
2
Where: X,- = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
85
-------
o
* ' *
O 00
d
evi
LO
(O
oi
^i
UJ
I
i
Q-
3
UJ
D-
o
o
CO
0)
_Q
as
s-
o
o.
o
s-
Q.
3
fj
o
OJ
CD
-O
O
o
NOIldOdOdd IVAIAUnS
86
-------
TABLE 4. CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
NaPCP Concentration fua/L)
Replicate
A
B
C
D
Control
0.082
0.082
-0.081
-0.081
32
-0.076
-0.076
0.229
-0.076
64
-0.122
0.041
0.041
0.041
128
-0.005
-0.005
-0.147
0.158
256
-0.118
0.140
0.303
-0.324
512
0.081
-0.024
0.081
-0.140
11.13.2.6.3 For this set of data: n = 24 ;
X = (0.000) =0.000
24 ;
D = 0.4265
11.13.2.6.4 Order the centered observations from smallest to largest
XC1) \
\
where XO) denotes the ith ordered observation. The ordered observations
for this example are listed in Table 5.
TABLE 5. ORDERED CENTERED OBSERVATIONS FOR THE SHAPIRO-WILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
11
12
x
-0.324
-0.147
-0.140
-0.122
-0.118
-0.081
-0.081
-0.076
-0.076
-0.076
-0.024
-0.005
i
13
14 '
15
16
17
18
19
20
21
22
23
24
xci)
-0.005
0.041
0.041
0.041
0.081
0.081
0.082
0.082
0.140
0.158
0.229
0.303
87
-------
11.13.2.6.5 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a,, a2, ... ak where k is n/2 if n is even and (n-l)/2
if n is odd. For the data in this example, n = 24 and k = 12. The a,- values
are listed in Table 6.
TABLE 6. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
^ v(r)~i+1)lv(i)
i a,- X - X
1
2
3
4
5
6
7
8
9
10
11
12
0.4493
0.3098
0.2554
0.2145
0.1807
0.1512
0.1245
0.0997
0.0764
0.0539
0.0321
0.'0107
0.627
0.376
0.298
0.262
0.200
0.163
0.162
0.157
0.117
0.117
0.065
0.000
X(24, .
Y<23)
A -
y{22)
A, -
Y<21>
A . ~
XC20) _
x(19) _
x<18> -
x(17) _
y(16)
ซ<15)
x!ซ! :
XC13> _
x<1>;
x(2)
x<3)
x"'
X
X2!
X *
x(ป
X
X
X
x<12>
11.13.2.6.6 Compute the test statistic, W, as follows:
1 ฃ 2
D i=i *
The differences x1> - X(i) are listed in Table 6. For the data in this
example,
W= (0.6444)2 = 0.974
0.4265
11.13.2.6.7 The decision rule for this test is to compare W as calculated in
Section 13.2.6.6 to a critical value found in Table 6, Appendix B. If the
computed W is less than the critical value, conclude that the data are not
normally distributed. For the data in this example, the critical value at a
significance level of 0.01 and n = 24 observations is 0.884. Since M = 0.974
is greater than the critical value, conclude that the data are normally
distributed.
11.13.2.7 Test for Homogeneity of Variance
11.13.2.7.1 The test used to examine whether the variation in mean proportion
surviving is the same across all toxicant concentrations including the
control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic
is as follows:
88
-------
B=
Where: V1- = degrees of freedom for each toxicant concentration and
control, V, = (n, - 1) !
nf = the number of replicates for concentration i
In = loge
i = 1, 2, ..., p where p is the number of concentrations
including the control ;
_ i-1
,
i-i
C =
11.13.2.7.2 For the data in this example (see Table 3), all toxicant
concentrations including the control have the same number of replicates
(n, = 4 for all i). Thus, V, - 3 for all i.
11.13.2.7.3 Bartlett's statistic is therefore:
B - [(18)1*2(0.0236) -3 ฃlrz(Si) 1/1.1296
2=1
= [18(-3.7465) - 3(-24.7516)3/1.1296
= 6.8178/1.1296 i
= 6.036
11.13.2.7.4 B is approximately distributed as chi-square with p - 1 degrees
of freedom, when the variances are in fact the same. Therefore, the
appropriate critical value for this test (from a table of chi-square
distribution), at a significance level of 0.01 with five degrees of freedom,
is 15.086. Since B = 6.036 is less than the critical value of 15.086,
conclude that the variances are not different. j
11.13.2.8 Dunnett's Procedure
11.13.2.8.1 To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 7.
89
-------
TABLE 7. ANOVA TABLE
Source
Between
Within
Total
df
P - 1
N - p
N - 1
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square(MS)
(SS/df)
Sg = SSB/(p-l)
S* = SSH/(N,p)
Where: p - number toxicant concentrations including the control
N - total number of observations n., + n2 ... + np
n,- = number of observations in concentration i
SSB = Y,Tl/nฑ-G2/N Between Sum of Squares
i-l
SST - ฃ T,Yij-G2/N Total Sum of Squares
SSW = SST-SSB Within Sum of Squares
G - the grand total of all sample observations,G = ฃ Tฑ
T,- = the total of the replicate measurements for concentration i
Y,-- - the jth observation for concentration i (represents the proportion
surviving for toxicant concentration i in test chamber j)
11.13.2.8.2 For the data in this example:
n, - n2 - n3 = n4 = n5 = n6 = 4 ;
N = 24
TI = YH + Y12 + Y13 + Y14 = 5.322 :
T2 = Y21 + Y22 + Y23 + Y24 - 4.733
T3 - Y31 + Y32 + Y33 + Y34 = 5.485
T4 - Y41 + Y42 H- Y43 + Y44 = 5.017
90
-------
T5 = Y51 + Y52 + Y53 + Y54 = 4.437
T6 = Y61 + Y62 + Y63 + Y64 = 2.414
G = T, + T2 + T3 + T4 + T5 + T6 = 27.408
SSB = f,Ti/ni-G2/N
= _1_(131.495) - (27. 408)2 = 1.574
4 24
SST =
i-1.7=1 ~" !
= 33.300 - (27.408)2 == 2.000 |
24
SSW = SST-SSB = 2.000 - 1.574 = 0.4260
S2 = SSB/(p-l) = 1.574/(6~1) = 0.3150
S2 = SSW/(N-p) = 0.426/(24-6) = 0.024 ,
11.13.2.8.3 Summarize these calculations in the ANOVA table (Table 8)
i
TABLE 8. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source df Sum of Squares Mean Square(MS)
(SS) (SS/df)
Between 5 1.574 0.315
Within 18 0.426 0.024
Total 23 2.002
91
-------
11.13.2.8.4 To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
Where: Y
= mean proportion surviving for concentration i
Y1 - mean proportion surviving for the control
Sw = square root of the within mean square
n., ~ number of replicates for the control
n,- - number of replicates for concentration i.
11.13.2.8.5 Table 9 includes the calculated t values for each concentration
and control combination. In this example, comparing the 32 ng/l concentration
with the control the calculation is as follows:
(1.330-1.183)
=1.341
[0.155^(1/4) + (1/4)]
TABLE 9. CALCULATED T VALUES
NaPCP Concentration
32
64
128
256
512
2
3
4
5
6
1.341
-0.374
0.693
2.016
6.624
11.13.2.8.6 Since the purpose of this test is to detect a significant
reduction in proportion surviving, a one-sided test is appropriate. The
critical value for this one-sided test is found in Table 5, Appendix C. For
an overall alpha level of 0.05, 18 degrees,of freedom for error and five
concentrations (excluding the control) the critical value is 2.41. The mean
proportion surviving for concentration i is considered significantly less than
the mean proportion surviving for the control if t,- is greater than the
critical value. Since t, is greater than 2.41, the 512 M9/L concentration has
significantly lower survival than the control. Hence the NOEC and the LOEC
for survival are 256 /zg/L and 512 /jg/L, respectively.
92
-------
11.13.2.8.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be detected statistically may be calculated.
MSD = d fl^d/nj + (1/73)
Where: d = the critical value for Dunnett's procedure
Sw = the square root of the within mean square
n = the common number of replicates at each concentration
(this assumes equal replication at each concentration)
n, = the number of replicates in the control .
11.13.2.8.8 In this example:
MSD = 2.41(0. 155)v/(l/4) + (1/4)
= 2.41 (0.155)(0.707)
= 0.264 I
11.13.2.8.9 The MSD (0.264) is in transformed units. To determine the MSD in
terms of percent survival, carry out the following conversion.
1. Subtract the MSD from the transformed control mean;
1.330 - 0.264 = 1.066 !
2. Obtain the untransformed values for the control mean and the difference
calculated in 1.
[Sine ( 1.330) ]* = 0.943
[Sine ( 1.066) ]2 = 0.766
3. The untransformed MSD (MSD ) is determined by subtracting the
untransformed values from 2. ! '
MSDU - 0.943 - 0.766 = 0.177
11.13.2.8.10 Therefore, for this set of data, the minimum difference in mean
proportion surviving between the control and any toxicant; concentration that
can be detected as statistically significant is 0.177.
11.13.2.8.11 This represents a decrease in survival of 19% from the control.
93
-------
11.13.2.9 Calculation of the LC50
11.13.2.9.1 The data used for the Probit Analysis is summarized in Table 10.
To perform the Probit Analysis, run the USEPA Probit Analysis Program.
An example of the program input and output is supplied in Appendix I. ;
TABLE 10. DATA FOR PROBIT ANALYSIS
NaPCP Concentration
Control
32
64
128
256
512
Number Dead
Number Exposed
2
40
6
40
1
40
4
40
9
40
27
40
11.13.2.9.2 For this example, the chi-square test for heterogeneity was not
significant, thus Probit Analysis appears appropriate for this data.
11.13.2.9.3 Figure 8 shows the output data for the Probit Analysis of the
data in Table 10 using the USEPA Probit Program.
11.13.3 EXAMPLE OF ANALYSIS OF FATHEAD MINNOW, PIMEPHALES PROMELAS, GROWTH
DATA
11.13.3.1 Formal statistical analysis of the growth data is outlined in
Figure 9. The response used in the statistical analysis is mean weight per
replicate. An 1C estimate can be calculated for the growth data via a point
estimation technique (see Section 9, Chronic Toxicity Test Endpoints and Data
Analysis). Hypothesis testing can be used to obtain the NOEC for growth.
Concentrations above the NOEC for survival are excluded from the hypothesis
test for growth effects.
11.13.3.2 The statistical analysis using hypothesis tests consists of a
parametric test, Dunnett's Procedure, and a nonparametric test, Steel's
Many-one Rank Test. The underlying assumptions of the Dunnett's Procedure,
normality and homogeneity of variance, are formally tested. The test for
normality is the Shapiro-Wilk's Test and Bartlett's Test is used to test for
homogeneity of variance. If either of these tests fails, the nonparametric
test, Steel's Many-one Rank Test, is used to determine the NOEC and LOEC
endpoints. If the assumptions of Dunnett's Procedure are met, the endpoints
are determined by the parametric test.
11.13.3.3 Additionally, if unequal numbers of replicates occur among the
concentration levels tested there are parametric and nonparametric alternative
analyses. The parametric analysis is a t test with the Bonferroni adjustment
(see Appendix D). The Wilcoxon Rank Sum Test with the Bonferroni adjustment
is the nonparametric alternative (see Appendix F).
94
-------
Probit Analysis of Fathead Minnow Larval Survival Data
Cone.
Control
32.0000
64.0000
128.0000
256.0000
512.0000
Number
Exposed
40
40
40
40
40
40
Number
Resp.
2
6
1
4
9
27
Observed
Proportion
Responding
0.0500
0.1500
0.0250
0.1000
0.2250
0.6750
Proportion
Responding
Adjusted for
Controls
0.0000
0.0779
-.0577
0.0237
0.1593
0.6474
Chi - Square for Heterogeneity (calculated)
Chi - Square for Heterogeneity
(Tabular value at 0.05 level)
4.522
7.815
Probit Analysis of Fathead Minnow Larval Survival Data
Estimated LC/EC Values and Confidence Limits
Point
LC/EC 1.00
LC/EC 50.00
Exposure
Cone.
127.637
422.696
Lower Upper
95% Confidence Limits
34.590
345.730
195.433
531.024
Figure 8. Output for USEPA Probit Analysis Program, Version 1.5
95
-------
STATISTICAL ANALYSIS OF FATHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
GROWTH
GROWTH DATA
MEAN DRY WEIGHT
POINT ESTIMATION
HYPOTHESIS TESTING
(EXCLUDING CONCENTRATIONS
ABOVE NOEC FOR SURVIVAL)
ENDPOINT ESTIMATE
IC25, IC50
SHAPIRO-MILK'S TEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
J
HETEROGENEOUS
VARIANCE
I
^
EQUAL NUME
REPUCAT
NO
1
JEROF
ES?
YES
T-TESTWITH DUNNETrs
BONFERRONI DUNNETTS
ADJUSTMENT lfctsl
\
EQ
YES
r
STEEL'S MANY-ONE
RANK TEST
*
ENDPOINT ESTIMATES
NOEC, LOEC
UAL NUMBER OF
REPLICATES?
I NO
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
Figure 9. Flowchart for statistical analysis of fathead minnow, Pimephales
promelas, larval growth data.
96
-------
11.13.3.4 The data, mean and variance of the observations; at each
concentration including the control are listed in Table 11. A plot of the
weight data for each treatment is provided in Figure 10. Since there is
significant mortality in the 512 vg/L concentration, its effect on growth is
not considered.
TABLE 11. FATHEAD MINNOW, PIMEPHALES PROMELAS, GROWTH DATA
Replicate Control
NaPCP Concentration C/zg/L)
32 64 128 256 512
A
B
C
D
0.711
0.662
. 0.646
0.690
0.517
0.501
0.723
0.560
0.602
0.669
0.694
0.676
0.566
0.612
0.410
0.672
0.455
0.502
0.606
0.254
-
-
MearKY,)
Si
i
0.677
0.00084
1
0.525 0.660 0.624 0.580
0.01032 0.00162 0.01256 0.0218
2 3 4 5
6
11.13.3.5 Test for Normality
11.13.3.5.1 The first step of the test for normality is to center the
observations by subtracting the mean of all the observations within a
concentration from each observation in that concentration. The centered
observations are summarized in Table 12.
TABLE 12. CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE
Replicate
NaPCP Concentration (tto/L)
Control
32
64
128
256
A
B
C
D
0.034
-0.015
-0.031
0.013
-0.058
-0.074
0.148
-0.015
-0.058
0.009
0.034
0.016
0.001
0.047
-0.155
0.107
0.001
0.048
0.152
-0.200
97
-------
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11.13.3.5.2 Calculate the denominator, D, of the test statistic:
i-i
Where: X,- = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations:
For this set of data, n = 20
X = _1_ (0.004) = 0.000
20 i
D = 0.1414
11.13.3.5.3 Order the centered observations from smallest to largest
X(1)
-0.200
-0.155
-0.074
-0.058
-0.058
-0.031
-0.015
-0.015
0.001
0.001
i
11
12
13
14
15
16
17
18
19
20
X(D
0.009
. 0.013
0.016
0.034
0.034
0..047
0..048
0.107
0.148
i 0.152
11.13.3.5.4 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a,, a2, ..., ak where k is n/2 if n is even and
(n-l)/2 if n is odd. For the data in this example, n = 20 and k = 10. The a,-
values are listed in Table 14.
99
-------
TABLE 14. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
i a,- X(n'i+1) - X(0
1
2
3
4
5
6
7
8
9
10
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
0.0711
0.0422
0.0140
0..352
0.303
0.131
0.106
0.105
0.065
0.049
0.031
0.012
0.008
v(20)
A
x<19)
xcia>
XC17)
X(16>
X(15>
X(H)
X(13)
x<12)
- Xm
x<2)
v(3)
- x<4)
- x<5)
- x(6)
- x(7)
- x(8)
- x(9)
- x(10)
11.13.3.5.5 Compute the test statistic, W, 'as follows:
W =
D
I
the differences x(n"i+1) - Xci) are listed in Table 14. For this set of data:
W = 1 (0.3666)2 = 0.9505
0.1414
11.13.3.5.6 The decision rule for this test is to compare W with the critical
value found in Table 6, Appendix B. If the computed W is less than the
critical value, conclude that the data are not normally distributed. For this
example, the critical value at a significance level of 0.01 and 20
observations (n) is 0.868. Since W = 0.959 is greater than the critical
value, the conclusion of the test is that the data are normally distributed.
11.13.3,6 Test for Homogeneity of Variance
11.13.3.6.1 The test used to examine whether the variation in mean dry weight
is the same across all toxicant concentrations including the control, is
Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as
fol1ows:
[ ( Vฑ) ln~S2 - 'Vฑ in
100
-------
Where: V,- = degrees of freedom for each toxicant concentration and
control, V,- = (n,- - 1)
n,- = the number of replicates for concentration i.
In = loge
i = 1, 2, ..., p where p is the number of concentrations
including the control
i=l
1=1 i=l
11.13.3.6.2 For the data in this example, (see Table 11) all toxicant
concentrations including the control have the same number of replicates
(n, = 4 for all i). Thus, V,- = 3 for all i.
11.13.3.6.3 Bartlett's statistic is therefore:
B= [(15) In (0.00947) -3 ฃ In (S?)]/1.133
= [15(-5.9145) - 3(-26.2842]/1.133
= 8.8911/1.133
= 7.847
11.13.3.6.4 B is approximately distributed as chi-square with p - 1 degrees
of freedom, when the variances are in fact the same. Therefore, the
appropriate critical value for this test, at a significance level of 0.01 with
four degrees of freedom, is 13.277. Since B = 7.847 is less than the critical
value of 13.277, conclude that the variances are not different.
101
-------
11.13.3.7 Dunnett's Procedure
11.13.3.7.1 To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 15.
TABLE 15. ANOVA TABLE
Source
Between
Within
Total
df
p - 1
N - p
N -, 1
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square(MS)
(SS/df)
SB = SSB/(p-l)
Sy = SSW/(N-p)
Where: p - number toxicant concentrations including the control
N = total number of observations n,, + n2 ... + n
n,- - number of observations in concentration i
SSB = r/j^-GV-W Between Sum of Squares
SST = ฃ ^yfi-G2/N Total Sum of Squares
jt-i;?=i
SSW = SST-SSB . Within Sum of Squares
G = the grand total of all sample observations, G = & T-
i=l :
T,- - the total of the replicate measurements for concentration i
YJJ = the jth observation for concentration i (represents the mean
dry weight of the fish for toxicant concentration i in test
chamber j)
102
-------
11.13.3.7.2 For the data in this example:
n., = n2 = n3 = n4 = n5 = 4
20
T - V -i- Y 4- V
T2 - Y2; + "ll + Yฃ
T _ Y >v i v
!; !!
Y14 = 2.709
Y,, = 2.301
Tc = Y
51
Y52 + Y53
33 + Y34 = 2.641
+ Y44 = 2.260
Y54 = 1.817
6 = T, + T2 + T3 + T4 + T5 = 11.728
SSB = f,Ti/nฑ-G2/N
1=1
= I (28.017) - (11.728)'
4 20
0.1270
SST =
= 7.146 - (11.728r = 0.2687
20
SSW = SST-SSB = 0.2687 - 0.1270 = 0.1417
S2 = SSB/(p-l) = 0.1270/(5-l) = 0.0318
S2 = SSW/(N-p) = 0.041/(20-5) = 0.0094
11.13.3.7.3 Summarize these calculations in the ANOVA table (Table 16)
TABLE 16. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
df
4
15
Sum of Squares
(SS)
0.1270
0.1417
Mean Square(MS)
(SS/df)
0.0318
0.0094
Total
19
0.2687
103
-------
11.13.3.7.4 To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
Where: Y
n
mean dry weight for toxicant concentration i
mean dry weight for the control
square root of the within mean square
number of replicates for the control
number of replicates for concentration i.
11.13.3.7.5 Table 17 includes the calculated t values for each concentration
and control combination. In this example, comparing the 32 jug/L concentration
with the control the calculation is as follows:
(0.677-0.575)
[0 . 097^(1/4) + (1/4)]
= 1.487
TABLE 17. CALCULATED T VALUES
NaPCP
Concentration
32
64
128
256
2
3
4
5
1.487
0.248
1.632
3.251
11.13.3.7.6 Since the purpose of this test is to detect a significant
reduction in mean weight, a one-sided test is appropriate. The critical value
for this one-sided test is found in Table 5, Appendix C. For an overall alpha
level of 0.05, 15 degrees of freedom for error and four concentrations
(excluding the control) the critical value is 2.36. The mean weight for
concentration "i" is considered significantly less than the mean weight for
the control if t, is greater than, the critical value. Since t4 and t5 are
greater than 2.36, the 128 /Ltg/L and 256 /*g/L concentrations have significantly
lower growth than the control. Hence the NOEC and the LOEC for growth are
128 ng/l and 256 ng/L, respectively.
104
-------
11.13.3.7.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be statistically detected may be calculated:,
MSD = d S^CL/nJ + (l/rz)
Where: d = the critical value for the Dunnett's Procedure
Sw = the square root of the within mean square
n = the common number of replicates at each concentration
(this assumes equal replication at each concentration)
n., = the number of replicates in the control.
11.13.3.7.8 In this example: ,
MSD = 2.36(0.052)^(1/4) + (1/4)
= 2.36 (0.097) (0.707)
= 0.162
11.13.3.7.9 Therefore, for this set of data, the minimum difference that can
be detected as statistically significant is 0.162 mg.
11.13.3.7.10 This represents a 24% reduction in mean weight from the control.
11.13.3.8 Calculation of the 1C
11.13.3.8.1 The growth data in Table 2 modified to be mean weights per
original number of fish are utilized in this example. As seen in Table 2 and
Figure 11, the observed means are not monotonically non-increasing with
respect to concentration (the mean response for each higher concentration is
not less than or equal to the mean response for the previous concentration,
and the responses between concentrations do not follow a linear trend).
Therefore, the means are smoothed prior to calculating the 1C. In the
following discussion, the observed means are represented, by Y1 and the
smoothed means by M,-.
11.13.3.8.2 Starting with the control mean, Yt = 0.677, we see that Y, > Y2.
Set M, - Yr Comparing Y2 to Y3, Y2 < Y3.
11.13.3.8.3 Calculate the smoothed means:
M2 = M3 - (Y2 + Y3)/2 = 0.618 ;
11.13.3.8.4 For the remaining observed means, M3 > Y4 :> Y,5 > Y6. Thus, M4
becomes Y4, M5 becomes Y5, etc., for the remaining concentrations. Table 18
contains the smoothed means, and Figure 11 provides a plot of the smoothed
concentration response curve.
105 >
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q
i
l*%
& z
LU
0_
5'
cฃ
,^J
Q
O
co
-co
01
c:
(0
OJ
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-a
OJ
JC
4->
O
o
E 00
CO 1
-o -a
(XJ (O
-CM
CO
C CO
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E-Q
T3 1
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JD +->
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73
"^U Q
s-
3 O)
c
-------
TABLE 18. FATHEAD MINNOW, PIMEPHALES PROHELAS.
MEAN GROWTH RESPONSE AFTER SMOOTHIN&
NaPCP
Cone
(pg/L)
Control
32
64
128
256
512
i
1
2
3
4
5
6
Response
means, Y.-
(mgj '
0.677
0.575
0.660
0.565
0.454
0.150
Smoothed
means. M.-
(>ng) '
0.677
0.618
0.1518
0.565
0.454
0.150
11.13.3.8.5 An IC25 and an IC50 can be estimated using the Linear
Interpolation Method. A 25% reduction in weight, compared to the controls,
would result in a mean dry weight of 0.508 mg, where M,,(l - p/100) = 0.677(1 -
25/100). A 50% reduction in weight, compared to the controls, would result in
a mean weight of 0.339 mg, where M.(l - p/100) = 0.677(1 --50/100). Examining
the smoothed means and their associated concentrations (Table 18), the
response 0.508 mg is bracketed by C4 = 128 ^g/L and C. = 2,56 //g/L. For the
50% reduction (0.339 mg), the response (0.339 /xg) is "bracketed by C5 =
256 /zg/L and C6 = 512 A*g/L. !
11.13.3.8.6 Using the equation in Section 4.2 from Appendix M, the estimate
of the IC25 is calculated as follows: ;
icp = c^U^U
IC25 = 128+[0.677 (1-25/100) -0.565] (256-128)
(0.454-0.565,1
= 194
11.13.3.8.7 Using the equation in Section 4.2 of Appendix M the estimate of
the IC50 is calculated as follows:.
ICp = C ~
IC50 = 256+ [0.677 (1-50/100) -0.454] (512-2561)
(0.150-0 .454)
= 353 fj.g/1
107
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11.13.3.8.8 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC25 was 193.9503 //g/L. The
empirical 95% confidence interval for the true mean was (54.9278 /*g/L,
340.6617 A*g/L). The computer program output for the IC25 for this data set is
shown in Figure 12.
11.13.3.8.9 When the ICPIN program was used to analyze this set of data for
the IC50, requesting 80 resamples, the estimate of the IC50 was 353.2884 M9/L-
The empirical 95% confidence interval for the true mean was 208.4723 //g/L and
418.5276 fig/L. The computer program output is shown in Figure 13.
11.14 PRECISION AND ACCURACY
11.14.1 PRECISION
11.14.1.1 Single-Laboratory Precision
11.14.1.1.1 Information on the single-laboratory precision of the fathead
minnow larval survival and growth test is presented in Table 19. The range of
NOECs was only two concentration intervals, indicating good precision.
TABLE 19. PRECISION OF THE FATHEAD MINNOW, PIMEPHALES PROMELAS, LARVAL
SURVIVAL AND GROWTH TEST, USING NAPCP AS A REFERENCE TOXICANT1'2
Test
1
2
3
4
5
n:
Mean:
NOEC
(/*9/L)
256
128
256
128
128
5
, NA
LOEC
(M9/L)
512
256
512
256
256
5
NA
Chronic
Value
U9/L)
362
181
362
181
181
5
253.4
From Pickering, 1988.
For a discussion of the precision of data from chronic toxicity tests,
(see Section 4, Quality Assurance).
108
-------
Cone. ID
Cone. Tested
Response
Response
Response
Response
1
2
3
4
0
0
0
0
1
0
.711
.662
.646
.690
0.
0.
0.
0.
2
32
517
501
723
560
0.
0.
0.
0.
3
64
602
669
694
676
4
128
0.566
0.612
0.410
0.672
0
0
0
"0
5
256
.,455
.502
..606
..254
6
512
0.143
0.163
0.195
0.099
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: NaPCP
Test Start Date: Example Test Ending Date:
Test Species: Fathead minnows
Test Duration: . 7-d
DATA FILE: fhmanual.icp
OUTPUT FILE: fhmanual.i25
Cone.
ID
1
2
3
4
5
6
Number
Replicates
4
4
4
4
4
4
Concentration
ug/i
0.000
32.000
64.000
128.000
256.000
512.000
Response
Means
0.677
0.575
0.660
0.565
0.454
0.150
Stcl. Pooled
Dev. Response Means
0.029
0.102
0.040
0.112
0.148
0.040
0.677
0.618
0.618
0.565
0.454
0.150
The Linear Interpolation Estimate: 193.9503 Entered:? Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean: 186.4935 Standard Deviation: 52.6094
Original Confidence Limits: Lower: 107.0613 Upper: , 285.6449
Expanded Confidence Limits: Lower: 54.9278 Upper: 340.6617
Resampling time in Seconds: 1.81 Random Seed: 1272173518
Figure 12. ICPIN program output for the IC25.
109
-------
Cone. ID
Cone. Tested
Response
Response
Response
Response
1
2
3
4
1
0
0.711
0.662
0.646
0.690
2
32
0.517
0.501
0.723
0.560
3
64
0.602
0.669
0.694
0.676
4
128
0.566
0.612
0.410
0.672
5
256
0.455
0.502
0.606
0.254
6
512
0.143
0.163
0.195
0.099
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: NaPCP
Test Start Date: Example Test Ending Date:
Test Species: Fathead minnows
Test Duration: 7-d
DATA FILE: fhmanual.icp
OUTPUT FILE: fhmanual.i50
Cone.
ID
1
2
3
4
5
6
Number
Replicates
4
4
4
4
4
4
Concentration
ug/l
0.000
32.000
64.000
128.000
256.000
512.000
Response
Means
0.677
0.575
0.660
0.565
0.454
0.150
Std. Pooled
Dev. Response Means
0.029
0.102
0.040
0.112
0.148
0.040
0.677
0.618
0.618
0.565
0.454
0.150
The Linear Interpolation Estimate: 353.2884 Entered P Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean: 345.1108 Standard Deviation: 37.0938
Original Confidence Limits: Lower: 262.7783 Upper: 394.0629
Expanded Confidence Limits: Lower: 208.4723 Upper: 418.5276
Resampling time in Seconds: 1.87 Random Seed: 1126354766
Figure 13. ICPIN program output for the IC50.
110
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11.14.1.2 Multilaboratory Precision
11.14.1.2.1 A multilaboratory study of Method 1000.0 described in the first
edition of this manual (USEPA, 1985e), was performed using seven blind samples
over an eight month period (DeGraeve et. al., 1988). In this study, each of
the 10 participating laboratories was to conduct two tests simultaneous with
each sample, each test having two replicates of 10 larvae for each of five
concentrations and the control. Of the 140 tests planned^ 135 were completed.
Only nine of the 135 tests failed to meet the acceptance criterion of 80%
survival in the controls. Of the 126 acceptable survival NOECs reported, an
average of 41% were median values, and 89% were within one concentration
interval of the median (Table 20). For the growth (weight) NOECs, an average
of 32% were at the median, and 84% were within one concentration interval of
the median (Table 21). Using point estimate techniques, the precision (CV) of
the IC50 was 19.5% for the survival data and 19.8% for the growth data. If
the mean weight acceptance criterion of 0.25 mg for the surviving control
larvae, which is included in this revised edition of the method, had applied
to the test results of the interlaboratory study, one third of the 135 tests
would have failed to meet the test criteria (Norberg-King, personal
communication and 1989 memorandum; DeGraeve et al., 1991),
11.14.2 ACCURACY
11.14.2.1 The accuracy of toxicity tests cannot be determined.
Ill
-------
TABLE 20. COMBINED FREQUENCY DISTRIBUTION FOR SURVIVAL NOECs
FOR ALL LABORATORIES1
NOEC Frequency (%)Distribution
Sample
Tests with Two Reps
Median ฑ lz>23
Tests with Four Reps
Median ฑ lz > 25
1.
2.
3.
4.
5.
6.
7.
Sodium Pentachlorophenate (A) 35
Sodium Pentachlorophenate (B) 42
Potassium
Potassium
Refinery
Refinery
Dichromate (A)
Dichromate (B)
Effluent 301
Effluent 401
Utility Waste 501
47
41
26
37
56
53
42
47
41
68
53
33
12
16
6
18
6
10
11
57
56
75
50
78
56
56
29
44
25
50
22
44
33
14
0
0
0
0
0
11
From DeGraeve et al., 1988.
Percent of values within one concentration intervals of the median.
Percent of values within two or more concentrations intervals of the
median.
112
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TABLE 21. COMBINED FREQUENCY DISTRIBUTION FOR WEIGHT NOECs FOR ALL
LABORATORIES1
NOEC Freauencv
Tests with Two
Sample Median ฑ I2
1.
2.
3.
4.
5.
6.
7.
Sodium Pentachlorophenate (A)
Sodium Pentachlorophenate (B)
Potassium Dichromate (A)
Potassium Dichromate (B)
Refinery Effluent 301
Refinery Effluent 401
Utility Waste 501
59
37
35
12
35
37
11
41
63
47
47
53
47
61
Reps
>23
0
0
18
41
12
16
28
(%) Distribution
Tests with Four Reps
Median ฑ I2 > 23
57
22
88
i 63
75
I 33
33
I
43
45
0
25
25
56
56
0
33
12
12
0
11
11
From DeGraeve et al., 1988.
Percent of values within one concentration intervals of the median.
Percent of values within two or more concentrations intervals of the
median.
113
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SECTION 12
TEST METHOD
FATHEAD MINNOW, PIHEPHALES PRONELAS,
EMBRYO-LARVAL SURVIVAL AND TERATOGENICITY TEST
METHOD 1001.0
12.1 SCOPE AND APPLICATION
12.1.1 This method estimates the chronic toxicity of whole effluents and
receiving water to the fathead minnow, Pimephales promelas, using embryos 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. The test is useful in screening for
teratogens because organisms are exposed during embryonic development.
12.1.2 Daily observations on mortality make it possible to also calculate the
acute toxicity for desired exposure periods (i.e., 24-h, 48-h, 96-h LCBOs).
12.1.3 Detection limits of the toxicity of an effluent or pure substance are
organism dependent.
12.1.4 Brief excursions in 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
degradable and highly volatile toxicants, in the source may not be detected in
the test.
12.1.5 This test method is commonly used in one of two forms: (1) a
definitive test, consisting of a minimum of five effluent concentrations and a
control, and (2) a receiving water test(s), consisting of one or more
receiving water concentrations and a control.
12.2 SUMMARY OF METHOD
12.2.1 Fathead minnow, Pimephales promelas, embryos are exposed in a static
renewal system to different concentrations of effluent or to receiving water
for seven days, starting shortly after fertilization of the eggs. Test
results are based on the total frequency of both mortality and gross
morphological deformities (terata).
12.3 INTERFERENCES
12.3.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).
114
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ฐI loV.5issolved ^en (DO), high concentrations of
toxic sbsUnces dlSSฐlved sollds' and extremes of PH may mask the presence of
12.3.3 Improper effluent sampling and sample handling may adversely affect
test results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests). ->amj>ie
ll'*-* Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival and confound test results.
12.4 SAFETY
12.4.1 See Section 3, Health and Safety. I
i
12.5 APPARATUS AND EQUIPMENT
Mathead "!1nnow and brine ^rimp culture units -- See Section 11
3J5hฐW'/7rPfa7^/rTla^ Urva1 Surv1val and Growth Test> and
.9?3b- UTฐ 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. In cases where
shipping is necessary, up to 48-h old embryos may be used for the test.
Ji!h?m!!niPl!;rf "" aut??atlc Ampler, preferably with sample cooling
capability, that can collect a 24-h composite sample of 5 L or more.
col?t?ine,rrs " tor Cample shipment and storage (see Section 8,
for Toxcny Teltl)!"9 SamDlin9> Sample Handling, and Sample Preparation
Chamber ฐr ea.uivalerit facility with temperature control
12.5.5 Water purification system -- MILLIPORE MILLI-Qฎ, deionized water or
equivalent (see Section 5, Facilities, Equipment, and Supplies)
12.5.6 Balance -- analytical, capable of accurately weighing to 0.00001 g.
on ^ wei?nts.' Class's -- for checking performance of balance
Weights should bracket the expected weights of material to :be weighed
12.5.8 Test chambers -- four (minimum of three) borosilicate glass or
fcP ฐsable non-toxic plastic labware, per test solution, such as: 500-mL
beakers; 100 mm x 15 mm or 100 mm x 20 mm glass or disposable polystyrene
SH ^heS; 01V2^ ฐDi stackable "Carolina" culture dishes The chambers
should be covered with safety glass plates or sheet plastic during the test to
aVd
115
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12 5 9 Dissecting microscope, or long focal length magnifying lens, hand or
sfend supported - for examining embryos and larvae in the test chambers.
12.5.10 Light box, microscope lamp, or flashlight - for illuminating
chambers during examination and observation of embryos and larvae.
12 5 11 Volumetric flasks and graduated cylinders -- Class A, borosilicate
glass or non-toxic plastic labware, 10-1000 ml, for making test solutions.
12.5.12 Volumetric pipets -- Class A, 1-100 ml.
12.5.13 Serological pipets -- 1-10 ml, graduated.
12.5.14 Pipet bulbs and fillers - PROPIPETฎ, or equivalent.
12 5 15 Droppers, and glass tubing with fire polished edges, 2-mm ID for
transferring embryos, and 4-mm ID - for transferring larvae.
12 5 16 Wash bottles -- for washing embryos from substrates and containers
and for rinsing small glassware and instrument electrodes and probes.;
12.5.17 Thermometers, glass or electronic, laboratory grade - for measuring
water temperatures.
12.5.18 Bulb-thermograph or electronic-chart type thermometers --for
continuously recording temperature.
12 5 19 Thermometer, National Bureau of Standards Certified (see USEPA Method
170.i, USEPA 1979b) -- to calibrate laboratory thermometers. ;
12.5.20 Meters, pH, DO, and specific conductivity - for routine physical and
chemical measurements.
12.6 REAGENTS AND CONSUMABLE MATERIALS
1? 6 1 Samole containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling and Sample Preparation
for Toxicity Tests).
12.6.2 Data sheets (one set per test) -- for recording data.
12.6.3 Tape, colored -- for labelling test chambers.
12.6.4 Markers, waterproof -- for marking containers, etc.
12 6 5 Reagents for hardness and alkalinity tests -- see USEPA Methods'130.2
and 310.1, USEPA 1979b.
12 6.6 Membranes and filling solutions for DO probe (see USEPA Method 360.1,
USEPA 1979b), or reagents -- for modified Winkler analysis.
116
-------
12.6.7 Standard pH buffers, pH 4, pH 7, and pH 10 (or as per instructions of
instrument manufacturer) -- for instrument calibration (see USEPA Method
150.1, USEPA 1979b).
12.6.8 Specific conductivity standards -- see USEPA Method 120.1, USEPA
1979b.
12.6.9 Laboratory quality control samples and standards -- for calibration of
the above methods.
12.6.10 Reference toxicant solutions -- see Section 4, Quality Assurance.
12.6.11 Reagent water defined as distilled or deionized water which does
not contain substances which are toxic to the test organisms (see Section 5,
Facilities, Equipment, and Supplies).
12.6.12 Effluent, receiving water, and dilutiorf water --' see Section 7,
Dilution Water; and Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.
12.6.13 TEST ORGANISMS, FATHEAD MINNOWS, PIMEPHALES PROMELAS
12.6.13.1 Fathead minnow embryos, less than 36-h old, are used for the test.
The test is conducted with four (minimum of three) test chambers at each
toxicant concentration and control. Fifteen (minimum of ten) embryos are
placed; in each replicate test chamber. Thus 60 (minimum of 30) embryos are
exposed at each test concentration and 360 (minimum of 180) embryos would be
needed for a test consisting of five effluent concentrations and a control.
12.6.13.2 Sources of Organisms
12.,6.13.2.1 It is recommended that the embryos be obtained from inhouse
cultures or other local sources if at all possible, because it is often
difficult to ship the embryos so that they will be less than 36 h old for
beginning the test. Receipt of embryos via Express Mail, air express, or
other carrier, from a reliable outside source is an acceptable alternative,
but they must not be over 48 h old when used to begin the test.
i
12.6.13.2.2 Culturing methods for fathead minnows, Pimephales promelas, are
described in Section 6, Section 11 and in USEPA, 1993b.
12.6.13.2.3 Fish obtained from outside sources (see Section 5, Facilities,
Equipment, and Supplies) such as commercial biological supply nouses for use
as brood stock should be guaranteed to be (1) of the correct species, (2)
disease free, (3) in the requested age range, and (4) in good condition. This
can be done by providing the record of the date on which the eggs were laid
and hatched, and information on the sensitivity of the contemporary fish to
reference toxicants.
12.6.13.3 Obtaining Embryos for Toxicity Tests from InhoUse Cultures.
12.6.13.3.1 Spawning substrates with the newly-spawned, fertilized embryos
are removed from the spawning tanks or ponds, and the embryos are separated
117
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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 combined and washed from the spawning substrate onto a
400 urn NITEXฎ screen, sprayed with a stream of deionized water to remove
detritus and food particles, and back-washed with dilution water into a
crystallizing dish for microscopic examination. Damaged and infertile eggs
are discarded.
12.6.13.3.2 The embryos from three or more spawns are pooled in a single
container to provide a sufficient number to conduct the tests. These embryos
may be used immediately to start a test inhouse or may be transported for use
at a remote location. When transportation is required, embryos should be
taken from the substrates within 12 h of spawning. This permits off-site
tests to be started with less than 36-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 2ฐC. Sudden changes in
pH, dissolved ions, osmotic strength, and DO should be avoided.
12.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE
12.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.
12.8 CALIBRATION AND STANDARDIZATION
12.8.1 See Section 4, Quality Assurance.
12.9 QUALITY CONTROL
12.9.1 See Section 4, Quality Assurance.
12.10 TEST PROCEDURES
12.10.1 TEST SOLUTIONS
12.10.1.1 Receiving Waters
12.10.1.1.1 The sampling point is determined by the objectives of the test.
Receiving water toxicity is determined with samples used directly as collected
or after samples are passed through a 60 urn NITEXฎ filter and compared without
dilution, against a control. Using four replicate chambers per test, each
containing 100 ml, and 400 ml for chemical analysis, would require
approximately one liter, or more, of sample per test day.
12.10.1.2 Effluents
12.10.1.2.1 The selection of the effluent test concentrations should be based
on the objectives of the study. A dilution factor of 0.5 is commonly used.
A dilution factor of 0.5 provides precision of ฑ 100%, and testing of
118
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concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%). Improvements in precision
decline rapidly if the dilution factor is increased beyond 0..5 and precision
declines rapidly if a smaller dilution factor is used. Therefore, USEPA
recommends the use of the > 0.5 dilution factor.
12.10.1.2.2 If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%). If a high rate of mortality is observed during the first
1 to 2 h of the test, additional dilutions should be added at the lower range
of effluent concentrations.
12.10.1.2.3 The volume of effluent required for daily renewal of four
replicates per concentration, each containing 100 ml of test solution, is
1.5 L. Sufficient test solution (approximately 1000 ml) iis prepared at each
effluent concentration to provide 400 ml additional volume for chemical
analyses. If the sample is used for more than one daily renewal of test
solutions, the volume must be increased proportionately.
12.10.1.2.4 Tests should begin as soon as possible, preferably within 24 h of
sample collection. The maximum holding time following retrieval of the sample
from the sampling device should not exceed 36 h for the off-site toxicity
tests unless permission is granted by the permitting authority. In no case
should the sample be used in a test more than 72 h after sample collection
(see Section 8, Effluent and Receiving Water Sampling, Sample Handling, and
Sample Preparation for Toxicity Tests). '
12.10.1.2.5 Just prior to test initiation (approximately 1 h) the temperature
of sufficient quantity of the sample to make the test solutions should be
adjusted to the test temperature and maintained at that temperature during the
addition of dilution water.
12.10.1.2.6 The DO of the test solutions should be checked prior to test
initiation. If any of the solutions are supersaturated with oxygen or any
solution has a DO below 4.0 mg/L, all of the solutions and the control must be
gently aerated. ;
12.10.1.3 Dilution Water
12.10.1.3.1 Dilution water may be uncontaminated receiving water, a standard
synthetic (reconstituted) water, or some other uncontamineited natural water
(see Section 7, Dilution Water).
12.10.1.3.2 If the hardness of the test solutions (including the control)
does not equal or exceed 25 mg/L as CaC03, it may be necessary to adjust the
hardness by adding reagents for synthetic softwater as listed in Table 3,
Section 7. In this case parallel tests should be conducted, one with the
hardness adjusted and one unadjusted.
119
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12.10.2 START OF THE TEST
12.10.2.1 Label the test chambers with a ma'rking pen and use color-coded tape
to identify each treatment and replicate. A minimum of five effluent
concentrations and a control are used for each effluent test. Each treatment
(including the control) should have four (minimum of three) replicates.
12.10.2.2 Tests performed in laboratories that have inhouse fathead minnow
breeding cultures must initiate tests with embryos less than 36 h old. When
the embryos must be shipped to the test site from a remote location, it may be
necessary to use embryos older than 36 h because of the difficulty of
coordinating test organism shipments with field operations. However, in the
latter case, the embryos must not be more than 48 h old at the start of the
test and should all be within 24 h of the same age.
12.10.2.3 Randomize the position of the test chambers at the beginning of the
test (see Appendix A). Maintain the chambers in this configuration throughout
the test. Preparation of a position chart may be helpful.
12.10.2.4 The test organisms should come from a pool of embryos consisting of
at least three separate spawnings. Gently agitate and mix the embryos to be
used in the test in a large container so that eggs from different spawns are
thoroughly mixed.
12.10.2.5 Using a small bore (2 mm ID) glass tube, the embryos are placed one
or two at a time into each randomly arranged test chamber or intermediate
container in sequential order, until each chamber contains 15 (minimum of 10)
embryos, for a total of 60 (minimum of 30) embryos for each concentration
(see Appendix A). The amount of water added to the chambers when transferring
the embryos to the compartments should be kept to a minimum to avoid
unnecessary dilution of the test concentrations.
12.10.2.6 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. Placing the test chambers on a light
table may facilitate examining and counting the embryos.
12.10.3 LIGHT, PHOTOPERIOD AND TEMPERATURE
12.10.3.1 The light quality and intensity should be at ambient laboratory
levels, which is approximately 10-20 /iE/m/s, or 50 to 100 foot 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 ฑ 1ฐC.
12.10.4 DISSOLVED OXYGEN (DO) CONCENTRATION
12.10.4.1 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 be measured in the new solutions at the start of the
test (Day 0) and before daily renewal of the new solutions on subsequent days.
The DO concentrations should not fall below 4.0 mg/l. (see Section 8, Effluent
and Receiving Water Sampling, Sample Handling, and Sample Preparation1 for
120
-------
Toxicity Tests). 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 KIMAXฎ 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 embryos.
12.10.5 FEEDING
12.10.5.1 Feeding is not required. <
12.10.6 OBSERVATIONS DURING THE TEST . | '
12.10.6.1 Routine Chemical and Physical Determinations
12.10.6.1.1 DO is measured at the beginning and end of each 24-h exposure
period in at least one test chamber at each test concentrations and in the
control.
12.10.6.1.2 Temperature and pH are measured at the end of each 24-h exposure
period in at least one test chamber at each test concentration and in the
control. Temperature should also be monitored continuously or observed and
recorded daily for at least two locations in the environmental control system
or the samples. Temperature should be measured in a sufficient number of test
vessels, at least at the end of the test, to determine temperature variation
in the environmental chamber.
12.10.6.1.3 The pH is measured in the effluent sample each clay before new,
test solutions are made.
12.10.6.1.4 Conductivity, alkalinity and hardness are measured in each new
sample (100% effluent or receiving water) and in the control.
12.10.6.2 Record all the measurements on the data sheet (Figure 1).
12.10.6.3 Routine Biological Observations
12.10.6.3.1 At the end of the first 24 h of exposure, before renewing the
test solutions,'examine the embryos. Remove the dead embryos (milky colored
and opaque) and record the number (Figure 2). If the rate of mortality
(including those with 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. -
i
12.10.6.3.2 At 25ฐC, hatching may begin on 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. Count and remove dead
embryos and larvae as previously discussed and record the numbers for all of
the test observations (Figure 2). Upon hatching, deformed larvae are counted
as dead.
121
-------
Discharger:
Location: _
Analyst:
Dates:
Dav
Control :
Temp.
D.O. Initial
Final
oH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
Remarks
Dav
Cone:
Temo.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
Remarks
Dav
Cone:
Terno.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
Remarks
Figure 1. Data form for the fathead minnow, Pimephales pro/ne/as, embryo-
larval survival and teratogenicity test. Routine chemical and
physical determinations.
122
-------
Discharger:
Location:
Analyst:
Dates:
Dav
Control :
Temp .
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
Remarks
Dav
Cone:
Temp.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
Remarks
Dav
Cone:
Temo.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
1
2
3
4
5
6
7
Remarks
Figure 1. Data form for the fathead minnow, Pimephales promelas, embryo-
larval survival and teratogenicity test. Routine chemical and
physical determinations (CONTINUED).
123
-------
Discharger:
Location:
Test Dates:.
Analyst:
Cone: Rep. Condition of
No. Embryos/larvae
Control: 1 Live/dead
Terata
2 Live/dead
Terata
3 Live/dead
Terata
4 Live/dead
Terata
Treatment: 1 Live/dead
Terata
2 Live/dead
Terata
3 Live/dead
Terata
4 Live/dead
Terata
Treatment: 1 Live/dead
Terata
2 Live/dead
Terata
3 Live/dead
Terata
4 Live/dead
Terata
Treatment: 1 Live/dead
Terata
2 Live/dead
Terata
3 Live/dead
Terata
4 Live/dead
Terata
Dav '
1
2
3
4
5
6
7
Figure 2. Data form for the fathead minnow, Pimephales promelas,
embryo-larval survival and teratogenicity test. Survival
and terata data.
124
-------
Discharger:
Location:
Test Dates:.
Analyst: _
Cone: Rep. Condition of
No. Embrvos/1 arvae
Treatment: 1 Live/dead
Terata
2 Live/dead
Terata
3 Live/dead
Terata
4 Live/dead
Terata
Treatment: 1 Live/dead
Terata
2 Live/dead
Terata
3 Live/dead
Terata
4 Live/dead
Terata
Dav
1 2 3 45 67
Comments:
Figure 2. Data form for the fathead minnow, Pimephales promelas,
embryo-larval survival and teratogenicity test. Survival
and terata data (CONTINUED).
125
-------
12.10.6.3.3 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 that the test
organisms remain immersed during the performance of the above operations.
12.10.7 DAILY CLEANING OF TEST CHAMBERS
12.10.7.1 Since feeding is not required, test chambers are not cleaned daily
unless accumulation of particulate matter at the bottom of the chambers causes
a problem.
12.10.8 TEST SOLUTION RENEWAL
12.10.8.1 Freshly prepared solutions are used to renew the tests daily. For
on-site toxicity studies, fresh effluent or receiving water samples should be
collected daily, and no more than 24 h should elapse between collection of the
samples and their use in the tests (see Section 8, Effluent and Receiving
Water Sampling, Sample Handling and Sample Preparation for Toxicity Tests).
For off-site tests, a minimum of three samples are collected, preferably on
days one, three, and five. Maintain the samples in the refrigerator at 4ฐC
until used.
12.10.8.2 The test solutions are renewed immediately after removing dead
embryos and/or larvae. During the daily renewal process, the water 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 should be added slowly by pouring down
the side of the test chamber to avoid excessive turbulence and possible injury
to the embryos or larvae.
12.10.9 TERMINATION OF THE TEST
12.10.9.1 The test is terminated after seven days of exposure. Count the
number of surviving, dead, and deformed larvae, and record the numbers of each
(Figure 2). The deformed larvae are treated as dead in the analysis of the
data. Keep a separate record of the total number and percent of deformed
larvae for use in reporting the teratogenicity of the test solution.
12.10.9.2 Prepare a summary of the data as illustrated in Figure 3.
12.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
12.11.1 A summary of test conditions and test acceptability criteria is
presented in Table 1.
12.12 ACCEPTABILITY OF TEST RESULTS
12.12.1 For the test results to be acceptable, survival in the controls must
be at least 80%.
126
-------
Discharger:
Location:
Test Dates:
Analyst:
Treatment
No. dead embryos
and larvae
No. terata
Total mortality
(dead and
deformed)
Total mortality
(%)
Terata (%)
Hatch (%)
Control
i
Comments:
Figure 3. Summary data for the fathead minnow, Pimephales promelas,
embryo-larval survival and teratogenicity test.
127
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TABLE 1. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
FATHEAD MINNOW, PIMEPHALES PROMELAS, EMBRYO-LARVAL SURVIVAL AND
TERATOGENICITY TOXICITY TESTS WITH EFFLUENTS AND RECEIVING WATERS
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 solutions:
9. Age of test organisms:
Static renewal
25 ฑ 1ฐC
Ambient laboratory illumination
10-20 //E/m2/s or 50-100 f.t-.c. (ambient
laboratory levels)
16 h light, 8 h dark ;
150 mL (Minimum)
70 mL (Minimum)
Daily
Less than 36-h old embryos (Maximum of
48-h if shipped)
10. No. embryos per test chamber: 15 (minimum of 10)
11. No. replicate test
chambers per concentration:
12. No. embryos per concentration:
13. Feeding regime:
14. Aeration:
15. Dilution water:
4 (minimum of 3)
60 (minimum of 30)
Feeding not required
None unless DO falls below 4.0 mg/L
Uncontaminated source of receiving or
other natural water, synthetic water
prepared using MILLIPORE MILLI-Q* or
equivalent deionized water and reagent
grade chemicals or DMW (see Section
7, Dilution Water). The hardness of the
test solutions should equal or exceed
25 mg/L (CaC03) to ensure hatching
success
128
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TABLE 1. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
FATHEAD MINNOW, PIMEPHALES PROMELAS, EMBRYO-LARVAL SURVIVAL AND
TERATOGENICITY TOXICITY TESTS WITH EFFLUENTS AND RECEIVING
WATERS (CONTINUED)
16. Test concentrations:
17. Dilution factor:
18. Test duration:
19. Endpoint:
20. Test acceptability criteria:
21. Sampling requirements:
22. Sample volume required:
Effluents: Minimum of 5 and a control
Receiving waters: 100% receiving water
or minimum of 5 and a control
Effluents: > 0.5
Receiving waters:
7 days
None, or > 0.5
Combined mortality (dead and deformed
organisms)
80% or greater survival in controls
For on-site tests, samples collected
daily and used within 24 h of the time
they are removed from the sampling
device. For off-site tests a minimum
of three samples collected on days one,
three, and five with a maximum holding
time of 36 h before first use (see
Section 8, Effluent and Receiving Water
Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests, and
Subsection 8.5.4)
1.5 to 2.5 L/day depending on the
volume of test solutions used
129
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12.13 DATA ANALYSIS
12.13.1 GENERAL
12.13.1.1 Tabulate and summarize the data (Figure 3).
12.13.1.2 The endpoints of this toxicity test are based on total mortality,
combined number of dead embryos, and dead and deformed larvae. The EC1 is
calculated using Probit Analysis (Finney, 1971; see Appendix I). Separate
analyses are performed for the estimation of LOEC and NOEC endpoints and for
the estimation of the EC1 endpoint. Concentrations at which there is no
survival in any of the test chambers are excluded from the statistical
analysis of the NOEC and LOEC, but included in the estimation of the EC1
endpoint. See the Appendices for examples of the manual computations and
examples of data input and output for the computer programs.
12.13.1.3 The statistical tests described here must be used with a knowledge
of the assumptions upon which the tests are contingent. The assistance of a
statistician is recommended for analysts who are not proficient in statistics.
12.13.2 EXAMPLE OF ANALYSIS OF FATHEAD MINNOW EMBRYO-LARVAL SURVIVAL AND
TERATOGENICITY DATA
12.13.2.1 Formal statistical analysis of the total mortality data is outlined
on the flowchart in Figure 4. The response used in the analysis is the total
mortality proportion in each test or control chamber. Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and for the
estimation of the EC endpoint. Concentrations at which there is 100% total
mortality in all of the test chambers are excluded from statistical analysis
of the NOEC and LOEC, but included in the estimation of the EC1 endpoint.
12.13.2.2 For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and LOEC endpoints
is made via a parametric test, Dunnett's Procedure, or a nonparametric test,
Steel's Many-one Rank Test, on the arc sine square root transformed data.
Underlying assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality is the Shapiro-Milk's
Test, and Bartlett's Test is used to test for homogeneity of variance. If
either of these tests fails, the nonparametric test, Steel's Many-one Rank
Test, is used to determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the parametric
procedure.
12.13.2.3 If unequal numbers of replicates occur among the concentration
levels tested, there are parametric and nonparametric alternative analyses.
The parametric analysis is a t test with the Bonferroni adjustment
(see Appendix D). The Wilcoxon Rank Sum Test with the Bonferroni adjustment
is the nonparametric alternative (see Appendix F).
12.13.2.4 Probit Analysis (Finney, 1971) is used to estimate the
concentration that causes a specified percent decrease in survival from the
control. In this analysis, the total mortality data from all test replicates
at a given concentration are combined.
130
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STATISTICAL ANALYSIS OF FATHEAD MINNOW EMBRYO-LARVAL
SURVIVAL, AND TERATOGENICITY TEST
TOTAL MORTALITY
TOTAL NUMBER OF DEAD EMBRYOS,
DEAD LARVAE, AND DEFORMED LARVAE
PROBIT ANALYSIS
1
r
ARC SINE
TRANSFORMATION
ENDPOINT ESTIMATE
EC1
SHAPIRO-WILK-STEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTS TEST
I
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
NO
YES
T-TESTWITH
BONFERRONI
ADJUSTMENT
EQUAL NUMBER OF
REPLICATES?
YES
NO
STEEL'S MANY-ONE
RANK TEST
T
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 4. Flowchart for statistical analysis of fathead minnow, Pimephales
promelas, embryo-larval data.
131
-------
12.13.2.5 The data for this example are listed in Table 2. Total mortality,
expressed as a proportion (combined total number of dead embryos, dead larvae
and deformed larvae divided by the number of embryos at start of test), is the
response of interest. The total mortality proportion in each replicate must
first be transformed by the arc sine square root transformation procedure
TABLE 2. DATA FROM FATHEAD MINNOW, PIMEPHALES PROMELAS,
EMBRYO-LARVAL TOXICITY TEST WITH GROUND WATER EFFLUENT
Effluent No.
Cone. Eggs at
(%) Start
Dead at Test
Termination
No. %
Deformed at Test
Termination
No. %
Dead + Deformed
at Termination
No. %
Control
3.125
6.25
12.5
25.0
50.0
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
0
2
0
1
0
0
0
1
0
0
0
0
0
0
0
1
1
2
2
1
4
3
5
3
0
20
0
10
0
0
0
10
0
0
0
0
0
0
0
10
10
20
20
10
40
30
50
30
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
9
8
8
4
6
7
5
7
0
0
0
0
0
10
0
0
0
0
0
10
0
0
0
0
90
80
80
40
60
70
50
70
0
2
0
1
0
1
0
1
0
0
0
1
0
0
0
1
10
10
10
5
10
10
10
10
0
20
0
10
0
10
0
10
0
0
0
10
0
0
0
10
100
100
100
50
100
100
100
100-
132
-------
described in Appendix B. The raw and transformed data, means and
variances of the transformed observations at each effluent concentration and
control are listed in Table 3. A plot of the data is provided in Figure 5.
Since there is 100% total mortality in replicates for the 50.0% concentration,
it is not included in this statistical analysis and is considered a
qualitative mortality effect.
TABLE 3. FATHEAD MINNOW, PIMEPHALES PROMELAS, EMBRYO-LARVAL TOTAL
MORTALITY DATA
Replicate Control
Effluent Concentration (%)
3.125 6.25
12.5
25.0 50.0
RAW
A
B
C
D
0.00
0.20
0.00
0.10
0.00
0.10
0.00
0.10
0.00
0.00
0.00
0.10
0.00
0.00
0.00
0.10
1.00
1.00
1.00
0.50
1.00
1.00
1.00
1.00
ARC SINE
TRANS-
FORMED
Mean(Y,)
s?
i
A
B
C
D
0.159
0.464
0.159
0.322
0.-276
0.022
1
0.159
0.322
0.159
0.322
0.241
0.009
2
0.159
0.159
0.159
0.322
0.200
0.007
3
0.159
0.159
0.159
0.322
0.200
0.007
4
1.412
1.412
1.412
0.785
1.255
0.098
5
12.13.2.6 Test for Normality
12.13.2.6.1 The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration. The centered
observations are summarized in Table 4.
12.13.2.6.2 Calculate the denominator, D, of the statistic:
D = t (X, -X)2
i=i '
Where: Xf = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
133
-------
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134
-------
TABLE 4. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Replicate
A
B
C
D
Control
-0.117
0.188
-0.117
0.046
3.125
-0.082
0.081
0.081
-0.082
Effluent
6.25
-0.041
-0.041
-0.041
0.122
Concentration (%)
12.5
-0.041
-0.041
-0.041
0.122
25.0 50.0
0.157
0.157
0.157
-0.470
12.13.2.6.3 For this set of data, n = 20
X =
1
(-0.003) =0.000
20
D = 0.4261
12.13.2.6.4 Order the centered observations from smallest to largest
< ...
-------
12.13.2.6.5 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a1? a2, ..., ak where k is n/2 if n is even and
(n-l)/2 if n is odd. For'the''data'in "this example,'n = 20 and'k
values are listed in Table 6.
10. The a,-
TABLE 6. COEFFICIENTS AND DIFFERENCES FOR THE SHAPIRO-WILK'S EXAMPLE
_ w(
1
2
3
4
5
6
7
8
9
10
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
. 0.0711
0.0422
0.0140
0.658
0.274
0.274
0.239
0.204
0.163
0.122
0.122
0.087
0.000
Y(20)
A
X(19)
x<18>
X(17)
X(16)
X(15>
X(14>
x<ซ)
X(12)
X(11)
- x(1)
- x<2)
- x(3)
- XC4)
- x(5)
- x<6)
- x<7)
- x(8)
- x<9>
- xฐฐ>
12.13.2.6.6 Compute the test statistic, W, as follows:
W = -
D
The differences x(n"l>1> - X(i) are listed in Table 6. For the data in this
example,
(0.6004)2
W
1
0.4261
= 0.846
12.13.2.6.7 The decision rule for this test is to compare W as calculated in
Section 13.2.6.6 to a critical value found in Table 6, Appendix B. If the
computed W is less than the critical value, conclude that the data are not
normally distributed. For the data in this example, the critical value at a
significance level of 0.01 and n = 20 observations is 0.868. Since W = 0.846
is less than the critical value, conclude that the data are not normally
distributed.
136
-------
12.13.2.6.8 Since the data do not meet the assumption of normality, Steel's
Many-one Rank Test will be used to analyze the total mortality data.
12.13.2.7 Steel's Many-one Rank Test
12.13.2.7.1 For each control and concentration combination, combine the data
and arrange the observations in order of size from smallest to largest.
Assign the ranks (1, 2, ..., 8) to the ordered observations with a rank of
1 assigned to the smallest observation, rank of 2 assigned to the next larger
observation, etc. If ties occur when ranking, assign the average rank to each
tied observation.
12.13.2.7.2 An example of assigning ranks to the combined data for the
control and 3.125% effluent concentration is given in Table 7. This ranking
procedure is repeated for each control/concentration combination. The
complete set of rankings is summarized in Table 8. The control group ranks
are next summed for each effluent concentration pairing, as shown in Table 9.
TABLE 7. ASSIGNING RANKS TO THE CONTROL AND 3.125% EFFLUENT CONCENTRATION
FOR STEEL'S MANY-ONE RANK TEST
Rank
2.5
2.5
2.5
2.5
6
6
6
8
Transformed
Proportion
Mortality
0.159
0.159
0.159
0.159
0.322
0.322
0.322
0.464
Ef f 1 uent
Concentration
(%)
Control
Control
3.125
3.125
Control
3.125
3.125
Control
12.13.2.7.3 For this example, we want to determine if the total mortality in
any of the effluent concentrations is significantly higher than the total
mortality in the control. If this occurs, the rank sum of the control would
be significantly less than the rank sum at that concentration. Thus we are
only concerned with comparing the control rank sum for each pairing with the
various effluent concentrations with some "minimum" or critical rank sum, at
or below which the concentration total mortality would be considered
significantly greater than the control. At a signficance level of 0.05, the
minimum rank sum in a test with four concentrations (excluding the control)
and four replicates per concentration is 10 (see Table 5, Appendix E).
12.13.2.7.4 Since the control rank sum for the 25.0% effluent concentration
pairing is equal to the critical value, the total proportion mortality in the
25.0% concentration is considered significantly greater than that in the
137
-------
TABLE 8. TABLE OF RANKS FOR STEEL'S MANY-ONE RANK TEST
Effluent Concentration (%)
Repl. Control
3.125
6.25
12.5
25.0
A
B
C
D
0.159 (2.5,3,3,1.5)
0.464 (8,8,8,4)
0.159 (2.5,3,3,1.5)
0.322 (6,6.5,6.5,3)
0.159 (2.5)
0.322 (6)
0.159 (2.5)
0.322 (6)
0.159 (3)
0.159 (3)
0.159 (3)
0.322 (3)
0.159 (3)
0.159 (3)
0.159 (3)
0.159 (3)
1.412 (7)
1.412 (7)
1.412 (7)
0.785 (5)
TABLE 9. RANK SUMS
Effluent
Concentration (%)
Control
Rank Sum
3.125
6.25
12.5
25.0
19
20.5
20.5
10
control. Since no other rank sums are less than or equal to the critical
value, no other concentrations have signficantly higher total proportion
mortality than the control. Hence the NOEC is 12.5% and the LOEC is 25.0%.
12.13.2.8 Calculation of the LC50
12.13.2.8.1 The data used for the Probit Analysis is summarized in Table 10,
To perform the Probit Analysis, run the USEPA Probit Analysis Program. An
example of the program input and output is supplied in Appendix I.
12.13.2.8.2 For this example, the chi-square test for heterogeneity was not
significant. Thus Probit Analysis appears appropriate for this data.
12.13.2.8.3 Figure 6 shows the output data for the Probit Analysis of the
data from Table 10 using the USEPA Probit Program.
138
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TABLE 10. DATA FOR PROBIT ANALYSIS
Effluent Concentration (%)
Control 3.125 6.25 12.5 25.0 50.0
Number Dead 3 1 0 1 6 15
Number Exposed 40 40 40 40 40 40
12.14 PRECISION AND ACCURACY
12.14.1 PRECISION
12.14.1.1 Single-laboratory Precision
12.14.1.1.1 Data shown in Tables 11 and 12 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 11) and 41% for Diquat (Table 12).
12.14.1.1.2 Precision data are also available from four embryo-larval
survival and teratogenicity tests on trickling filter pilot plant effluent
(Table 13). Although the data could not be analyzed by Probit Analysis, the
NOECs and LOECs obtained using Dunnett's Procedure were the same for all four
tests, 7% and 11% effluent, respectively, indicating maximum precision in
terms of the test design.
12.14.1.2 Multilaboratory Precision
12.14.1.2.1 Data on the multilaboratory precision of this test are not yet
available.
12.14.2 ACCURACY
12.14.2.1 The accuracy of toxicity tests cannot be determined.
139
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USEPA PROBIT ANALYSIS PROGRAM
USED FOR CALCULATING LC/EC VALUES
Version 1.5
Probit Analysis of Fathead Minnow Embryo-Larval Survival
and Teratogenicity Data
Cone.
Control
0.5000
.0000
.0000
.0000
1,
2.
4.
8.0000
Number
Exposed
20
20
20
20
20
20
Number
Resp.
2
2
1
4
16
20
Observed
Proportion
Responding
0.1000
0.1000
0.0500
0.2000
0.8000
1.0000
Proportion
Responding
Adjusted for
Controls
0.0000
0.0174
-.0372
0.1265
0.7816
1.0000
Chi - Square for Heterogeneity (calculated) = 0.441
Chi - Square for Heterogeneity (tabular value) = 7.815
Probit Analysis of Fathead Minnow Embryo-Larval Survival
and Teratogenicity Data
Estimated LC/EC Values and Confidence Limits
Point
LC/EC 1.00
LC/EC 50.00
Exposure
Cone.
1.346
3.018
Lower Upper
95% Confidence Limits
0.453
2.268
1.922
3.672
Figure 6. Output for USEPA Probit Program, Version 1.5.
140
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TABLE 11.
PRECISION OF THE FATHEAD MINNOW, PIMEPHALES PROMELAS,
EMBRYO-LARVAL SURVIVAL AND TERAJOGENICITY TEST, USING
ranMTiiM flc A Dctrcocwrr TnvirAMT1'2
CADMIUM AS A REFERENCE
Test
1
2
3
4
5
N
Mean
SD
CV(%)
LCI3
(mg/L)
0.014
0.006
0.005
0.003
0.006
5
0.0068
0.0042
62
95% Confidence NOEC4
Limits (mg/L)
0.009 - 0.018 0.012
0.003 - 0.010 0.012
0.003 - 0.009 0.013
0.002 - 0.004 0.011
0.003 - 0.009 0.012
5
NA
NA
Tests conducted by Drs. Wesley Birge and Jeffrey Black, University
of Kentucky, Lexington, under a cooperative agreement with the
Bioassessment and Ecotoxicology Branch, EMSL, USEIPA, Cincinnati, OH.
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 dilution water was reconstituted water
with a hardness of 100 mg/L as calcium carbonate, and a pH of 7.8.
Determined by Probit Analysis.
Highest no-observed-effect concentration determined by independent
statistical analysis (2X2 Chi-square Fisher's Exact Test). NOEC
range of 0.011 - 0.013 represents a difference of one exposure
concentration.
141
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TABLE 12. PRECISION OF THE FATHEAD MINNOW, PIMEPHALES
PROMELAS, EMBRYO-LARVAL, SURVIVAL AND
TERATOGENICITY TOXICITYTEST, USING DIQUAT
AS A REFERENCE TOXICANT1'2
Test
1
2
3
4
5
N
Mean
SD
CV(%)
LCI3
(mg/L)
0.58
2.31
1.50
1.71
1.43
5
1.51
0.62
41.3
95% Confidence
Limits
0.32 - 0.86
--4
1.05 - 1.87 ;
1.24 - 2.09
0.93 - 1.83
Tests conducted by Drs. Wesley Birge and Jeffrey Black, Uni-
versity of Kentucky, Lexington, under a cooperative agreement
with the Bioassessment and Ecotoxicology Branch, EMSL, USEPA,
Cincinnati, OH.
The Diquat concentrations were determined by chemical analysis.
The dilution water was reconstituted water with a hardness of
100 mg/L as calcium carbonate, and a pH of 7.8.
Determined by Probit Analysis.
Cannot be calculated.
142
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TABLE 13. PRECISION OF THE FATHEAD MINNOW, PIMEPHALES PROMELAS,
EMBRYO-LARVAL SURVIVAL AND TERATOGENICITY STATIC-RENEWAL
TEST CONDUCTED WITH TRICKLING FILTER EFFLUENT1'^'3
Test
No.
1
2
3
4
NOEC
(% Effluent)
7
7
7
7
LOEC
(% Effluent)
11
: 11
11
; n
Data provided by Timothy Neiheisel, Bioassessrnent and
Ecotoxicology Branch, EMSL, USEPA, Cincinnati, OH.
Effluent 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).
143
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SECTION 13
TEST METHOD
DAPHNID, CERIODAPHNIA DUBIA,
SURVIVAL AND REPRODUCTION TEST
METHOD 1002.0
13.1 SCOPE AND APPLICATION
13.1.1 This method measures the chronic toxicity of effluents and receiving
water to the daphnid, Cen'odaphm'a dubia, using less than 24 h old neonates
during a three-brood (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.
13.1.2 Daily observations on mortality make it possible to also calculate
acute toxicity for desired exposure periods (i.e., 24-h, 48-h, and 96-h
LCBOs).
13.1.3 Detection limits of the toxicity of an effluent or pure substance are
organism dependent.
13.1.4 Brief excursions in 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
degradable or highly volatile toxicants in the source may not be detected in
the test.
13.1.5 This test method is commonly used in one of two forms: (1) a
definitive test, consisting of a minimum of five effluent concentrations and a
control, and (2) a receiving water test(s), consisting of one or more
receiving water concentrations and a control.
13.2 SUMMARY OF METHOD
13.2.1 Cen'odaphm'a dubia are exposed in a static renewal system to different
concentrations of effluent, or to receiving water, until 60% of surviving
control organisms have three broods of offspring. Test results are based on
survival and reproduction. If the test is conducted as described, the
surviving control organisms should produce 15 or more young in three broods.
If these criteria are not met at the end of 8 days, the test must be repeated.
13.3 INTERFERENCES
13.3.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).
144
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13.3.2 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).
13.3.3 Pathogenic and/or predatory organisms in the dilution water and
effluent may affect test organism survival and confound test results.
13.3.4 The amount and type of natural food in the effluent or dilution water
may confound test results.
13.3.5 Food added during the test may sequester metals and other toxic
substances and confound test results. Daily renewal of solutions, however,
will reduce the probability of reduction of toxicity caused by feeding.
13.4 SAFETY
13.4.1 See Section 3, Health and Safety.
13.5 APPARATUS AND EQUIPMENT
13.5.1 Ceriodaphnia and algal culture units -- See Ceriodaphnia and algal
culturing methods below and algal culturing methods in Section 14 and USEPA,
1993b.
13.5.2 Samplers -- automatic sampler, preferably with sample cooling
capability, capable of collecting a 24-h composite sample of 5 L or more.
13.5.3 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
13.5.4 Environmental chambers, incubators, or equivalent facilities with
temperature control (25 ฑ 1ฐC). ,
13.5.5 Water purification system -- MILLIPORE MILLI-Qฎ, deionized water or
equivalent (see Section 5, Facilities, Equipment, and Supplies).
13.5.6 Balance -- analytical, capable of accurately weighing 0.00001 g.
13.5.7 Reference weights, Class S -- for checking performance of balance.
Weights should bracket the expected weights of the material to be weighed.
13.5.8 Test chambers --10 test chambers are required for each concentration
and control. Test chambers such as 30-mL borosilicate glass beakers or
disposable polystyrene cups are recommended because they will fit in the
viewing field of most stereoscopic microscopes. The glass beakers and plastic
cups are rinsed thoroughly with dilution water before use. To avoid potential
contamination from the air and excessive evaporation of the test solutions
during the test, the test vessels should be covered with safety glass plates
or sheet plastic (6 mm thick).
13.5.9 Mechanical shaker or magnetic stir plates --for algal cultures.
145
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13.5.10 Light meter -- with a range of 0-200 /iE/m2/s (0-1000 ft-c).
13.5.11 Fluorometer (optional) -- equipped with chlorophyll detection light
source, filters, and photomultiplier tube (Turner Model 110 or equivalent).
13.5.12 UV-VIS spectrophotometer (optional) -- capable of accommodating 1-5
cm cuvettes.
13.5.13 Cuvettes for spectrophotometer -- 1-5 cm light path.
13.5.14 Electronic particle counter (optional) -- Coulter Counter, ZBI, or
equivalent, with mean cell (particle) volume determination.
13.5.15 Microscope with 10X, 45X, and 100X objective lenses, 10X ocular
lenses, mechanical stage, substage condenser, and light source (inverted or
conventional microscope) -- for determining sex and verifying identification.
13.5.16 Dissecting microscope, stereoscopic, with zoom objective,
magnification to 50X -- for examining and counting the neonates in the test
vessels.
13.5.17 Counting chamber -- Sedgwick-Rafter, Palmer-Maioney, or
hemocytometer.
13.5.18 Centrifuge (optional) -- plankton, or with swing-out buckets having a
capacity of 15-100 ml.
13.5.19 Centrifuge tubes -- 15-100 ml, screw-cap.
13.5.20 Filtering apparatus -- for membrane and/or glass fiber filters.
13.5.21 Racks (boards) -- to hold test chambers. It is convenient to use a
piece of styrofoam insulation board, 50 cm x 30 cm x 2.5 cm (20 in x 12 in x 1
in), drilled to hold 60 test chambers, in six rows of 10 (see Figure 1).
13.5.22 Light box -- for illuminating organisms during examination.
13.5.23 Volumetric flasks and graduated cylinders -- class A, borosilicate
glass or non-toxic plastic labware, 10-1000 mL, for culture work and
preparation of test solutions.
13.5.24 Pipettors, adjustable volume repeating dispensers -- for feeding.
Pipettors such as the Gilson REPETMANฎ, Eppendorf, Oxford, or equivalent,
provide a rapid and accurate means of dispensing small volumes (0.1 mL) of
food to large numbers of test chambers.
13.5.25 Volumetric pipets -- class A, 1-100 mL.
13.5.26 Serological pipets -- 1-10 mL, graduated.
13.5.27 Pipet bulbs and fillers -- PROPIPETฎ, or equivalent.
146
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13.5.28 Disposable polyethylene pipets, droppers, and glass tubing with
fire-polished edges * > 2mm ID -- for transferring organisms.
13.5.29 Wash bottles -- for rinsing small glassware and instrument electrodes
and probes.
13.5.30 Thermometer, glass or electronic, laboratory grade, -- for measuring
water temperatures.
13.5.31 Bulb-thermograph or electronic-chart type thermometers -- for
continuously recording temperature.
13.5.32 Thermometer, National Bureau of Standards Certified (see USEPA Method
170.1, USEPA 1979b) --to calibrate laboratory thermometers.
13.5.33 Meters, DO, pH, and specific conductivity -- for routine physical and
chemical measurements.
13.6 REAGENTS AND CONSUMABLE MATERIALS
13.6.1 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
13.6.2 Data sheets (one set per test) -- for recording the data.
13.6.3 Vials, marked -- for preserving specimens for verification (optional).
13.6.4 Tape, colored -- for labeling test vessels.
13.6.5 Markers, waterproof -- for marking containers.
13.6.6 Reagents for hardness and alkalinity tests -- see USEPA Methods 130.2
and 310.1, USEPA, 1979b.
13.6.7 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument .
manufacturer) -- for instrument calibration check (see USEPA Method 150.1,
USEPA, 1979b).
13.6.8 Specific conductivity standards -- see USEPA Method 120.1, USEPA,
1979b.
13.6.9 Membranes and filling solutions for DO probe (see USEPA Method 360.1*
USEPA, 1979b), or reagents -- for modified Winkler analysis.
13.6.10 Laboratory quality control samples and standards -- for calibration
of the above methods.
13.6.11 Reference toxicant solutions -- see Section 4, Quality Assurance.
147
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13.6.12 Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms (see Section 5,
Facilities, Equipment, and Supplies).
13.6.13 Effluent, surface water, and dilution water -- see Section 7,
Dilution Water; and Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests.
13.6.14 Trout chow, yeast, and CEROPHYLฎ food (or substitute food) --for
feeding the cultures and test organisms.
13.6.14.1 Digested trout chow, or substitute flake food (TETRAMINฎ, BIORILฎ,
or equivalent), is prepared as follows:
1. Preparation of trout chow or substitute flake food requires one week.
Use starter or No. ltpellets prepared according to current U.S. Fish and
Wildlife Service specifications. Suppliers of trout chow include
Zeigler Bros., Inc., P.O. Box 95, Gardners, PA, 17324 (717-780-9009);
Glencoe Mills, 1011 Elliott, Glencoe, MN, 55336 (612-864-3181); and
Murray Elevators, 118 West 4800 South, Murray, UT 84107 (800-521-9092).
2. Add 5.0 g of trout chow pellets or substitute flake food to 1 L.of
MILLI-Qฎ water. Mix well in a blender and pour into a 2-L separatory
funnel. Digest prior to use by aerating continuously from the bottom of
the vessel for one week at ambient laboratory temperature. Water lost
due to evaporation is replaced during digestion. Because of the
offensive odor usually produced during digestion, the vessel should be
placed in a fume hood or other isolated, ventilated area.
3. At the end of digestion period, place in a refrigerator and allow to
settle for a minimum of 1 h. Filter the supernatant through a fine mesh
screen (i.e., NITEXฎ 110 mesh). Combine with equal volumes of
supernatant from CEROPHYLLฎ and yeast preparations (below). The
supernatant can be used fresh, or frozen until use. Discard the
sediment.
13.6.14.2 Yeast is prepared as follows:
1. Add 5.0 g of dry yeast, such as FLEISCHMANN'Sฎ Yeast, Lake State Kosher
Certified Yeast, or equivalent, to 1 L of MILLI-Qฎ water.
2. Stir with a magnetic stirrer, shake vigorously by hand, or mix with a
blender at low speed, until the yeast is well dispersed.
3. Combine the yeast suspension immediately (do not allow to settle) with
equal volumes of supernatant from the trout chow (above) and CEROPHYLLฎ
preparations (below). Discard excess material.
13.6.14.3 CEROPHYLLฎ is prepared as follows:
1. Place 5.0 g of dried, powdered, cereal or alfalfa leaves, or rabbit
pellets, in a blender. Cereal leaves are available as "CEREAL LEAVES,"
from Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178;
800-325-3010; or as CEROPHYLLฎ, from Ward's Natural Science
Establishment, Inc., P.O. Box 92912, Rochester, NY 14692-9012;
148
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716-359-2502. Dried, powdered, alfalfa leaves may be obtained from
health food stores, and rabbit pellets are available at pet shops.
2. Add 1 L of MILLI-Qฎ water.
3. Mix in a blender at high speed for 5 min, or stir overnight at medium
speed on a magnetic stir plate.
4. If a blender is used to suspend the .material, place in a refrigerator
overnight to settle. If a magnetic stirrer is used, allow to settle for
1 h. Decant the supernatant and combine with equal volumes of
supernatant from trout chow and yeast preparations (above). Discard
excess material.
13.6.14.4 Combined yeast-cerophyl-trout chow (YCT) is mixed as follows:
1. Thoroughly mix equal (approximately 300 ml) volumes of the three foods
as described above.
, 2. Place aliquots of the mixture in small (50 ml to 100 ml) screw-cap
plastic bottles and freeze until needed.
3. Freshly prepared food can be used immediately, or it can be frozen until
needed. Thawed food is stored in the refrigerator between feedings, and
is used for a maximum of two weeks. Do not store frozen over three
months.
4. It is advisable to measure the dry weight of solids in each batch ;of YCT
before use. The food should contain 1.7-1.9 g solids/I. Cultures or
test solutions should contain 12-13 mg solids/L.
13.6.15 Algal food -- for feeding the cultures and test organisms.
13.6.15.1 Algal Culture Medium is prepared as follows:
1. Prepare (five) stock nutrient solutions using reagent grade chemicals as
described in Table 1.
2. Add 1 ml of each stock solution, in the order listed in Table 1, to
approximately 900 ml of MILLI-Qฎ water. Mix well after the addition of
each solution. Dilute to 1 L, mix well. The final concentration of
macronutrients and micronutrients in the culture medium is qiven in
Tab!e 2. .
3. Immediately filter the medium through a 0.45 urn 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 with 500
mL deionized water prior to use.
4. If the filtration is carried out with sterile apparatus, filtered medium
can be used immediately, 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 vessels.
5. Unused sterile medium should not be stored more than one week prior to
use, because there may be substantial loss of water by evaporation.
13.6.15.2 Algal Cultures
13.6.15.2.1 See Section 6, Test Organisms, for information on sources of
starter cultures of Selenastrum capricornutum, 5. minutum, and Chlamydomonas
reinhardti.
149
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TABLE 1. NUTRIENT STOCK SOLUTIONS FOR MAINTAINING ALGAL STOCK CULTURES
STOCK
SOLUTION
COMPOUND
AMOUNT DISSOLVED IN
500 mL MILLI-Qฎ WATER
1. MACRONUTRIENTS
A.
B.
C.
D.
2. MICRONUTRIENTS
MgCl2.6H20
CaCl2'2H20
NaN03
MgS04-7H20
K2HP04
NaHCO,
H,BO,
MnCl2.4H20
ZnCl2
FeCl,.6H20
CoCl2.6H20
Na2MoO,.2H20
CuCl2.2H20
Na2EDTA-2H20
Na2Se04
6.08 g
2.20 g
12.75 g
7.35 g
0.522 g
7.50 g
92.8
208.0
1.64
mg
mg
79.9
mg
mg_
0.714 ing,
3.63 mg
0.006 mg
150.0 mg
1.196 mg5
Add 1 mL of this
Add 1 mL of
Add 1 mL
ZnCl2 - Weigh out 164 mg and dilute to 100 mL.
solution to Stock 2, micronutrients.
CoCl2-6H20 - Weigh out 71.4 mg and dilute to 100 mL.
this solution to Stock 2, micronutrients.
NaJloO/^H^ - Weigh out 36.6 mg and dilute to 10 mL.
of this solution to Stock 2, micronutrients.
CuCV2H?0 - 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 2, micronutrients. ^ .
Na2Se04 - Weigh out 119.6 mg and dilute to 100 mL. Add 1 mL or this
solution to Stock 2, micronutrients.
150
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TABLE 2. FINAL CONCENTRATION OF MACRONUTRIENTS AND MICRONUTRIENTS
IN THE CULTURE MEDIUM
MACRONUTRIENT
NaN03
MgCT2.6H20
CaCl2-2H20
MgS04-7H20
K2HP04
NaHC03
MICRONUTRIEN.T
H3B03
MnCT2.4H20
ZnC12
CoCl2ซ6H20
CuCl2-2H20
Na2Mo04-2H20
FeCl3.6H20
Na2EDTA-2H20
Na2Se04
CONCENTRATION
fma/U
25.5
12.2
4.41
14.7
1.04
15.0
CONCENTRATION
fua/n
185.0
416.0
3.27
1.43
0.012
7.26
160.0
300.0
2.39
ELEMENT
N \
\
Mg
Ca !
i
S
P
Na
K
C i
ELEMENT
B
Mn
Zn
Co
Cu
Mo
Fe
--
Se
CONCENTRATION
(ma/L)
4.20
2.90
1.20
1.91
0.186
11.0
0.469
2.14
CONCENTRATION
lua/L}
32.5
115.0
1.57
0.354
0.004
2.88
33.1
0.91
151
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13.6.15.2.2 Two types of algal cultures are maintained: "stock" cultures, and
"food" cultures.
13.6.15.2.2.1 Establishing and Maintaining Stock Cultures of Algae:
1 Upon receipt of the "starter" culture (usually about 10 ml), a stock
' culture is initiated by aseptically transferring one milliliter to each
of several 250-mL culture flasks containing 100 ml algal culture medium
(prepared as described above). The remainder of the starter culture can
be held in reserve for up to six months in a refrigerator (in the dark)
at 4 ฐ C.
2 The stock cultures are used as a source of algae to initiate "food"
cultures for Ceriodaphnia dubia toxicity tests. The volume of stock
culture maintained at any one time will depend on the amount of algal
food required for the Ceriodaphnia dubia cultures and tests, stock
culture volume may be rapidly "scaled up" to several liters, if.
necessary, using 4-L serum bottles or similar vessels, each containing 6
I of growth medium. . . . .
3 Culture temperature is not critical. Stock cultures may be maintained
at 25ฐC in environmental chambers with cultures of other organisms if
the illumination is adequate (continuous "cool-white" fluorescent
lighting of approximately 86 ฑ 8.6 ^E/m/s, or 400 ft-c).
4. Cultures are mixed twice daily by hand.
5. Stock cultures can be held in the refrigerator until used to start
"food" cultures, or can be transferred to new medium weekly.
One-to-three milliliters of 7-day old algal stock culture, containing
approximately 1.5 X 106 cells/ml, are transferred to each 100 ml of
fresh culture medium. The inoculum should provide an initial cell
density of approximately 10,000-30,000 cells/ml in the new stock
cultures. Aseptic techniques should be used in maintaining the stocK
algal cultures, and care should be exercised to avoid contamination by
other microorganisms. _-
6 Stock cultures should be examined microscopically weekly, at transfer,
for microbial contamination. Reserve quantities of culture organisms
can be maintained for 6-12 months if stored in the dark at 4ฐC. It is
advisable to prepare new stock cultures from "starter" cultures obtained
from established outside sources of organisms (see Section 6, Test
Organisms) every four to six months.
13.6.15.2.2.2 Establishing and Maintaining "Food" Cultures of Algae:
1 "Food" cultures are started seven days prior to use for Ceriodaphnia
dubia cultures and tests. Approximately 20 ml of 7-day-old algaljtock
culture (described in the previous paragraph), containing 1.5 X 10
cells/ml, are added to each liter of fresh algal culture medium (i.e.,
3 L of medium in a 4-L bottle, or 18 L in a 20-L bottle). The inoculum
should provide an initial cell density of approximately 30,000 cells/ml.
Aseptic techniques should be used in preparing and maintaining the
cultures, and care should be exercised to avoid contamination by other
microorganisms. However, sterility of food cultures is not as critical
as in stock cultures because the food cultures are terminated in
152
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7-10 days. A one-month supply of algal food can be grown at one time,
and stored in the refrigerator.
2. Food cultures may be maintained at 25ฐC in environmental chambers with
the algal stock cultures or cultures of other organisms if the
illumination is adequate (continuous "cool-white" fluorescent lighting
of approximately 86 ฑ 8.6 //E/m/s or 400 ft-c).
3. Cultures .are mixed continuously on a magnetic stir plate (with a medium
size stir bar) or in a moderately aerated separatory funnel, or are
mixed twice daily by hand. If the cultures are placed on a magnetic
stir plate, heat generated by the stirrer might elevate the culture
temperature several degrees. Caution should be exercised to prevent the
culture temperature from rising more than 2-3ฐC.
13.6.15.2.3 Preparing Algal Concentrate for Use as Ceriodaphnia dubia Food:
1. An algal concentrate containing 3,0 to 3.5 X 107 cells/ml is prepared
from food cultures by centrifuging the algae with a plankton or
bucket-type centrifuge, or by allowing the cultures to settle in a
refrigerator for at least three weeks and siphoning off the supernatant.
2. The cell density (cells/mL) in the concentrate is measured with an
electronic particle counter, microscope and hemocytometer, fluorometer,
or spectrophotometer (see Section 14, Green Alga, Selenastrum
capn'cornutum Growth Test), and used to determine the dilution (or
further concentration) required to achieve a final cell count of 3.0 to
3.5 X loVmL.
3. Assuming a cell density of approximately 1.5 X 106 cells/ml in the
algal food cultures at 7 days, and 100% recovery in the concentration
process, a 3-L, 7-10 day culture will provide 4.5 X 109 algal cells.
This number of cells would provide approximately 150 ml of algal cell
concentrate (1500 feedings at 0.1 ml/feeding) for use as food. This
would be enough algal food for four Ceriodaphm'a dubia tests.
4. Algal concentrate may be stored in the refrigerator for one month.
13.6.15.3 Food Quality
13.6.15.3.1 USEPA recommends Fleishmann'sฎ yeast, Cerophyllฎ, trout chow, and
Selenastrum capn'cornutum as the preferred Ceriodaphnia dubia food
combination. This recommendation is based on extensive data developed by many
laboratories which indicated high Ceriodaphnia dubia survival and reproduction
in culturing and testing. The use of substitute food(s) is acceptable only
after side-by-side tests are conducted to determine that the quality of the
substitute food(s) is equal to the USEPA recommended food combination based on
survival and reproduction of Ceriodaphnia dubia.
\
13.6.15.3.2 The quality of food prepared with newly acquired supplies of
yeast, trout chow, dried cereal leaves, algae, and/or any substitute food(s)
should be determined in side-by-side comparisons of Ceriodaphnia dubia
survival and reproduction, using the new food and food of known, acceptable
quality, over a seven-day period in control medium.
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13.6.16 TEST ORGANISMS, DAPHNIDS, CERIODAPHNIA DUBIA
13.6.16.1 Cultures of test organisms should be started at lea:st three weeks
before the brood animals are needed, to ensure an adequate supply of neonates
for the test. Only a few individuals are needed to start a culture because of
their prolific reproduction.
13.6.16.2 Neonates used for toxicity tests must be obtained from individually
cultured organisms. Mass cultures may be maintained, however, to serve as a
reserve source of organisms for use in initiating individual cultures and in
case of loss of individual cultures.
13.6.16.3 Starter animals may be obtained from commercial sources and may be
shipped in polyethylene bottles. Approximately 40 animals and 3 ml of food
are placed in a 1-L bottle filled full with culture water for shipment.
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.
13.6.16.4 It is best to start the cultures with one animal, which is
sacrificed after producing young, mounted on a microscope slide, and retained
as a permanent 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 Cen'odaphm'a dubia (modified from Beckett and Lewis, 1982):
1. Pi pet the animal onto a watch glass.
2. Reduce the water volume by withdrawing excess water with the pi pet.
3. Add a few drops of carbonated water.(club soda or seltzer water) or
70% 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 mounting medium is CMCP-9/10
Medium , prepared by mixing two parts of CMCP-9 with one part of
CMCP-10 stained with enough acid fucKsin dye to color the mixture a
light pink. For more viscosity and faster drying, CMC-10 stained
with acid fuchsin may be used.
5. Using forceps or a pipet, transfer the animal to the drop of mounting
medium on the microscope slide.
6. Cover with a 12 mm round 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 varnish around the edges of the
covers!ip.
CMCP-9, CMCP-10 and Acid Fuchsin are available from Polysciences, Inc.,
Paul Valley Industrial Park, Harrington, PA, 18976, 215-343-6484.
Neonates from mass cultures are not to be used directly in toxicity
tests (see Subsection 13.10.2.3).
154
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9. Identify to species (see Pennak, 1978; Pennak, 1989:; and Berner,
1986).
10. Label with waterproof ink or diamond pencil.
11. Store for permanent record.
13.6.16.5 Mass Culture
13.6.16.5.1 Mass cultures are used only as a "backup" reservoir of organisms.
13.6.16.5.2 One-liter or 2-1 glass beakers, crystallization dishes, "battery
jars," or aquaria may be used as culture vessels. Vessels are commonly filled
to three-fourths capacity. Cultures are fed daily. Four or more cultures are
maintained in separate vessels and with overlapping ages to serve as back-up
in case one culture is lost due to accident or other unanticipated problems,
such as low DO concentrations or poor quality of food or laboratory water.
13.6.16.5.3 Mass cultures which will serve as a source of brood organisms for
individual culture should be maintained in good condition by frequent renewal
with new culture medium at least twice a week for two weeks. At each renewal,
the adult survival is recorded, and the offspring and the old medium are
discarded. After two weeks, the adults are also discarded, and the culture is
re-started with neonates in fresh medium. Using this schedule, 1-L cultures
will produce 500 to 1000 neonate Cen'odaphm'a dubia each week.
13.'6.16.6 Individual Culture - ;
13.6.16.6.1 Individual cultures are used as the immediate source of neonates
for toxicity tests.
13.6.16.6.2 Individual organisms are cultured in 15 mL of culture medium in
30-mL (1 oz) plastic cups or 30-mL glass beakers. One neonate is placed in
each cup. It is convenient to place the cups in the same type of board used
for toxicity tests (see Figure 1).
13.6.16.6.3 Organisms are fed daily (see Subsection 13.6.16.9) and are
transferred to fresh medium a minimum of three times a week, typically on
Monday, Wednesday, and Friday. On the transfer days, food is added to the new
medium immediately Before or after the organisms are transferred.
13.6.16.6.4 To provide cultures of overlapping ages, new boards are started
weekly, using neonates from adults which produce at least eight young in their
third or fourth brood. These adults can be used as sources of neonates until
14 days of age. A minimum of two boards are maintained concurrently to
provide backup supplies of organisms in case of problems.
13.6.16.6.5 Cultures which are properly maintained should produce at least
20 young per adult in three broods (seven days or less). Typically, 60 adult
females (one board) will produce more than the minimum number of neonates
(120) required for two tests.
13'.6.16.6.6 Records should be maintained on the survival of brood organisms
and number of offspring at each renewal. Greater than 20% mortality of
i
155
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adults, or less than an average of 20 young per female would indicate
problems, such as poor quality of culture media or food. Cultures that do not
meet these criteria should not be used as a source of test organisms.
13.6.16.7 Culture Medium
13.6.16.7.1 Moderately hard synthetic water prepared using MILLIPORE MILLI-Qฎ
or equivalent deionized water and reagent grade chemicals or 20% DMW is
recommended as a standard culture medium (see Section 7, Dilution Water).
13.6.16.8 Culture Conditions
13.6.16.8.1 The daphnid, Cenodaphnia dubia, should be cultured at a
temperature of 25 ฑ 1ฐC.
13.6.16.8.2 Day/night cycles prevailing in most laboratories will provide
adequate illumination for normal growth and reproduction. A photoperiod of
16-h of light and 8-h of darkness is recommended. Light intensity should be
10-20 /iE/mz/s or 50 to 100 ft-c.
13.6.16.8.3 Clear, double-strength safety glass or 6 mm plastic panels are
placed on the culture vessels to exclude dust and dirt, and reduce
evaporation.
13.6.16.8.4 The organisms are delicate and should be handled as carefully and
as little as possible so that they are not unnecessarily stressed. They are
transferred 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.
13.6.16.9 Food and Feeding
13.6.16.9.1 Feeding the proper amount of the right food is extremely
important in Ceriodaphnia dubia culturing. The key is to provide sufficient
nutrition to support normal reproduction without adding excess food which may
reduce the toxicity of the test solutions, clog the animal's filtering
apparatus, or greatly decrease the DO concentration and increase mortality.
A combination of Yeast, CEROPHYLLฎ, and Trout chow (YCT), along with the
unicellular green alga, Selenastrum capn'cornutum, will provide suitable
nutrition if.fed daily.
13.6.16.9.2 Other algal species (such as S. minutum or Ch1amydomonas
reinhardti), other substitute food combinations (such as Flake Fish Food), or
different feeding rates may be acceptable as long as performance criteria are
met and side-by-side comparison tests confirm acceptable quality (see
Subsection 13.6.15.3).
13.6.16.9.3 Cultures should be fed daily to maintain the organisms in optimum
condition so as to provide maximum reproduction. Stock cultures which are
stressed because they are not adequately fed may produce low numbers of young,
large numbers of males, and/or ephippial females. Also, their offspring may
produce few young when used in toxicity tests.
157
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13.6.16.9.4 Feed as follows:
1. If YCT is frozen, remove a bottle of food from the freezer 1 h before
feeding time, and allow to thaw.
2. YCT food mixture and algal concentrates should both be thoroughly mixed
by shaking before dispensing.
3. Mass cultures are fed daily at the rate of 7 ml YCT and 7 ml algae
concentrate/L culture.
4. Individual cultures are fed at the rate of 0.1 ml YCT and 0.1 ml algae
concentrate per 15 ml culture.
5. Return unused YCT food mixture and algae concentrate to the refrigerator.
Do not re-freeze YCT. Discard unused portion after two weeks.
13.6.16.10 It is recommended that chronic toxicity tests be performed monthly
with a reference toxicant. Daphnid, Cen'odaphnia dubia, neonates less than'
24 h old, and all within 8 h of the same age are used to monitor the chronic
toxicity of the reference toxicant to the Cen'odaphnia dubia produced by the
culture unit (see Section 4, Quality Assurance).
13.6.16.11 Record Keeping
13.6.16.11.1 Records, kept in a bound notebook, include (1) source of
organisms used to start the cultures, (2) type of food and feeding times, (3)
dates culture were thinned and restarted, (4) rate of reproduction in
individual cultures, (5) daily observations of the condition and behavior of
the organisms in the cultures, and (6) dates and results of reference toxicant
tests performed (see Section 4, Quality Assurance).
13.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE
13.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.
13.8 CALIBRATION AND STANDARDIZATION
13.8.1 See Section 4, Quality Assurance.
13.9 QUALITY CONTROL
13.9.1 See Section 4, Quality Assurance.
13.10 TEST PROCEDURES
13.10.1 TEST SOLUTIONS
13.10.1.1 Receiving Waters
13.10.1.1.1 The sampling point is determined by the objectives of the test.
Receiving water toxicity is determined with samples used.directly as collected
or after samples are passed through a 60 urn NITEXฎ filter and compared without
dilution, against a control. For a test consisting of single receiving water
and control, approximately 600 ml of sample would be required for each test,
158
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assuming 10 replicates of 15 ml, and sufficient additional sample for chemical
analysis.
13.10.1.2 Effluents
13.10.1.2.1 The selection of the effluent test concentrations should be based
on the objectives of the study. A dilution factor of 0.5 is commonly used.
A dilution factor of 0.5 provides precision of ฑ 100%, and testing of
concentrations between 6.25% and 100% effluent using only five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%). Improvements in precision
decline rapidly if the dilution factor is increased beyond 0.5, and precision
declines rapidly if a smaller dilution factor is used. Therefore, USEPA
recommends the use of the > 0-5 dilution factor.
13.10.1.2.2 If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%). If a high rate of mortality is observed during the first
1 to 2 h of the test, additional dilutions should be added at the lower range
of effluent concentrations.
13.10.1.2.3 The volume of effluent required for daily renewal of 10
replicates per concentration, each containing 15 ml of test solution, with a
dilution series of 0.5, is approximately 1 L/day. 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 under a stereomicroscope with a minimum of
re-focusing. Ten test chambers are used for each effluent dilution and for
the control. Sufficient test solution (approximately 550 ml) is prepared at
each effluent concentration to provide 400 ml additional volume for chemical
analyses at the high, medium, and low test concentrations.
13.10.1.2.4 Tests should begin as soon as possible, preferably within 24 h of
sample collection. The maximum holding time following retrieval of the sample
from the sampling device should not exceed 36 h for off-site toxicity tests
unless permission is granted by the permitting authority. In no case should
the sample be used in a test more than 72 h after sample collection (see
Section 8, Effluent and Receiving Water Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests).
13.10.1.2.5 Just prior to test initiation (approximately one h) the
temperature of sufficient quantity of the sample to make the test solutions
should be adjusted to the test temperature and maintained at that temperature
during the preparation of the test solutions.
13.10.1.2.6 The DO of the test solutions should be checked prior to test
initiation. If any of the solutions are supersaturated with oxygen or any
solution has a DO concentration below 4.0 mg/L, all the solutions and the
control must be gently aerated.
159
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13.10.1.3 Dilution Water
13.10.1.3.1 Dilution water may be uncontaminated receiving water, a standard
synthetic (reconstituted) water, or some other uncontaminated natural water
(see Section 7, Dilution Water).
13.10.2 START OF THE TEST
13.10.2.1 Label the test chambers with a marking pen. Use of color-coded
tape to identify each treatment and replicate is helpful. A minimum of five
effluent concentrations and a control are used for each effluent test. Each
treatment (including the control) must have ten replicates.
13.10.2.2 The test solutions can be randomly assigned to a board using a
template (Figure 1) or by using a table of random numbers (see Appendix A).
When using the randomized block design, test chambers are randomized only
once, at the beginning of the test. A number of different templates should be
prepared, so that the same template is not used for every test.
13.10.2.3 Neonates less than 24 h old, and all within 8 h of the same age,
are required to begin the test. The neonates are obtained from individual
cultures using brood boards, as described above (see Section 6,, Test
Organisms). Neonates are taken only from adults that have eight or more young
in their third or subsequent broods. These adults can be used as brood stock
until they are 14 days old. If the neonates are held more than one or two
hours before using in the test, they should be fed (0.1 ml YCT and 0.1 ml
algal concentrate/15 ml of media).
13.10.2.4 Ten brood cups, each with 8 or more young, are randomly selected
from a brood board for use in setting up a test. To start the test, one
neonate from the first brood cup is transferred to each of the six test
chambers in the first row on the test board (Figure 1). One neonate from the
second brood cup is transferred to each of the six test chambers in the second
row on the test board. This process is continued until each of the 60 test
chambers contains one neonate. . ,
13.10.2.4.1 The cups and test chambers may be placed on a light table to
facilitate counting the neonates. However, care must be taken to avoid
temperature increase due to heat from the light table.
13.10.2.5 This blocking procedure allows the performance of each female to be
tracked. If a female produces one weak offspring or male, the likelihood of
producing all weak offspring or all males is greater. By using this known
parentage technique, poor performance of young from a given female can be
omitted from all concentrations.
13.10.3 LIGHT, PHOTOPERIOD, AND TEMPERATURE
13.10.3.1 The light quality and intensity should be at ambient laboratory
levels, approximately 10-20 nE/m/s, or 50 to 100 ft-c, with a photoperiod of
16 h of light and 8 h of darkness.
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13.10.3.2 It is critical that the test water temperature be maintained at
25 ฑ 1ฐC to obtain three broods in seven days.
13.10.4 DISSOLVED OXYGEN (DO) CONCENTRATION
13.10.4.1 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 be measured in the new solutions at the start of the
test (Day 0) and before daily renewal of the test solutions on subsequent
days. The DO concentration should not fall below 4.0 mg/L (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests). Aeration is generally not practical during the daphnid,
Cen'odaphm'a dubia, test. If the DO in the effluent and/or dilution water is
low, aerate gently before preparing the test solutions. 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 KIMAXฎ 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 organisms.
13.10.5 FEEDING
13.10.5.1 The organisms are fed when the test is initiated, and daily
thereafter. Food is added to the fresh medium immediately before or
immediately after the adults are transferred. Each feeding consists of 0.1 ml
YCT and 0.1 mL Selenastrum capn'cornutum concentrate/15 ml test solution
(0.1 ml of algal concentrate containing 3.0-3.5 X 10 cells/ml will provide
2-2.3 X 10 cells/ml in the test chamber).
13.10.5.2 The YCT and algal suspension can be added accurately to the test
chambers by using automatic pipettors, such as Gil son, Eppendorf, Oxford, or
equivalent.
13.10.6 OBSERVATIONS DURING THE TEST
13.10.6.1 Routine Chemical and Physical Determinations
13.10.6.1.1 DO is measured at the beginning and end of each 24-h exposure
period in at least one test chamber at each test concentration and in the
control.
13.10.6.1.2 Temperature and pH are measured at the end of each 24-h exposure
period in at least one test chamber at each test concentration and in the
control. Temperature should be monitored continuously or observed and
recorded daily for at least two locations in the environmental control system
or the samples. Temperature should be measured in sufficient number of test
vessels at least at the end of the test to determine the temperature variation
in the environmental chamber.
13.10.6.1.3 The pH is measured in the effluent sample each day before new
test solutions are made.
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13.10.6.1.4 Conductivity, alkalinity and hardness are measured in each new
sample (100% effluent or receiving water) and in the control.
13.10.6.1.5 Record the data on data sheet (Figure 2).
13.10.6.2 Routine Biological Observations
13.10.6.2.1 Three or four broods are usually obtained in the controls in a
7-day test conducted at 25 ฑ 1ฐC. A brood is a group of offspring released
from the female over a short period of time when the carapace is discarded
during molting. In the controls, the first brood of two-to-five young is
usually released on the third or fourth day of the test. Successive broods
are released every 30 to 36 h thereafter. The second and third broods usually
consist of eight to 20 young each. The total number of young produced by a
healthy control organism in three broods often exceeds 30 per female.
13.10.6.2.2 The release of a brood may be inadvertently interrupted during
the daily transfer of organisms to fresh test solutions, resulting in a split
in the brood count between two successive days. For example, four neonates of
a brood of five might be released on Day 3, just prior to test solution
renewal, and the fifth released just after renewal, and counted on Day 4.
Partial broods, released over a two-day period, should be counted as one
brood.
13.10.6.2.3 Each day, the live adults are transferred to fresh test
solutions, and the numbers of live young are recorded (see data form,
Figure 3). The young can be counted with the aid of a stereomicroscope with
substage lighting. Place the test chambers on a light box over a strip of
black tape to aid in counting the neonates. The young are discarded after
counting.
13.10.6.2.4 Some of the effects caused by toxic substances include, (1) a
reduction in the number of young produced, (2) young may develop in the brood
pouch of the adults, but may not be released during the exposure period, and
(3) partially or fully developed young may be released, but are all dead at
the end of the 24-h period. Such effects should be noted on the data sheets
(Figure 3).
13.10.6.2.5 Protect the daphnids, Ceriodaphm'a dubia, from unnecessary
disturbance during the test by carrying out the daily test observations,
solution renewals, and transfer of females carefully. Make sure the females
remain immersed during the performance of these operations.
13.10.7 DAILY PREPARATION OF TEST CHAMBERS
13.10.7.1 The test is started (Day 0) with new disposable polystyrene cups or
precleaned 30-mL borosilicate glass beakers that are labeled and color-coded
with tape. Each following day, a new set of plastic cups or precleaned glass
beakers is prepared, labeled, and color-coded with tape similar to the
original set. New solutions are placed in the new set of test chambers, and
the test organisms are transferred from the original test chambers to the new
ones with corresponding labels and color-codes. Each day, previously used
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Discharger: _
Location:
Template No.:
Analyst:
Dates:
Food:
Day
Control :
Temp.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
0
1
2
3
4
5
6
7
Remarks
Day
Cone:
Temp.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
0
1
2
3
4
5
6
7
Remarks
Day
Cone:
Temp.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
0
1
2
3
4
5
6
7
Remarks
Figure 2. Data form for the daphnid, Ceriodaphnia dubia, survival and
reproduction test. Routine chemical and physical
determinations.
163
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Discharger: _
Location:
Template No.:
Analyst:
Dates: _
Food:
Day
Cone:
Temp.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
0
1
2
3
4
5
6
7
Remarks
Day
Cone:
Temp.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
0
1
2
3
4
5
6
7
Remarks
Day
Cone:
Temp.
D.O. Initial
Final
oH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
0
1
2
3
4
5
6
7
Remarks
Figure 2. Data form for the daphnid, Ceriodaphm'a dubia, survival and
reproduction test. Routine chemical and physical determinations
(CONTINUED).
164
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glass beakers are recleaned (see Section 5, Facilities, Equipment, and
Supplies) for the following day, and previously used plastic cups are
discarded.
13.10.8 TEST SOLUTION RENEWAL
13.10.8.1 Freshly prepared solutions are used to renew the test daily. For
on-site toxicity studies, fresh effluent or receiving water samples should be
collected daily, and no more than 24 h should elapse between collection of the
samples and their use in the tests (see Section 8, Effluent and Receiving
Water Sampling, Sample Handling, and Sample Preparation for Toxicity Tests).
For off-site tests, a minimum of three samples are collected, preferably on
days one, three, and five. No more than 36. h should elapse between collection
of the sample and the first use in the test. Maintain the samples in the
refrigerator at 4ฐC until used. '
13.10.8.2 New test solutions are prepared daily, and the test organisms are
transferred to the freshly prepared solutions using a small-bore (2 mm) glass
or polyethylene dropper or pipet. The animals are released under the surface
of the water so that air is not trapped under the carapace. Organisms that
are dropped or injured are discarded.
13.10.9 TERMINATION OF THE TEST
13.10.9.1 Tests should be terminated when 60% of the control organisms have
produced their third brood, or at the end of 8 days, whichever occurs first.
Because of the rapid rate of development of Cen'odaphnia dubia, at test
termination all observations on organism survival and numbers of offspring
should be completed within two hours. 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.
13.10.9.2 Count the young, conduct required chemical measurements, and
complete the data sheets (Figure 3).
13.10.9.3 Any animal not producing young should be examined to determine if
it is a male (Berner, 1986). In most cases, the animal will need to be placed
on a microscope slide before examining (see Subsection 13.6.16.4).
13.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
13.11.1 A summary of test conditions and test acceptability criteria is
presented in Table 3.
13.12 ACCEPTABILITY OF TEST RESULTS
13.12.1 For the test results to be acceptable, at least 80% of the control
organisms must survive, and 60% of surviving adults in the controls must have
had at least three broods, with an average total number of 15 or more
offspring per surviving adult.
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Discharger:
Location:
Date Sample Collected:
Analyst:
Test Start-Date/Time:
Test Stop -Date/Time:
Re
1
Cone.
Dav
1
2
3
4
5
6
7
Total
2
3
4
5
lica
6
te
7
8
9
10
Number
of
Younq
Number
of
Adults
Young
per
Adult
Replicate
Cone.
1
Dav
1
2
3
4
5
6
7
Total
2
3
4
5
6
7
8
9
10
Number
of
Younq
Number
of
Adults
Young
per
Adult
Re
Cone.
1
Dav
1
2
3
4
5
6
7
Total
2
3
4
plic
5
ate
6
7
8
9
10
Number
of
Younq
Number
of
Adults
Young
per
Adult
Figure 3. Data form for the daphnid, Cen'odaphm'a dubia, survival and
reproduction test. Daily summary of data.
166
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Discharger:
Location:
Date Sample Collected:
Analyst:
Test Start-Date/Time:
Test Stop -Date/Time:
Re
1
Cone.
Dav
1
2
3
4
5
6
7
Total
?
3
4
R
ica
6
te
7
R
9
10
Number
of
Younq
Number
of
Adults
Young
per
Adult
Replicate
Cone.
1
Dav
1
2
3
4
5
6
7
Total
?
3
4
5
6
7
8
9
10
Number
of
Younq
Number
of
Adults
Young
per
Adult
Re
Gone.
1
Dav
1
2
3
4
5
6
7
Total
?
3
4
plic
5
ate
6
7
8
9
-10
Number
of
Younq
Number
of
Adults
Young
per
Adult
Figure 3. Data form for the daphnid, Ceriodaphm'a dubia, survival and
reproduction test. Daily summary of data (CONTINUED).
167
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
DAPHNID, CERIODAPHNIA DUBIA, SURVIVAL AND REPRODUCTION TOXICITY
TESTS WITH EFFLUENTS AND RECEIVING WATERS
1. Test type:
2. Temperature (ฐC):
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test chamber size:
7. Test solution volume:
8. Renewal of test solutions:
9. Age of test organisms:
10. No. neonates per
test chamber:
11. No. replicate test
chambers per concentration:
12. No. neonates per
test concentration:
13. Feeding regime:
14. Cleaning:
15. Aeration:
16. Dilution water:
Static renewal
25 ฑ 1ฐC
Ambient laboratory illumination
10-20 /^E/m2/s, or 50-100 ft-c
(ambient laboratory levels)
16 h light, 8 h dark
30 mL (minimum)
15 mL (minimum)
Daily
Less than 24 h; and all released
within a 8-h period
10
10
Feed 0.1 mL each of YCT and algal
suspension per test chamber daily
Use freshly cleaned glass beakers or
new plastic cups daily
None
Uncontaminated source of receiving or
other natural water, synthetic water
prepared using MILLIPORE MILLI-Qฎ or
equivalent deionized water and reagent
grade chemicals or DMW (see Section
7, Dilution Water)
168
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
FOR DAPHNID, CERIODAPHNIA DUBIA, SURVIVAL AND REPRODUCTION
TOXICITY TESTS WITH EFFLUENTS AND RECEIVING WATERS (CONTINUED)
17. Test concentrations:
18. Dilution factor:
19. Test duration:
20. Endpoints:
21. Test acceptability criteria:
22. Sampling requirements:
Effluents: Minimum of 5 and a control
Receiving Water: 100% receiving water
or minimum of 5 and a control
Effluents: > 0.5
Receiving Waters:
None or > 0.5
22. Sample volume required:
Until 60% of surviving control
organisms have three broods (maximum
test duration 8 days)
Survival and reproduction
80% or greater survival and an average
of 15 or more young per surviving
female in the control solutions. 60%
of surviving control organisms must
produce three broods.
For on-site tests, samples collected
daily, and used within 24 h of the
time they are removed from the
sampling device. For off-site tests,
a minimum of three samples collected
on days one, three, and five with a
maximum holding time of 36 h before
first use (see Section 8, Effluent and
Receiving Water Sampling, Sample
Handling, and Sample Preparation for
Toxicity Tests, Subsection 8.5.4)
1 L/day i
169
-------
13.13 DATA ANALYSIS '
13.13.1 GENERAL
13.13.1.1 Tabulate and summarize the data. A sample set of survival and
reproduction data is listed in Table 4.
TABLE 4. SUMMARY OF SURVIVAL AND REPRODUCTION DATA FOR THE DAPHNID,
CERIODAPHNIA DUBIA, EXPOSED TO AN EFFLUENT FOR SEVEN DAYS
Effluent
Concentration
(X)
No. of Young per Adult
Replicate
10
No.
Live
Adults
Control
1.56
3.12
6.25
12.5
25.0
27
32
39
27
10
0
30
35
30
34
13
0
29
32
33
36
7
0
31
26
33
34
7
0
16
18
36
31
7
0
15
29
33
27
10
0
18
27
33
33
10
0
17
16
27
31
16
0
14
35
38
33
12
0
27
13
44
31
2
0
10
10
10
10
10
3
13.13.1.2 The endpoints of toxicity tests using the daphnid, Cen'odaphm'a
dubia, are based on the adverse effects on survival and reproduction. The
LC50, the IC25, the IC50 and the EC50 are calculated using point estimation
techniques, and LOEC and NOEC values for survival and reproduction are
obtained using a hypothesis test approach such as Fisher's Exact Test (Finney,
1948; Pearson and Hartley, 1962), Dunnett's Procedure (Dunnett, 1955) or
Steel's Many-one Rank Test (Steel, 1959; Miller, 1981) (see Section 9, Chronic
Toxicity Test Endpoints and Data Analysis). Separate analyses are performed
for the estimation of the LOEC and NOEC endpoints and for the estimation of
the LC50, IC25, IC50 and EC50. Concentrations at which there is no survival
in any of the test chambers are excluded from the statistical analysis of the
NOEC and LOEC for reproduction, but included in the estimation of the LC50,
IC25, IC50, and EC50. See the Appendices for examples of the manual
computations, program listings, and examples of data input and program output.
13.13.1.3 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 Appendix B. The assistance of a
statistician is recommended for analysts who are not proficient in statistics.
170
-------
13.13.2 EXAMPLE OF ANALYSIS OF THE DAPHNID, CERIODAPHNIA DUBIA, SURVIVAL DATA
13.13.2.1 Formal statistical analysis of the survival data is outlined on the
flowchart in Figure 4. The response used in the analysis,is the number of
animals surviving at each test concentration. Separate analyses are performed
for the estimation of the NOEC and LOEC endpoints and for the estimation of
the EC50, LC50, IC25, or IC50 endpoints. Concentrations at which there is no
survival in any of the test chambers are excluded from the statistical
analysis of the NOEC and LOEC, but included in the estimation of the LC, EC,
and 1C endpoints.
13.13.2.2 Fisher's Exact Test is used to determine the NOEC and LOEC
endpoints. It provides a conservative test of the equality of any two
survival proportions assuming only the independence of responses from a
Bernoulli (binomial) population. Additional information on Fisher's Exact
Test is provided in Appendix G.
13.13.2.3 Probit Analysis (Finney, 1971; Appendix I) is used to estimate the
concentration that causes a specified percent decrease in survival from the
control. In this analysis, the total number dead at a given concentration is
the response.
j
13.13.2.4 Example of Analysis of Survival Data
13.13.2.4.1 The data in Table 4 will be used to illustrate the analysis of
survival data from the daphnid, Cen'odaphm'a dubia, Survival and Reproduction
Test. As can be seen from the data in Table 4, there were no deaths in the
1.56%, 3.12%, 6.25%, and 12.5% concentrations. These concentrations are
obviously not different from the control in terms of survival. This leaves
only the 25% effluent concentration to be tested statistically for a
difference in survival from the control.
13.13.2.5 Fisher's Exact Test
13.13.2.5.1 The basis for Fisher's Exact Test is a 2x2 contingency table.
From the 2x2 table prepared by comparing the control and the effluent
concentration, determine statistical significance by look-ing up a value in the
table provided in Appendix G (Table G.5). However, to use this table the
contingency table must be arranged in the format illustrated in Table 5.
13.13.2.5.2 Arrange the table so that the total number of observations for
row one is greater than or equal to the total for row two (A > B). Categorize
a success such that the proportion of successes for row one is greater than or
equal to the proportion of successes for row two (a/A > b/B)., For these data,
a success may be 'alive' or 'dead' 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 the contingency table. The table of
significance levels of b is included in Appendix G, Table G.5. Enter
Table G.5 in the section for A, subsection for B, and the line for a. If the
b value of the contingency table is equal to or less than the integer in the
column headed 0.05 in Table G.5, then the survival proportion for the effluent
concentration is significantly different from that of the control. A dash or
171
-------
STATISTICAL ANALYSIS OF CERIODAPHNIA
SURVIVAL AND REPRODUCTION TEST
SURVIVAL
MORTALITY DATA
#DEAD
TWO OR MORE
PARTIAL MORTALITIES?
FISHER'S EXACT
TEST
NO
IS PROBIT MODEL
APPROPRIATE?
(SIGNIFICANT X2 TEST)
^
YES
r
NO
ENDPOINT ESTIMATES
NOEC, LOEC
ONE OR MORE
PARTIAL MORTALITIES?
i
YES
r
NO
GRAPHICAL METHOD
LC50
PROBIT METHOD L
M
H
fe
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONG.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONC.?
NO
YES
SPEARMAN-KARBER
METHOD
I
LC50AND95%
CONFIDENCE
INTERVAL
ซ
TRIMMED SPEARMAN
KARBER METHOD
Figure 4. Flowchart for statistical analysis of the daphnid, Ceriodaphm'a
dubia, survival data.
172
-------
TABLE 5. FORMAT OF THE 2x2 CONTINGENCY TABLE
Condition 1
Condition 2
Total
i
Number of
Successes Failures
a A - a
b B - b
a + b [(A+B) - a - b]
Number of
Observations
A
B
A + B
absence of entry in Table G.5 indicates that no contingency table in that
class is significant.
13.13.2.5.3 To compare the control and the effluent concentration of 25%, the
appropriate contingency table for the test is given in Table 6.
13.13.2.5.4 Since 10/10 > 3/10, the category 'alive' is regarded as a
success. For A = 10, B = 10 and, a = 10, under the column headed 0.05, the
value from Table G.5 is b = 6. Since the value of b (b = 3) from the
contingency table (Table 6), is less than the value of b (b = 6) from
Table G.5 in Appendix G, the test concludes that the proportion surviving in
the 25% effluent concentration is significantly different from the control.
Thus the NOEC for survival is 12.5% and the LOEC is 25%.
TABLE 6. 2x2 CONTINGENCY TABLE FOR CONTROL AND 25% EFFLUENT
Number of Number of
Observations
. . . Alive Dead
Control 10 0 10
25% Effluent 3 7 10
Total 13 7 20
173
-------
13.13.2.6 Calculation of the LC50
13.13.2.6.1 The data used for the Trimmed Spearman-Karber Method are
summarized in Table 7. To perform the Trimmed Spearman-Karber Method, run the
USEPA Trimmed Spearman-Karber Program. An example of the program input and
output is supplied in Appendix J.
TABLE 7. DATA FOR TRIMMED SPEARMAN-KARBER ANALYSIS
Effluent Concentration (%)
Control 1.56 3.12 6.25 12.5 25.0
Number Dead
Number Exposed
0
10
0
10
0
10
0
10
0
10
8
10
13.13.2.6.2 For this-example, with only one partial mortality, Trimmed
Spearman-Karber analysis appears appropriate for this data.
13.13.2.6.3 Figure 5 shows the output for the Trimmed Spearman-Karber
Analysis of the data in Table 7 using the USEPA Program.
13.13.3 EXAMPLE OF ANALYSIS OF THE DAPHNID, CERIODAPHNIA DUBIA, REPRODUCTION
DATA
13.13.3.1 Formal statistical analysis of the reproduction data is outlined on
the flowchart in Figure 6. The response used in the statistical analysis is
the number of young produced per adult female, which 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, if it has not been identified as a male, would be
included in the analysis with zero entered as the number of young produced.
The subsequent calculation of the mean number of live young produced per adult
female for each toxicant concentration provides a combined measure of the
toxicant's effect on both mortality and reproduction. An 1C estimate can be
calculated for the reproduction data using a point estimation technique (see
Section 9, Chronic Toxicity Test Endpoints and Data Analysis). Hypothesis
testing can be used to obtain an NOEC for reproduction. Concentrations above
the NOEC for survival are excluded from the hypothesis test for reproduction
effects.
13.13.3.2 The statistical analysis using hypothesis tests consists of a
parametric test, Dunnett's Procedure, and a nonparametric test, Steel's
Many-one Rank Test. The underlying assumptions of the Dunnett's Procedure,
normality and homogeneity of variance, are formally tested using the Shapiro
174
-------
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5
DATE: 1 TEST NUMBER: 2
TOXICANT: effluent
SPECIES: ceriodaphnia dubia
RAW DATA: Concentration
DURATION:
7 Days
.00
1.25
3.12
6.25
12.5
25.0
SPEARMAN-KARBER TRIM:
SPEARMAN-KARBER ESTIMATES:
Number
Exposed
10
10
10
10
10
10
Mortal!
0
0
0
0
0
8
20.41 %
LC50: 77.28
95% CONFIDENCE LIMITS
ARE NOT RELIABLE,:
NOTE: MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING.
ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION.
Figure 5. Output for USEPA Trimmed Spearman-Karber program.
175
-------
STATISTICAL ANALYSIS OF CERIODAPHNIA
SURVIVAL AND REPRODUCTION TEST
REPRODUCTION DATA
NO. OF YOUNG PRODUCED
POINT ESTIMATION
1
F
HYPOTHESIS TESTING
(EXCLUDING CONCENTRATIONS
ABOVE NOEC FOR SURVIVAL)
ENDPOINT ESTIMATE
IC25, IC50
SHAPIRO-WILK'S TEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
HETEROGENEOUS
VARIANCE
EQUAL NUMBER OF
REPLICATES?
NO
YES
T-TESTWITH
BONFERRONI
ADJUSTMENT
EQUAL NUMBER OF
REPLICATES?
YES
NO
STEEL'S MANY-ONE
RANK TEST
T
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 6. Flowchart for the statistical analysis of the daphnid, Ceriodaphnia
dubia, reproduction data.
176
-------
t -?d Bartlett/s Test fฐr homogeneity of variance.
these tests fails, a nonparametric test, Steel's Many-one Rank
rnH. to de+um1n! the NOEC and LOEC- If th* assumptions of Dunnett's
Procedure are met, the endpoints are determined by the parametric test
13.13.3.3 Additionally, if unequal numbers of replicates occur amona the
anSlvses ^Kp^6"615 ?6?ted ^ are ^metric and nonparametr?c alterative
analyses. The parametric analysis is a t test with the Bonferroni adjustment
(see Appendix D) The Wilcoxon Rank Sum Test with the Boinferroni adjustment
is the nonparametric alternative (see Appendix F) . aajusiment
13.13.3.4 The data, mean, and variance of the observations at each
concentration including the control are listed in Table 8. A plot of the
number of young per adult female for each concentration is pro! ded in
Figure 7. Since there is significant mortality in the 25% effluent
concentration, its effect on reproduction is not considered
TABLE 8. THE DAPHNID, CERIODAPHNIA DUBIA, .REPRODUCTION DATA
Replicate Control
Effluent Concentratinn (%)
1.56
3.12
6.25
12.5
1
2
3
4
6
7
8
9
10
Mean(Y,.)
?.i
i
27
30
29
31
16
15
18
17
14
27
22.4
48.0
1
32
35
32
26
18
29
27
16
35
13
26.3
64.0
2
39
30
33
33
36
33
33
27
38
44
34.6
23.4
3
27
34
36
34
31
27
33
31
33
31
31.7
8.7
4
10
13
7
7
7
10
10
16
12
2
9.4
15.1
5
13.13.3.5 Test for Normality
13.13.3 5.1 The first step of the test for normality is to center the
observations by subtracting the mean of all the observatio s witMn a
concentration from each observation in that concentration T e centered
observations are summarized in Table 9 centered
177
-------
*
s-
=3
CO
to
I
ns
c
c
Q.
O
C
Z
O
Z
UJ
8
u.
LLI
a
ns
O)
d)
rC
s-
o>
o.
en
o c
o
O) +->
JO U
i-g
c o
<4- O.
O O)
ro
-O
O)
3
dO
178
-------
TABLE 9. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Effluent Concentration (%)
Repl i cate
1
2
3
4
5
6
7
8
9
10
Control
4.6
7.6
6.6
8.6
-6.4
-7.4
-4.4
-5.4
-8.4
4.6
1.56
5.7
8.7
5.7
-0.3
-8.3
2.7
0.7
-10.3
8.7
-13.3
3.12
4.4
-4.6
-1.6
-1.6
1.4
-1.6
-1.6
-7.6
3.4
9.4
6.25
-4.7
2.3
4.3
2.3
-0.7
-4.7
1.3
-0.7
1.3
-0.7
12.5
0.6
3.6
-2.4
-2.4
-2.4
0.6
0.6
6.6
2.6
-7.4
13.13.3.5.2 Calculate the denominator, D, of the test statistic:
D = ฃ (Xฑ-X)2
i=l
Where: Xf = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations.
For this set of data,
n = 50
X = -^ (0.0) = 0.0
50
D = 1433.4
' i
13.13.3.5.3 Order the centered observations from smallest to largest
Where X(O is the ith ordered observation. These ordered observations are
listed in Table 10.
179
-------
TABLE 10. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-MILK'S EXAMPLE.
ci>
(D
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
-13.3
-10.3
-8.4
-8.3
-7.6
-7.4
-7.4
-6.4
-5.4
-4.7
-4.7
-4.6
-4.4
-2.4
-2.4
-2.4
-1.6
-1.6
-1.6
-1.6
-0.7
-0.7
-0.7
-0.3
0.6
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
0.6
0.6
0.7
1.3
1.3
1.4
2.3
2.3
2.6
2.7
3.4
3.6
4.3
4.4
4.6
4.6
5.7
5.7
6.6
6.6
7.6
8.6
8.7
8.7
9.4
13.13.3.5.4 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a15 a2, ..., ak where k is n/2 if n is even and
(n-l)/2 if n is odd. For the data in this example, n = 50, k = 25. The a,-
values are listed in Table 11.
13.13.3.5.5 Compute the test statistic, W, as follows:
W= -i [ฃaAX(n-*+V-X(1))]2
D i-i *
The differences xcn"i"'1> - X
-------
TABLE 11. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
ai
0.3751
0.2574
0.2260
0.2032
0.1847
0.1691
0.1554
0.1430
0.1317
0.1212
0.1113
0.1020
0.0932
0.0846
0.0764
0.0685
0.0608
0.0532
0.0459
0.0386
0.0314
0.0244
0.0174
0.0104
0.0035
X<,M, . ซ
22.7
19.0
17.1
16.9
15.2
14.0
14.0
12.1
11.1
9.3
9.3
9.0
8.7
6.0
5.8
5.1
4.2
3.9
3.9
3.0
2.0
2.0
1.4
0.9
0.0
X(50)
X(49)
x(48)
XC47>
Y<46>
A
x<45)
X(44)
X(43)
x<42)
X(41)
x<40)
X(39,
X(38)
X(37)
X(36)
x<35>
X(34)
Y<33)
A
X(32)
X(31)
X(30)
Y(29)
A
X(28)
X(27>
Y<26)
A
X
- XC6)
- x(7>
- x<8)
- XC9)
x(10)
- x<11>
- x<12)
- x(13)
- x<14>
- x(15)
- x(16)
- XC17)
- x(18>
- x<19)
YC20)
*" A
- x<21)
- XC22)
- x(23>
- x<24)
y(25)
"" A
13.13.3.5.6 The decision rule for this test is to compare W with the critical
value found in Table 6, Appendix B. If the computed W is less than the
critical value, conclude that the data are not normally distributed. For this
example, the critical value at a significance level of 0.01 and 50
observations (n) is 0.930. Since W = 0.97 is greater than the critical value,
the conclusion of the test is that the data are normally distributed.
13.13.3.6 Test for Homogeneity of Variance
13.13.3.6.1 The test used to examine whether the variation in number of young
produced is the same across all effluent concentrations including the control,
is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as
follows:
181
-------
riins;]
B--JS^ _J-
Where: V, = degrees of freedom for each effluent concentration and
control, V,- = (n,- - 1)
p = number of levels of effluent concentration and control
nf - the number of replicates for concentration i
In - loge
i - 1, 2, ...ป P where p is the number of concentrations
including the control
-32 _
13.13.3.6.2 For the data in this example (see Table 8), all effluent
concentrations including the control have the same number of replicates
(nf = 10 for all i). Thus, V,- = 9 for all i.
13.13.3.6.3 Bartlett's statistic is therefore:
B- [(45)ln(31.8) -9 f) ln(.sf) ]/1. 04
= [45(3.5) - 9(16.!)]/!.04
- 12.6/1.04
- 12.1
13.13.3.6.4 B is approximately distributed as chi-square with p - 1 degrees
of freedom, when the variances are in fact the same. Therefore, the
appropriate critical value for this test, at a significance level of 0.01 with
four degrees of freedom, is 13.3. Since B = 12.1 is less than the critical
value of 13.3, conclude that the variances are not different.
13.13.3.7 Dunnett's Procedure
13.13.3.7.1 To obtain an estimate of the pooled variance for the Dunnett's
Procedure, construct an ANOVA table as described in Table 12.
182
-------
TABLE 12. ANOVA TABLE
Source
df
Sum of Squares
(SS)
Mean Square(MS)
(SS/df)
Between
Within
P - 1
N - p
SSB
SSW
SB = SSB/(p-l)
Sy = SSW/(N-p)
Total
N - 1
SST
Where: p = number effluent concentrations including
N = total number of observations n, + n2 ... -
n,- = number of observations in concentration i
SSB = T,Tl/ni-Gz/N Between Sum of Squares
SST = f, ^YlJ-G2/N Total Sum of Squares
the control
h n..
1=1.7=1
SSW = SST-SSB
Within Sum of Squares
G = the grand total of all sample observations, G= ฃ r,
; i-i
Tf =. the total of the replicate measurements for concentration i
,-j = the jth observatioh for concentration i (represents the
number of young produced by female j in effluent
concentration i)
183
-------
13.13.3.7.2 For the data in this example:
n, = n2 = n3 = nA = n5 = 10
N
T,
T2
T'
T5
G
= 50
= Y -
-Jป-
" Y31 '
~ Y41 '
= Y51-
= T, -t
* Y12 + '
f Y22 + .
f Y32 + .
f Y42 + .
f Y52 + .
- T, + T,
. . +
. . +,
. . +
. . +
. . +
+ T. +
YHO
Y
Y210
Y310
'410
YSIO
T, =
= 224
= 263
= 346
= 317
= 94
1244
SSB = &Tl/nฑ-G2/N
t =
2
(22.4-26.3)
[5.64^(1/10)+(1/10)]
P ni
SST if 2-i Yi-<G
= 36,272- =5321.28
SSW = SST-SSB = 5321.28 - 3887.88 = 1433.40
SB = SSB/(p-l) = 3887.88/(5-l) = 971.97
Sj = SSW/(N-p) = 1433.40/(50-5) = 31.85
13.13.3.7.3 Summarize these calculations in an ANOVA table (Table 13).
13.13.3.7.4 To perform the individual comparisons, calculate the t statistic
for each concentration and control combination as follows:
Where: Y, ซ mean number of young produced for effluent concentration i
Y, ซ= mean number of young produced for the control
Sw = square root of within mean square
nt - number of replicates for the control
n,- ป number of replicates for concentration i.
184
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TABLE 13. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source df Sum of Squares Mean Square(MS)
(SS) (SS/df)
Between
Within
4
45
3887.88
1433.40
971.97
i 31.85
Total 49 5321.28
Since we are looking for a decrease in reproduction from the control, the mean
for concentration i is subtracted from the control mean in the t statistic
above. However, if we were looking for an increased response over the
control, the control mean would be subtracted from the mean at a
concentration.
13.13.3.7.5 Table 14 includes the calculated t values for each concentration
and control combination. In this example, comparing the 1.56% concentration
with the control the calculation is as follows:
t2 = _ (22.4-26.3) _ .C.
[5.64v/(l/10)
TABLE 14. CALCULATED T VALUES
Effluent Concentration (%) i t,-
1.56
3.12
6.25
12.5
2
3
4
5
-1.55
-4.84
-3.69
5.16
13.13.3.7.6 Since the purpose of this test is to detect a significant ,
reduction in mean reproduction, a one-sided test is appropriate. The critical
value for this one-sided test is found in Table 5, Appendix C. Since an entry
for 45 degrees of freedom for error is not provided in the table, the entry
for 40 degrees of freedom for error, an alpha level of 0.05 and four
concentrations (excluding the control) will be used, 2.23. The mean
185
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reproduction for concentration "i" is considered significantly less than the
mean reproduction for the control if t= is greater than the critical value.
Since t5 is greater than 2.23, the 12.5% concentration has significantly lower
reproduction than the control. Hence the NOEC and the LOEC for reproduction
are 6.25% and 12.5%, respectively.
13.13.3.7.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be statistically detected may be calculated:
MSD = d
Where: d - the critical value for the Dunnett's Procedure
Sw = the square root of the within mean square
n - the common number of replicates at each concentration
(this assumes equal replication at each concentration)
n., - the number of replicates in the control.
13.13.3.7.8 In this example:
MSD = 2.23(5.64)^(1/10)+(1/10)
= 2.23 (5.64) (0.447)
= 5.62
13.13.3.7.9 Therefore, for this set of data, the minimum difference that can
be detected as statistically significant is 5.62.
13.13.3.7.10 This represents a 25% decrease in mean reproduction from the
control.
13.13.3.8 Calculation of the 1C
13.13.3.8.1 The reproduction data in Table 4 are utilized in this example.
As can be seen from Figure 8, the observed means are not monotonically
non-increasing with respect to concentration. Therefore, the means must be
smoothed prior to calculating the 1C.
13.13.3.8.2 Starting with the observed control mean,_Y.,= 22.4, and the
observed mean_for the lowest effluent concentration, Y2 = 26.3, we see that Y\
is less than Y2.
13.13.3.8.3 Calculate the smoothed means:
M, = M2 = (7, + Y2)/2 = 24.35
186
-------
8
u>
(O
&
o
tj
ป~
I
c:
i-
S-
0)
4 ' ' o
4-> S-
o o.
i 0)
0_ i.
00
0)
187
-------
13.13.3.8.4 Since Y3 = 34.6 is larger than M2, average Y3 with the previous
concentrations:
M1 = M2 = M3 = (M, + M2 + Y3)/3 = 27.7.
13.13.3.8.5 Additionally, Y4 = 31.7 is larger than M3, and is pooled with the
first three means. Thus:
(M, + M2
M3 + Y4)/4 = 28.7 = M1 = M2 = M3 = M4
13.13.3.8.6 Since M4 > Y5 = 9.4, set M5 = 9.4. Likewise, M5 > Y6 = 0, and M6
becomes 0. Table 15 contains the smoothed means and Figure 8 gives a plot of
the smoothed means and the interpolated response curve.
TABLE 15. DAPHNID, CERIODAPHNIA DUBIA, REPRODUCTION
MEAN RESPONSE AFTER SMOOTHING
Effluent
Cone. (%)
Control
1.56
3.12
6.25
12.5
25.0
i
1
2
3
4
5
6
Response
Means, Y,
(young/ female)
22.4
26.3
34.6
31.7
9.4
0.0
Smoothed
Means, M,-
(young/female)
28.75
28.75
28.75
28.75
9.40
0.00
13.13.3.8.7 Estimates of the IC25 and IC50 can be calculated using the Linear
Interpolation Method. A 25% reduction in reproduction, compared to the
controls, would result in a mean reproduction of 21.56 young per adult, where
M,(l - p/100) = 28.75(1 - 25/100). A 50% reduction in reproduction, compared
to the controls, would result in a mean reproduction of 14.38 young per adult,
where Mt(l - p/100) = 28.75(1 - 50/100). Examining the smoothed means and
their associated concentrations (Table 15), the two effluent concentrations
bracketing 21.56 young per adult are C, = 6.25% effluent and C5 = 12.5%
effluent. The two effluent concentrations bracketing a response of 14.38
young per adult are also C4'= 6.25% and C5 = 12.5%.
188
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13.13.3.8.8 Using equation from Section 4.2 in Appendix M, the estimate of
the IC25 is as follows:
ICp = C..
= 6. 25+ [28. 75 (1-25/100) -28.75]
_
(9.40-28.75)
= 8.57% effluent
13.13.3.8.9 The estimate of the IC50 is as follows:
ICp = C+CM ~
IC50 =6.25+128.75(1-50/100) -28.75] (12-5-6.25)
(9.40-28.75)
= 10.89% effluent
13.13.3.8.10 When the ICPIN program was used to analyze this data set for
the IC25, requesting 80 resamples, the estimate of the IC25 was 8.5715%
effluent. The empirical 95% confidence interval for the true mean was 8.3112%
and 9.0418% effluent. The computer output for this data set is provided in
Figure 9.
13.13.3.8.11 When the ICPIN program was used to analyze this data set for
the IC50, requesting 80 resamples, the estimate of the IC50 was 10.8931%
effluent. The empirical 95% confidence interval for the true mean was
10.4373% and 11.6269% effluent. The computer output for this data set is
provided in Figure 10.
13.14 PRECISION AND ACCURACY
13.14.1 PRECISION
13.14.1.1 Single- Laboratory Precision
13.14.1.1.1 Information on the single-laboratory precision of the daphnid,
Cenodaphma dubia, Survival and reproduction test based on the NOEC and LOEC
yM^n>from nine tests with the reference toxicant sodium pentachlorophenate
(NaPCP) 1S provided in Table 16. The NOECs and LOECs of all tests fell in the
same concentration .range, indicating maximum possible precision Table 17
gives precision data for the IC25 and IC50 values for seven tests with the
reference toxicant NaPCP. Coefficient of variation was 41% for the IC25 and
2o/o for the IC50.
13.14.1.1.2 Ten sets of data from six laboratories met the acceptability
criteria, and were statistically analyzed using nonparametric procedures to
determine NOECs and LOECs.
189
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Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
Response 9
Response 10
1
0
27
30
29
31
16
15
18
17
14
27
2
1.56
32
35
32
26
18
29
27
16
35
13
3
3.12
39
30
33
33
36
33
33
27
38
44
4
6.25
27
34
36
34
31
27
33
31
33
31
5
12.5
10
13
7
7
7
10
10
16
12
2
6
25.0
0
0
0
0
0
0
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date: Example Test Ending Date:
Test Species: Ceriodaphnia dubia
Test Duration: 7-d
DATA FILE: cdmanual.icp
OUTPUT FILE: cdmanual.i25
Cone.
ID
1
2
3
4
5
6
Number
Replicates
10
10
10
10
10
10
Concentration
%
0.000
1.560
3.120
6.250
12.500
25.000
Response
Means
22.400
26.300
34.600
31.700
9.400
0.000
Std.
Dev. F
6.931
8.001
4.835
2.946
3.893
0.000
Pooled
Response Means
28.750
28.750
28.750
28.750
9.400
0.000
The Linear Interpolation Estimate: 8.5715 Entered P Value: 25
Number of Resamplings: 80 .,.,. n 1001
The Bootstrap Estimates Mean: 8.5891 Standard Deviation: ฐ;1831
Original Confidence Limits: Lower: 8.3112 Upper: 9.0418
Resampling time in Seconds: 2.53 Random Seed: -641671986
Figure 9. Example of ICPIN program output for the IC25.
190
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Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
Response 9
Response 10
1
0
27
30
29
31
16
15
18
17
14
27
2
1.56
32
35
32
26
18
29
27
16
35
13
3
3.12
39
30
33
33
36
33
33
27
38
44
4
6.25
27
34
36
34
31
27
33
31
33
31
5
12.5
10
13
7
7
7
10
10
16
12
2
6
25.0
0
0
0
0
0
0
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date: Example Test Ending Date:
Test Species: Ceriodaphnia dubia
Test Duration: 7-d
DATA FILE: cdmanual.icp
OUTPUT FILE: cdmanual.i50
Cone.
ID
1
2
3
4 .
5
6
Number
Replicates
10
10
10
10
10
10
Concentration
%
0.000
1.560
3.120
6.250
12.500
25.000
Response
Means
22.400
26.300
34.600
31.700
9.400
0.000
Std.
Dev . F
6.931
8.001
4.835
2.946
3.893
0.000
Pool ed
Response Means
28.750
28.750
28.750
28.750
9.400
0.000
The Linear Interpolation Estimate: 10.8931 Entered P Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean: 10.9316 Standard Deviation: 0.3357
Original Confidence Limits: Lower: 10.4373 Upper: 11.6269
Resampling time in Seconds: 2.58 Random Seed: 172869646
Figure 10. Example of ICPIN program output for the IC50.
191
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TABLE 16. SINGLE LABORATORY PRECISION OF THE DAPHNID, CERIODAPHNIA
DUBIA, SURVIVAL AND REPRODUCTION TEST, USING NAPCP AS A
REFERENCE TOXICANT1'2
Test
I3
24
3
45
5
6
7
8
9
1 For a discussion of
tests see Section 4,
" Pi ซs 4* ป ฃ\j* n %* 4* *ป ** 4* <ป **i i*i\stฃ
NOEC
(mg/L)
0.25
0.20
0.20
0.30
0.30
0.30
0.30
0.30
0.30
the precision
LOEC
(mg/L)
0.50
0.60
0.60
0.60
0.60
0.60
0.60
0.60
0.60
of data from
Chronic
Value
(mg/L)
6.35
0.35
0.35
0.42
0.42
0.42
0.42
0.42
0.42
chronic toxicity
Quality Assurance.
'*ป i/ttM/ssJ ts\i fil^ T T T t^ 1 xM.t*irซ Ami<^4ป4^ป Q A r\l r\i~i\i D Vi *\ i^ /^ I
EMSL-Cincinnati, OH. Tests were conducted in reconstituted hard water
(hardness - 180 mg CaCO,/L;'pH = 8.1).
Concentrations used in Test 1 were: 0.03, 0.06, 0.12, 0.25, 0.50,
1.0 mg NaPCP/L.
2 and 3 were: 0.007, 0.022, 0.067, 0.20,
Concentrations used in Tests
0.60 mg NaPCP/L.
Concentrations used in Tests 4
0.30, 0.60 mg NaPCP/L.
through 9 were: 0.0375, 0.075, 0.150,
13.14.1.2 Multilaboratory Precision
13.14.1.2.1 A multilaboratory study was performed by the Aquatic Biology
Branch, EMSL-Cincinnati in 1985e, involving a total of 11 analysts in 10
different laboratories (Neiheisel et. a!., 1988; USEPA, 1988e). Each analyst
performed one-to-three seven-day tests using aliquots of a copper-spiked
effluent sample, for a total of 25 tests. The tests were performed on the
same day in all participating laboratories, using a pre-publication draft of
Method 1002.0. The NOECs and LOECs for these tests were within one
concentration interval which, with a dilution factor of 0.5, is equivalent to
a two-fold range in concentration (Table 18).
192
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TABLE 17. THE DAPHNID, CERIODAPHNIA DUBIA, SEVEN-DAY SURVIVAL AND
REPRODUCTION TEST PRECISION FOR A SINGLE LABORATORY
USING NAPCP AS THE REFERENCE TOXICANT (USEPA, 1991a)
Test Number NOEC (mg/L) IC25 (mg/L) IC50 (mg/L)
19
46A
46B
49
55
56
n
Mean
CV(%)
0.30
0.20
0.20
0.20
0.20
0.10
7
NA
NA
0.3754
0.0938
0.2213
0.2303
0.2306
0.2241
7
0.2157
41.1
0.4508
0.2608
0.2879
0.2912
0.3177
0.2827
7
0.2953
27.9
13.14.1.2.2 A second multilaboratory study of Method 1002.0 (using the first
edition of this manual; USEPA, 1985c), was coordinated by Battelle, Columbus
Division, and involved 11 participating laboratories (Table 19) (DeGraeve et
a]., 1989). All participants used 10% DMW (10% PERRIERฎ'Water) as the culture
and dilution water, and used their own formulation of food for culturing and
testing the Ceriodaphnia dubia. Each laboratory was to conduct at least one
test with each of eight blind samples. Each test consisted of 10 replicates
of one organism each for five toxicant concentrations and a control. Of the
116 tests planned, 91 were successfully initiated, and 70 (77%) met the
survival and reproduction criteria for acceptability of the results (80%
survival and nine young per initial female). If the reproduction criteria of
15 young/female, used in this edition of the method, had been applied to the
results of the interlaboratory study, 22 additional tests would have been
unacceptable. The overall precision (CV) of the test was 27% for the survival
data (7-day LC50s) and 37.5% and 39.0% for the reproduction data (IC50s and
IC25s, respectively).
i
13.14.2 ACCURACY
13.14.2.1 The accuracy of toxicity tests cannot be determined.
193
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TABLE 18. INTERLABORATORY PRECISION FOR THE DAPHNID, CERIODAPHNIA
DUBIA, SURVIVAL AND REPRODUCTION TEST WITH COPPER SPIKED
EFFLUENT (USEPA, 1988e)
Endooints (%
Analyst
3
4
4
5
5
6
6
10
10
11
Test
1
1
2
1
2
1
2
1
2
1
Reproduction
NOEC LOEC
12
6
6
6
12
12
6
6
6
12
25
12
12
12
25
25
12
12
12
25
Effluent)
Survival
NOEC LOEC
25
12
25
12
12
25
25
12
12
25
50
25
50
25
25
50
50
25
25
50
194
-------
TABLE 19. INTERLABORATORY PRECISION DATA FOR THE DAPHNID, CERIODAPHNIA
DUBIA, SUMMARIZED FOR EIGHT REFERENCE TOXICANTS AND EFFLUENTS
(USEPA, 1991a)
Test Material
Sodium chloride
Industrial
Sodium chloride
Pulp and Paper
Potassium dichromate
Pulp and Paper
Potassium dichromate
Industrial
n
Mean
Standard Deviation
Mean IC50
1.34
3.6
0.96
60.0
35.8
70.2
53.2
69.8
CV%
29.9
83.3
57.4
28.3
30.8
7.5
25.9
37.0
8
37.5
23.0
Mean IC25
1.00
3.2
0.09
47.3
23.4
55.7
29.3
67.3
i
i
CV%
34.3
78.1
44.4
27.0
32.7
12.2
46.8
36.7
8
39.0
19.1
195
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SECTION 14
TEST METHOD
GREEN ALGA, SELENASTRUM CAPRICORNUTUH, GROWTH TEST
METHOD 1003.0
14.1 SCOPE AND APPLICATION
14.1.1 This method measures the chronic toxicity of effluents and receiving
water to the freshwater green alga, Selenastrum capn'cornutum, in a four-day
static 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.
*
14.1.2 Detection limits of the toxicity of an effluent or pure substance are
organism dependent.
14.1.3 Brief excursions in 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
degradable or highly volatile toxicants present in the source may not be
detected in the test.
14.1.4 This test method is commonly used in one of two forms: (1) a
definitive test, consisting of a minimum of five effluent concentrations and a
control, and (2) a receiving water test(s), consisting of one or more
receiving water concentrations and a control.
14.1.5 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.
14.2 SUMMARY OF METHOD
14.2.1 A green alga, Selenastrum capricornutum, 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.
14.3 INTERFERENCES
14.3.1 Toxic substances may be introduced by contaminants in dilution water,
glassware, sample hardware, and testing equipment (see Section 5, Facilities,
Equipment, and Supplies).
14.3.2 Adverse effects of high concentrations of suspended and/or dissolved
solids, color, and extremes of pH may mask the presence of toxic substances.
196
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14.3.3 Improper effluent sampling and handling may adversely affect test
results (see Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Tests).
14.3.4 Pathogenic organisms and/or planktivores in the dilution water and
effluent may affect test organism survival and growth, and confound test
results.
14.3.5 Nutrients in the effluent or dilution water may confound test results.
14.4 SAFETY
14.4.1 See Section 3, Safety and Health.
14.5 APPARATUS AND EQUIPMENT
14.5.1 Laboratory Selenastrum capricornutum culture unit. see culturing
methods below and USEPA, 1993b. To test effluent toxicity, sufficient numbers
of log-phase-growth organisms must be available.
14.5.2 Samplers -- automatic sampler, preferably with sample cooling
capability, that can collect a 24-h composite sample of 5 L or more.
14.5.3 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
14.5.4 Environmental chamber, incubator, or equivalent facility -- with
"cool-white" fluorescent illumination (86 ฑ 8.6 fiE/m /s, 400 ฑ 40 ft-c, or
4306 lux) and temperature control (25 ฑ 1ฐC).
14.5.5 Mechanical shaker -- capable of providing orbital motion at the rate
of 100 cycles per minute (cpm).
14.5.6 Light meter -- with a range of 0-200 /*E/m2/s (0-1000 ft-c).
14.5.7 Water purification system -- MILLIPORE MILLI-Qฎ, deionized water or
equivalent (see Section 5, Facilities, Equipment, and Supplies).
14.5.8 Balance -- analytical, capable of accurately weighing 0.00001 g.
14.5.9 Reference weights, class S -- for checking performance of balance.
14.5.10 Volumetric flasks and graduated cylinders -- class A, 10-1000 mL,
borosilicate glass, for culture work and preparation of test solutions.
14.5.11 Volumetric pipets -- class A, 1-100 mL.
14.5.12 Serological pipets -- 1-10 mL, graduated.
14.5.13 Pipet bulbs and fillers -- PROPIPETฎ, or equivalent.
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14.5.14 Wash bottles -- for rinsing small glassware, instrument electrodes,
and probes.
14.5.15 Test chambers -- four (minimum of three) 125 or 250 mL borosilicate,
Erlenmeyer flasks, with foam plugs or stainless steel or Shumadzu closures.
For special glassware cleaning requirements (see Section 5, Facilities,
Equipment, and Supplies).
14.5.16 Culture chambers -- 1-4 L borosilicate, Erlenmeyer flasks.
14.5.17 Thermometers, glass or electronic, laboratory grade -- for measuring
water temperatures.
14.5.18 Bulb-thermograph or electronic-chart type thermometers -- for
continuously recording temperature.
14.5.19 Thermometer, National Bureau of Standards Certified, (see USEPA
Method 170.1, USEPA, 1979b) -- to calibrate laboratory thermometers.
14.5.20 Meters, pH and specific conductivity -- for routine physical and
chemical measurements.
14.5.21 Tissue grinder -- for chlorophyll extraction.
14.5.22 Fluorometer (Optional) -- equipped with chlorophyll detection light
source, filters, and photomultiplier tube (Turner Model 110 or equivalent).
14.5.23 UV-VIS spectrophotometer -- capable of accommodating 1-5 cm cuvettes.
14.5.24 Cuvettes for spectrophotometer -- 1-5 cm light path.
14.5.25 Electronic particle counter (Optional) -- Coulter Counter, Model ZBI,
or equivalent, with mean cell (particle) volume determination.
14.5.26 Microscope -- with 10X, 45X, and 100X objective lenses, 10X ocular
lenses, mechanical stage, substage condenser, and light source (inverted or
conventional microscope).
14.5.27 Counting chamber -- Sedgwick-Rafter, Palmer-Maioney, or
hemocytometer.
14.5.28 Centrifuge -- with swing-out buckets having a capacity of 15-100 ml.
14.5.29 Centrifuge tubes -- 15-100 ml, screw-cap.
14.5.30 Filtering apparatus -- for membrane and/or glass fiber filters.
14.6 REAGENTS AND CONSUMABLE MATERIALS
14.6.1 Sample containers -- for sample shipment and storage (see Section 8,
Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation
for Toxicity Tests).
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14.6.2 Data sheets (one set per test) -- for recording data.
14.6.3 Tape, colored -- for labeling test chambers.
14.6.4 Markers, waterproof -- for marking containers, etc.
14.6.5 Reagents for hardness and alkalinity tests -- see USIEPA Methods 130.2
and 310.1, USEPA, 1979b.
14.6.6 Buffers pH 4, pH 7, and pH 10 (or as per instructions of instrument
manufacturer) for instrument calibration (see USEPA Method 150.1, USEPA,
1979b).
14.6.7 Specific conductivity standards (see USEPA Method 120.1, USEPA,
1979b). :
14.6.8 Standard particles -- such as chicken or turkey fibroblasts or polymer
microspheres, 5.0 ฑ 0.03 urn diameter, 65.4 /*m volume, for calibration of
electronic particle counters (available from Duke Scientific Co., 1135D, San
Antonio Road, Palo Alto, CA 94303).
14.6.9 Membranes and filling solutions for DO probe (see USEPA Method 360.1,
USEPA, 1979b), or reagents -- for modified Winkler analysis.
14.6.10 Laboratory quality control samples and standards -- for calibration
of the above methods.
14.6.11 Reference toxicant solutions -- see Section 4, Quality Assurance.
14.6.12 Reagent water -- defined as distilled or deionized water that does
not contain substances which are toxic to the test organisms (see Section 5,
Facilities, Equipment, and Supplies).
14.6.13 Effluent or receiving water and dilution water -- see Section 7,
Dilution Water; and Section 8, Effluent and Receiving Water Sampling, Sample
Handling, and Sample Preparation for Toxicity Testing.
14.6.14 Acetone -- pesticide-grade or equivalent.
14.6.15 Dilute (10%) hydrochloric acid -- carefully add 10 ml of concentrated
HC1 to 90 ml of MILLI-Qฎ water. :
14.6.16 TEST ORGANISMS, GREEN ALGA, SELENASTRUM CAPRICORNUTUM
14.6.16.1 Selenastrum capn'cornutum, a unicellular coccoid green alga is the
test organism.
14.6.16.2 Algal Culture Medium is prepared as follows:
1. Prepare (five) stock nutrient solutions using reagent grade chemicals as
described in Table 1. Cautionary note: EDTA may affect metal toxicity.
It is recommended that tests be conducted with and without EDTA in the
199
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TABLE 1. NUTRIENT STOCK SOLUTIONS FOR MAINTAINING ALGAL STOCK CULTURES
AND TEST CONTROL CULTURES
STOCK COMPOUND AMOUNT DISSOLVED IN
SOLUTION 500 mL MILLI-Qฎ WATER
1. MACRONUTRIENTS
A. MgCl2.6H20 6.08 g
CaCl,-2H20 2.20 g
NaN03 12.75 g
B. MgS04-7H20 7.35 g
C. K2HP04 0.522 g
D. NaHC03 . 7.50 g
2. MICRONUTRIENTS
H,B03 92.8 mg
MnCl2.4H20 208.0 mg
ZnC12 1.64 mg1
FeCl3.6H20 79.9 nra
CoCl2-6H20 0.714 mg,
Na2MoO,-2H20 3.63 mg,
CuCl2-2H20 0.006 mg4
Na2EDTA.2H20 150.0 mg
Na2Se04 1.196 mg5
ZnCl? - Weigh out 164 mg and dilute to 100 mL. Add 1 mL of this
solution to Stock 2, micronutrients.
CoCl2*6H20 - Weigh out 71.4 mg and dilute to 100 mL. Add 1 mL of
this solution to Stock 2, micronutrients.
NajjMoO^HjjO - Weigh out 36.6 mg and dilute to 10 mL. Add 1 mL
of this solution to Stock 2, micronutrients.
CuCl2-2H20 - 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 2, micronutrients.
Na?Se04 - Weigh out 119.6 mg and dilute to 100 mL. Add 1 mL of this
solution to Stock 2, micronutrients.
200
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culture media if metals are suspected in the effluent or receiving
water.
2. Add 1 ml of each stock solution, in the order listed in Table 1, to
approximately 900 ml of MILLI-Qฎ water. Mix well after the addition of
each solution. Dilute to 1 L, mix well, and adjust the pH to 7.5 ฑ 0.1,
using 0.1N NaOH or HC1, as appropriate. The final concentration of
macronutrients and micronutrients in the culture medium is given in
Table 2.
3. Immediately filter the pH-adjusted medium through a 0.45 /zm pore
diameter membrane at a vacuum of not more than 380 imm (15 in.) mercury,
or at a pressure of not more than one-half atmosphere (8 psi). Wash the
filter with 500 mL deionized water prior to use.
4. If the filtration is carried out with sterile apparatus, filtered medium
can be used immediately, 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 vessels. If a 0.22 ^g
filter is used no sterilization is needed.
5. Unused sterile medium should not be stored more than one week prior to
use, because there may be substantial loss of water by evaporation.
14.6.16.3 Stock Algal Cultures
14.6.16.3.1 See Section 6, Test Organisms, for information on sources of
"starter" cultures of the green alga, Selenastrum capricornutum.
14.6.16.3.2 Upon receipt of the "starter" culture (usually about 10 ml), a
stock culture is initiated by aseptically transferring 1 rnL 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 in a 4-1. flask. The
remainder of the starter culture can be held in reserve for up to six months
in a refrigerator (in the dark) at 4ฐC.
14.6.16.3.3 Maintain the stock cultures at 25 ฑ 1ฐC, under continuous
"Cool-White" fluorescent lighting of 86 ฑ 8.6 /*E/m2/s (400 ฑ 40 ft-c). Shake
continuously at 100 cpm or twice daily by hand.
14.6.16.3.4 Transfer 1 to 2 mL of stock culture weekly to 50 - 100 ml 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. Examine the stock
cultures with a microscope for contaminating microorganisms at each transfer.
14.6.16.3.5 Viable unialgal culture material may be maintained for long
periods of time if placed in a refrigerator at 4ฐC.
14.6.16.4 It is recommended that chronic toxicity tests be performed monthly
with a reference toxicant. Algal cells four to seven days; old are used to
monitor the chronic toxicity (growth) of the reference toxicant to the algal
stock produced by the culture unit (see Section 4, Quality Assurance,
Subsection 4.17).
201 i
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TABLE 2. FINAL CONCENTRATION OF MACRONUTRIENTS AND MICRONUTRIENTS
IN THE CULTURE MEDIUM
MACRONUTRIENT
NaN03
MgCl2-6H20
CaCl2.2H20
MgS04-7H20
K2HPOA
NaHC03
MICRONUTRIENT
H3B03
MnCl2-4H20
ZnCl2
CoCl2-6H20
CuCl2-2H20
Na2Mo04-2H20
FeCT3.6H20
Na2EDTA-2H20
Na2Se04
CONCENTRATION
Ima/D
25.5
12.2
4.41
14.7
1.04
15.0
CONCENTRATION
(ua/Ll
185.0
416.0
3.27
1.43
0.012
7.26
160.0
300.0
2.39
ELEMENT
N
Mg
Ca
S
P
Na
K
C
ELEMENT
B
Mn
Zn
Co
Cu
Mo
Fe
--
Se
CONCENTRATION
lm/L}
4.20
2.90
1.20
1.91
0.186
11.0
0.469
2.14
CONCENTRATION
fua/L)
32.5
115.0
1.57
0.354
0.004
2.88
33.1
0.91
202
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14.6.16.5 Record Keeping ',.
14.6.16.5.1 Records, kept in a bound notebook, include (1) dates culture
media was prepared, (2) source of "starter" cultures, (3) date stock cultures
were started, (4) cell density in stock cultures, and (5) dates and results of
reference toxicant tests performed (see Section 4, Quality Assurance).
14.7 EFFLUENT AND RECEIVING MATER COLLECTION, PRESERVATION, AND STORAGE
14.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests.
14.8 CALIBRATION AND STANDARDIZATION
14.8.1 See Section 4, Quality Assurance.
14.9 QUALITY CONTROL
14.9.1 See Section 4, Quality Assurance.
14.10 TEST PROCEDURES
14.10.1 TEST SOLUTIONS
14.10.1.1 Receiving Waters
14.10.1.1.1 The sampling point is determined by the objectives of the test.
Receiving water toxicity is determined with samples used directly as collected
or after samples are passed through a 60 urn NITEXฎ filter and compared without
dilution against a control. Using four replicate chambers per test, each
containing 100 ml and 400 ml for chemical analyses, would require
approximately 1 L or more of sample for the test.
14.10.1.2 Effluents
14.10.1.2.1 The selection of the effluent test concentrations should be based
on the objectives of the study. A dilution factor of 0.5 is commonly used. A
dilution factor of 0.5 provides precision of ฑ 100%, and testing of
concentrations between 6.25% and 100% effluent using five effluent
concentrations (6.25%, 12.5%, 25%, 50%, and 100%). Improvements in precision
decline rapidly if the dilution factor is increased beyond 0.5 and precision
declines rapidly if a smaller dilution, factor is used, therefore, USEPA
recommends using a > 0.5 dilution factor.
14.10.1.2.2 If the effluent is known or suspected to be highly toxic, a lower
range of effluent concentrations should be used (such as 25%, 12.5%, 6.25%,
3.12%, and 1.56%). If a high rate of mortality is observed during the first
1 to 2 h of the test, additional dilutions should be added at the lower range
of the effluent concentrations.
203
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14.10.1.2.3 The volume of effluent required for the test is 1 to 2 L.
Sufficient test solution (approximately 900 or 1500 ml) is prepared at each
effluent concentration to provide 400 ml additional volume for chemical
analyses at the high, medium, and low test concentrations. There is no daily
renewal of test solution.
14.10.1.2.4 Tests should begin as soon as possible, preferably within 24 h of
sample collection. The maximum holding time following retrieval of the sample
from the sampling device should not exceed 36 h for off-site toxicity tests
unless permission is granted by the permitting authority. In no case should
the sample be used in a test more than 72 h after sample collection (see
Section 8, Effluent and Receiving Water Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests). :
14.10.1.2.5 Just prior to test initiation (approximately 1 h) the temperature
of sufficient quantity of the sample to make test solutions should be adjusted
to the test temperature and maintained at that temperature during the addition
of dilution water.
14.10.1.2.6 The DO of the test solutions should be checked prior to test
initiation. If any of the solutions are supersaturated with oxygen or any
solution has a DO concentration below 4.0 mg/L, all of the solutions and the
control must be gently aerated.
14.10.1.2.7 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 is added per liter of
effluent prior to use in preparing the test dilutions. Thus, all test
treatments and controls will contain at a minimum the concentration of
nutrients in the stock culture medium.
14.10.1.2.8 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.
14.10.1.3 Dilution Water
14.10.1.3.1 Dilution water may be stock culture medium, any uncontaminated
receiving water, a standard synthetic (reconstituted) water, or some other
natural water (see Section 7, Dilution Water). However, if water other than
the stock culture medium is used for dilution water, 1 ml of each stock
nutrient solution should be added per liter of dilution water. Natural waters
used as dilution water must be filtered through a prewashed filter, such as a
6F/A, GF/C, or equivalent filter, that provides 0.45 ^m particle size
retention.
14.10.1.3.2 If the growth of the algae in the test solutions is to be
measured with an electronic particle counter, the effluent and dilution water
must be filtered through a GF/A or GF/C filter, or other filter providing
0.45 urn particle size retention, and checked for "background" particle count
204
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before it is used in the test. Glass-fiber filters generally provide more
rapid filtering rates and greater filtrate volume before plugging.
14.10.1.4 Preparation of Inoculum
14.10.1.4.1 The inoculum is prepared no more than 2 to 3 h prior to the
beginning of the test, using SeTenastrum capricornutum harvested from a four-
to-seven-day stock culture. Each milliliter of inoculum must contain enough
cells to provide an initial cell density of approximately 10,000 cells/ml
(ฑ 10%) 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.
14.10.1.4.2 Estimate the volume of stock culture required to prepare the
inoculum. As an example, if the four-to-seven-day-old stock culture used as
the source of the inoculum has a cell density of 2,000,000 cells/ml, a test
employing 24 flasks, each containing 100 ml of test medium and inoculated with
a total of 1,000,000 cells, would require 24,000,000 cells or 15 ml of stock
solution (24,000,000/2,000,000) to provide sufficient inoculum. It is
advisable to prepare a volume 20% to 50% in excess of the minimum volume
required, to cover accidental loss in transfer and handling.
14.10.1.4.3 Prepare the inoculum as follows:
1. Centrifuge 15 ml of stock culture at 1000 x g for 5 min. This volume
will provide a 50% excess in the number of cells.
2. Decant the supernatant and resuspend the cells in 10 ml of control
medium.
3. Repeat the centrifugation and decantation step, and resuspend the cells
in 10 ml control medium.
4. Mix well and determine the cell density in the algal concentrate. Some
cells will be lost in the concentration process.
5. Determine the density of cells (cells/ml) in the stock culture (for this
example, assume 2,000,000 per ml).
6. Calculate the required volume of stock culture as follows:
i
Volume (ml) of Number test flasks Volume of test 10,000
Stock Culture = to be used. x Solutions/flask x cells/ml
Required Cell density (cells/ml) in the stock culture
= 24 flasks x 100 ml/flask x 10.000 cells/ml
2,000,000 cells/ml ;
= 12.0 ml Stock Culture ,
7. 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.
8. The volume of the algal inoculum should be considered in calculating the
dilution of toxicant in the test flasks. ;
205
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14.10.2 START OF THE TEST
14.10.2.1 Label the test chambers with a marking pen and use the color-coded
tape to identify each treatment and replicate. A minimum of five effluent
concentrations and a control are used for each effluent test. Each treatment
(including the control) should have four (minimum of three) replicates.
14.10.2.2 Randomize the position of the test flasks at the beginning of the
test (see Appendix A). Preparation of a position chart may be helpful.
14.10.2.3 The test begins when the algae are added to the test flasks. Mix
the inoculum well, and add 1 ml to the test solution in each randomly arranged
flask. Make a final check of the cell density in three of the test solutions
at time "zero" (within 2 h of the inoculation).
14.10.2.3.1 Alkalinity, hardness, and conductivity are measured at the
beginning of the test in the high, medium, and low effluent concentrations and
control before they are dispensed to the test chambers and the data recorded
on the data sheet (Figure 1).
Discharger:
Location:
Test Dates:
Analyst:
Effluent Concentration
Parameter
Temperature
DH
Alkalinitv
Hardness
Conductivity
Chlorine
Control
Remarks
Figure 1. Data form for the green alga, Selenastrum capn'cornutum,
growth test. Routine chemical and physical determinations.
14.10.3 LIGHT, PHOTOPERIOD, AND TEMPERATURE
14.10.3.1 Test flasks are incubated under continuous illumination at 86 ฑ
8.6 nE/m/s (400 ฑ 40 ft-c), at 25 ฑ 1ฐC, and should be shaken continuously at
100 cpm on 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 specifications are met at all positions, this need not be
done.
206
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14.10.4 DISSOLVED OXYGEN (DO) CONCENTRATION
14.10.4.1 Because of the continuous illumination of the test flasks, DO
concentration should never be a problem during the test and no aeration will
be required.
14.10.5 OBSERVATIONS DURING THE TEST
14.10.5.1 Routine Chemical and Physical Determinations
14.10.5.1.1 Temperature should be monitored continuously or observed and
recorded daily for at least two locations in the environmental control system
or the samples. Temperature should be checked in a sufficient number of test
vessels at least at the end of the test to determine variability in the
environmental chamber.
14.10.5.1.2 Temperature and pH are measured at the end of each 24-h exposure
period in at least one test flask at each concentration and in the control.
14.10.5.1.3 Record all the measurements on the data sheet (Figure 1).
14.10.5.2 Biological Observations
14.10.5.2.1 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 or two days in the test. It may be desirable, therefore, to
determine the algal growth response daily. Otherwise, biological observations
are not required until the test is terminated and the test solutions are not
renewed during the test period.
14.10.6 TERMINATION OF THE TEST
I
14.10.6.1 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 (c) turbidity (light absorbance).
14.10.6.2 Cell counts
14.10.6.2.1 Automatic Particle Counters
14.10.6.2.1.1 Several types of automatic electronic and optical particle
counters are available for use in the rapid determination of cell density
(cells/ml) and mean cell volume (MCV) in MN /cell. The Coulter Counter is
widely used and is discussed in detail in USEPA (1978b).
14.10.6.2.1.2 If biomass data are desired for algal growth potential
measurements, a Model ZM Coulter Counter is used. However, the instrument
must be calibrated with a reference sample of particles of known volume.
14.10.6.2.1.3 When the Coulter Counter is used, an aliquot (usually 1 mL) of
the test culture is diluted 10X to 20X with a 1% sodium chloride electrolyte
solution, such as ISOTONฎ, to facilitate counting. The resulting dilution is
207
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counted using an aperture tube with a lOO-y^m diameter aperture. 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 direction; 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 pi pet.
3. Place the aliquot in a counting beaker, and add 9 ml (or 19 ml) of
electrolyte solution (such as Coulter ISOTONฎ).
4. Determine the cell density (and MCV, if desired).
14.10.6.2.2 Manual microscope counting method
14.10.6.2.2.1 Cell counts may be determined using a Sedgwick-Rafter,
Palmer-Maioney, hemocytometer, inverted microscope, or similar methods. For
details on microscope counting methods, see APHA (1992) and USEPA (1973).
Whenever feasible, 400 cells per replicate are counted to obtain ฑ 10%
precision at the 95% confidence level. This method has the advantage of
allowing for the direct examination of the condition of the cells.
14.10.6.3 Chlorophyll Content
14.10.6.3.1 Chlorophyll may be estimated 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).
14.10.6.3.2 The in-vivo chlorophyll 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 /j,m 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 pi pet.
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.
14.10.6.3.3 For additional information on chlorophyll measurement methods,
(see APHA, 1992).
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14.10.6.4 Turbidity (Absorbance)
14.10.6.4.1 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 nm. 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.
14.10.6.4.2 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.
i
14.10.6.5 Record the data as indicated in Figure 2.
Discharger:
Location:
Test Dates:
Analyst: _
Concentration
Control
Cone:
Cone:
Cone:
Cone:
Cone:
Cell
1
Density Measurement
Replicate
2
3
4
Treatment
Mean
Comments
Comments:
Figure 2.
Data form for the
growth test, cell
green alga, Selenastrunt capricornutum,
density determinations.
14.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
14.11.1 A summary of test conditions and test acceptability criteria is
presented in Table 3.
14.12 ACCEPTABILITY OF TEST RESULTS
14.12.1 For the test results to be acceptable, the algal cell density in the
control flasks must exceed 1 X 106 cells/ml with EDTA or 2 X 105 cells/ml
without EDTA at the end of the test, and not vary more than 20% among
replicates.
209
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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
GREEN ALGA, SELENASTRUM CAPRICORNUTUM, GROWTH TOXICITY TESTS
WITH EFFLUENTS AND RECEIVING WATERS
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 solutions:
9. Age of test organisms:
9. Initial cell density in
test chambers:
10. No. replicate chambers
per concentration:
11. Shaking rate:
12. Aeration:
13. Dilution water:
Static non-renewal
25 ฑ 1ฐC
"Cool white" fluorescent
lighting
86 ฑ 8.6 /.E/m2/s (400 ฑ 40 ft-c
or 4306 lux)
Continuous i11umination
125 mL or 250 mL
50 mL or 100 mL1
None
4 to 7 days
10,000 cells/mL
4 (minimum of 3)
100 cpm continuous, or twice
daily by hand
None
Algal stock culture medium,
enriched uncontaminated source
of receiving or other natural
water, synthetic water prepared
using MILLIPORE MILLI-Qฎ or
equivalent deionized water and
reagent grade chemicals, or DMW
(see Section 7, Dilution Water)
For tests not continuously shaken use 25 mL in 125 mL flasks and 50 mL
in 250 mL flasks.
210
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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
GREEN ALGA, SELENASTRUM CAPRICORNUTUM, GROWTH TOXICITY TESTS
WITH EFFLUENTS AND RECEIVING WATERS (CONTINUED)
14. Test concentrations:
15. Test dilution factor:
16. Test duration:
17. Endpoint:
18. Test acceptability
criteria:
19. Sampling requirements:
20. Sample volume required:
Effluents: Minimum of 5 and a control
Receiving Water: 100% receiving water
or minimum of 5 and a control
Effluents: > 0.5
Receiving Waters: None or > 0.5
96 h
Growth (cell counts, chlorophyll
fluorescence, absorbance, biomass)
1 X 106 cells/mL with EDTA or 2 X 10s
cells/mL without EDTA in the controls:
Variability of controls should not
exceed 20%
For on-site tests, one sample collected
at test initiation, and used within 24
h of the time it is removed from the
sampling device. For off-site tests,
holding time must not exceed 36 h (see
Section 8, Effluent and Receiving Water
Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests,
Subsection 8.5.4)
1 or 2 L depending on test volume
211
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14.13 DATA ANALYSIS
14.13.1 GENERAL
14.13.1.1 Tabulate and summarize the data. A sample set of algal growth
response data is shown in Table 4.
TABLE 4. GREEN ALGA, SELENASTRUM CAPRICORNUTUM, GROWTH RESPONSE DATA
Toxicant Concentration
Cd/L)
Replicate Control
10
20
40
80
A
B
C
1209
1180
1340
1212
1186
1204
826
628
816
493
416
413
127
147
147
49.3
40.0
44.0
Log10
" 1 U
Trans-
formed
A
B
C
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
Mean(Y,.) 3.094 3.080 2.876 2.643 2.146
1.646
14.13.1.2 The endpoints of toxicity tests using the green alga, Selenastrum
capricornutum, are based on the adverse effects on cell growth (see Section 9,
Chronic Toxicity Test Endpoints and Data Analysis). The EC50, the IC25, and
the IC50 are calculated using the point estimation techniques, and LOEC and
NOEC values for growth are obtained using a hypothesis testing approach such
as Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank Test (Steel,
1959; Miller, 1981). Separate analyses are performed for the estimation of
the LOEC and NOEC endpoints and for the estimation of the EC50, IC25, and
IC50. See the Appendices for examples of the manual computations, and
examples of data input and program output.
14,13.1.3 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 Appendix B. The assistance of a
statistician is recommended for analysts who are not proficient in statistics.
14.13.2 EXAMPLE OF ANALYSIS OF ALGAL GROWTH DATA
14.13.2.1 Formal statistical analysis of the growth data is outlined on the
flowchart in Figure 3. The response used in the statistical analysis is the
number of cells per millilHer per replicate. Separate analyses are performed
for the estimation of the NOEC and LOEC endpoints and for the estimation* of'
the IC25 and IC50 endpoints.
212
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STATISTICAL ANALYSIS OF ALGAL GROWTH TEST
GROWTH RESPONSE DATA
CELLS/ML
POINT ESTIMATION
ENDPOINT ESTIMATE
IC25, IC50
SHAPIRO-WILK-STEST
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETTSTEST
HETEROGENEOUS
VARIANCE
1
NO
r1 .
EQUAL NUMI
REPLJCAT
T-TESTWITH
BONFERRONI
ADJUSTMENT
JEROF
ES?
i YES
DUNNETTS
TEST
YES
EQUAL NUMBER OF
REPLICATES?
STEEL'S MANY-ONE
RANK TEST
i
NO
WII.COXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 3. Flowchart for statistical analysis of the green alga, Selenastrum
capricornutum, growth response data.
213
-------
14.13.2.2 The statistical analysis using hypothesis tests consists of a
parametric test, Dimnett's Procedure, and a nonparametric test, Steel's
Many-one Rank Test. The underlying assumptions of the Dunnett's Procedure,
normality and homogeneity of variance, are formally tested. The test for
normality is the Shapiro-Wilk's Test, and Bartlett's Test is used to test for
homogeneity of variance. If either of these tests fails, the nonparametric
test, Steel's Many-one Rank Tests, is used to determine the NOEC and LOEC
endpoints. If the assumptions of Dunnett's Procedure are met, the endpoints
are determined by the parametric test.
14.13.2.3 Additionally, if unequal numbers of replicates occur among the
concentration levels tested there are parametric and nonparametric alternative
analyses. The parametric analysis is a t test with the Bonferroni adjustment
(see Appendix D). The Wilcoxon Rank Sum Test with the Bonferroni adjustment
is the nonparametric alternative (see Appendix F).
14.13.2.4 Data from an algal growth test with cadmium chloride will be used
to illustrate the statistical analysis. The cell counts were Iog10
transformed in an effort to stabilize the variance for the ANOVA analysis.
The raw data, Iog10 transformed data, mean and standard deviation of the
observations at each concentration including the control are listed in
Table 4. A plot of the Iog10 transformed cell counts for each treatment is
provided in Figure 4.
14.13.2.5 Test for Normality
14.13.2.5.1 The first step of the test for normality is to center the
observations by subtracting the mean of all the observations within a
concentration from each observation in that concentration. The centered
observations are summarized in Table 5.
TABLE 5. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Toxicant Concentration
Cd/L)
Replicate Control
10
20
40
80
A
B '
C
-0.012
-0.022
0.033
0.004
-0.006
0.001
0.041
-0.078
0.036
0.050
-0.024
-0.027
-0.042
0.021
0.021
0.047
-0.044
-0.003
214
-------
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5 oc-piij
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8
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M SSSS
uj uJ2iE
z E>z
^^ ^~ -^ /n
S
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i -i
P Is
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**" o.
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22
03
01
0 S-
i m
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-------
14.13.2.5.2 Calculate the denominator, D, of the test statistic:
D =
Where: X,-
the ith centered observation
X - the overall mean of the centered observations
n = the total number of centered observations.
For this set of data, n = 18
x = (o.ooo) = o.ooo
18
D = 0.0214
14.13.2.5.3 Order the centered observations from smallest to largest:
X<1) is the ith ordered observation. These ordered observations are
listed in Table 6.
TABLE 6. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
X(i)
-0.078
-0.044
-0.042
-0.027
-0.024
-0.022
-0.012
-0.006
-0.003
i
10
11
12
13
14
15
16
17
18
X(i>
0.001
0.004
0.021
0.021
0.033
0.036
0.041
0.047
0.050
14.13.2.5.4 From Table 4, Appendix B, for the number of observations, n,
obtain the coefficients a,, a2, ..., ak where k is n/2 if n is even and
(n-l)/2 if n is odd. For the data in this example, n = 18, k = 9. The a,-
values are listed in Table 7.
216
-------
14.13.2.5.5 Compute the test statistic, W, as follows:
,
D 1=1
The differences x(n"i+1) - X(i) are listed in Table 7.
TABLE 7. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S EXAMPLE
XC i) '
-
1
2
3
4
5
6
7
8
9
0.4886
0.3253
0.2553
0.2027
0.1587
0.1197
0.0837
0.0496
0.0163
0.128
0.091
0.083
0.063
0.057
0.043
0.033
0.010
0.004
X(18)
XC17)
x(16)
X(15)
x<14)
x(13>
X(12)
x(
X(10)
- X(1>
T A
- X(3)
- X(4)
- x(5)
- xฃ
- x(8)
- x(9)
For this set of data:
W =
0.0214
(0.1436)2 = 0.964
14.13.2.5.6 The decision rule for this test is to compare W with the critical
value found in Table 6, Appendix B. If the computed W is less than the
critical value, conclude that the data are not normally distributed. For this
example, the critical value at a significance level of 0.01 and 18
observations (n) is 0.858. Since W = 0.964 is greater than the critical
value, the conclusion of the test is that the data are normally distributed.
14.13.2.6 Test for Homogeneity of Variance
14.13.2.6.1 The test used to examine whether the variation in mean cell count
is the same across all toxicant concentrations including the control, is
Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as
follows:
B =
217
-------
Where: V,- ป degrees of freedom for each toxicant concentration and
control, V,- = (n,- - 1)
p = number of levels of toxicant concentration including the
control
nf - the number of replicates for concentration i
In = loge
i = 1, 2, ..., p, where p is the number of concentrations
including the control
- i=i
14.13.2.6.2 For the data in this example, (see Table 4) all toxicant
concentrations including the control have the same number of replicates
(n{ - 3 for all i). Thus, V, = 2 for all i.
14.13.2.6.3 Bartlett's statistic is therefore:
B = [(12) in (0.0018) -2 112(5-1)] /1. 194
i-l
= [12(-6.3200) - 2(-41.9082)]/1.194
= 7.9764/1.194
= 6.6804
14.13.2.6.4 B is approximately distributed as chi-square with p - 1 degrees
of freedom, when the variances are in fact the same. Therefore, the
appropriate critical value for this test, at a significance level of 0.01 with
five degrees of freedom, is 15.09. Since B = 6.6804 is less than the critical
value of 15.09, conclude that the variances are not different.
218
-------
14.13.2.7 Dunnett's Procedure
14.13.2.7.1 To obtain an estimate of the pooled variance for Dunnett's
Procedure, construct an ANOVA table as described in Table 8.
TABLE 8. ANOVA TABLE
Source df
Between p - 1
Within N - p
Total N - 1
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
S* = SSE!/(p-l)
Sj = SSW/(N-p)
i
Where: p = number of toxicant concentrations including the control
N = total number of observations n1 + n2 ... 4- n
n,- = number of observations in concentration I
i
SSB = 'L,Tl/ni-G2/N Between Sum of Squares;
SST = E TYlj-Gz/N Total Sum of Squares
SSW = SST-SSB Within Sum of Squares
6 = the grand total of all sample observations, G = ฃ TV
TJ = the total of the replicate measurements for
concentration i
,-J = the jth observation for concentration i (represents
the cell count for toxicant concentration i in test
chamber j)
219
-------
14.13.2.7.2 For the data in this example:
= n2 = n3 = n4 = n5 = n6 = 3
18
TI = Y,, + Y12 + Y13 = 9.281
_i ..n ..12 yซ = g>23g
= 2 22 23
= Y3 + Y32 + Y33
' + Y* + Y = 7.928
S
- Y61 + Y62 + Y63
+ Y 3 = 6.438
8.627
7.928
6.438
4.938
6 =
SSB =
T, + T2 + T3 + T4 + T5 + T6 = 46.451
= _i_(374.606) - (46.451)2 = 4.997
3 18
SST =
i=1:7=1
= 124.890 - (46.451T = 5.018
18
SSW = SST-SSB = 5.018 - 4.997 = 0.0210
S2B = SSB/(p-l) = 4.996/(6-l) - 0.9990
SSW/(N-p) = 0.021/(18-6) = 0.0018
14.13.2.7.3 Summarize these calculations in the ANOVA table (Table 9)
TABLE 9. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Total
df
17
Sum of Squares
(SS)
5.017
Mean Square(MS)
(SS/df)
Between
Within
5
12
4.997
0.021
0.999
0.0018
220
-------
1
14.13.2.7.4 To perform the individual comparisons, calculate the t statistic
for each concentration, and control combination as follows:
t -
L ~
+ (1/nฑ)
Where: Y,- = mean cell count for toxicant concentration i
Y1 = mean cell count for the control
SH = square root of the within mean square
n., = number of replicates for the control
n,- = number of replicates for concentration i.
14.13.2.7.5 Table 10 includes the calculated t values for each concentration
and control combination. In this example, comparing the 5 /*g/L concentration
with the control the calculation is as follows:
ta -
(3.094-3.080)
[0.0424^(1/3) +(1/3) ]
TABLE 10. CALCULATED T VALUES
Toxicant Concentration
(M9 Cd/L)
5
10
20
40
80
i
2
3
4
5
6
tf'
0.405
6.300
13.035
27.399
41.850
14.13.2.7.6 Since the purpose of this test is to detect a significant
reduction in mean cell count, a one-sided test is appropriate. The critical
value for this one-sided test is found in Table 5, Appendix C. For an overall
alpha level of 0.05, 12 degrees of freedom for error and five concentrations
(excluding the control) the critical value is 2.50. The mean count for
concentration i is considered significantly less than the mean count for the
control if t, is greater than the critical value. Since t,, t,, t, and t, are
greater than 2.50, the 10, 20, 40 and 80 Mg/L concentrations have
significantly lower mean cell counts than the control. Hence the NOEC and the
LOEC for the test are 5 /j.g/1 and 10 yttg/L, respectively.
221 '
-------
14.13.2.7.7 To quantify the sensitivity of the test, the minimum significant
difference (MSD) that can be statistically detected may be calculated.
MSD = d S^CL/n^ + (1/12)
Where: d = the critical value for Dunnett's Procedure
Sw - the square root of the within mean square
n - the common number of replicates at each concentration
(this assumes equal replication at each concentration)
n! - the number of replicates in the control.
14.13.2.7.8 In this example:
MSD = 2. 50 (0.0424) Vd/3)+ (1/3)
= 2.50 (0.0424)(0.8165)
= 0.086
14.13.2.7.9 The MSD (0.086) is in transformed units. An approximate MSD in
terms of cell count per 100 ml may be calculated via the following
conversion.
1. Subtract the MSD from the transformed control mean.
3.094 - 0.086 = 3.008
2. Obtain the untransformed values for the control mean and the difference
calculated in 1.
10(3.094>
10C3.008)
3. The untransformed MSD (MSD ) is determined by subtracting the
untransformed values from 2.
MSUU = 1241.6 - 1018.6 = 223
14.13.2.7.10 Therefore, for this set of data, the minimum difference in mean
cell count between the control and any toxicant concentration that can be
detected as statistically significant is 223.
14.13.2.7.11 This represents a decrease in growth of 18% from the control.
14.13.2.8 Calculation of the ICp
222
-------
1
14.13.2.8.1 The growth data in Table 4 are utilized in this example.
Table 11 contains the means for each toxicant concentration. As can be seen,
the observed means are monotonically non-increasing with respect to
concentration. Therefore, it is not necessary to smooth the means prior to
calculating the ICp. See Figure 5 for a plot of the response curve.
TABLE 11. ALGAL MEAN GROWTH RESPONSE AFTER SMOOTHING
Toxicant
Cone.
(/*ง Cd/L)
Control
5
10
20
40
80
i
1
2
3
4
5
6
Response
means,Y-
(cells/mL)
1243
1201
757
441
140
44
Smoothed
mean, M-
( cells/ml)
1243
1201
757
441
140
44
14.13.2.8.2 An IC25 and IC50 can be estimated using the Linear Interpolation
Method (Appendix M). A 25% reduction in cell count, compared to the controls
would result in a mean count of 932 cells, where M^l-p/100) = 1243(1-25/100)
A 50% reduction in cell count, compared to the controls, would result in a
mean count of 622 cells. Examining the means and their associated
concentrations (Table 11), the response, 932 cells, is bracketed by C, = 5 Mg
Cd/L and C3 = 10 /*g Cd/L. The response, 622 cells, is bracketed by C, = 10 aq
Cd/L and C4 = 20 //g Cd/L. 3
14.13.2.8.3 Using the equation from section 4.2 of Appendix M, the estimate
of the IC25 is calculated as follows:
ICp =
IC25 = 5+[1243 (1-25/100)-1201] (10-5)
(757-1201)
= 8 /*g Cd/L.
14.13.2.8.4 The IC50 estimate is 14 /^g Cd/L:
IC25 = 6.25+[28.75(1-25/100)-28.75]
(9.40-28.75)
IC50 = 10+ [1243 (1-50/100) -757]
__
(441-757)
14 Mg Cd/L.
223
-------
LU
I
LU
0
a. LU
LU X
CE f=
Q LU
II
t-J
o
S-
cn
o
o
Q.
fO
O
s
TO
rt
O
LU
O
O
o
o
ง
-o
88888888ฐ^
CM
o> oo
0)
s-
cn
OJ
O)
a
5-
0)
00
-a
lNnO0113ONV3W
S-
<4-
O
O -f->
ol -a
IT)
CD
s_
224
-------
1
I
14.13.2.8.5 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC25 was 8.0227 /*g Cd/L. The
empirical 95% confidence interval for the true mean was 6.4087 ปg Cd/L and
10.0313 /zg Cd/L. The ICPIN computer program output for the IC25 for this data
set is shown in Figure 6.
.
14.13.2.8.6 When the ICPIN program was used to analyze this set of data,
requesting 80 resamples, the estimate of the IC50 was 14.2774 /*g Cd/L. The
empirical 95% confidence interval for the true mean was 9.7456 /ig Cd/L and
18.5413 ng Cd/L. The computer program output for the IC50 for this data set
is shown in Figure 7.
14.13.3 BIOSTIMULATION
14.13.3.1 Where the growth response in effluent (or surface water) exceeds
growth in the control flasks, the percent stimulation, S(%), is 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.,
1980)i
T-C
x 100
Where: T = Mean effluent or surface water response
C = Mean control response
14.14 PRECISION AND ACCURACY
14.14.1 PRECISION
14.14.1.1 Single-Laboratory Precison
14.14.1.1.1 Data from repetitive 96-h toxicity tests conducted with cadmium
chloride as the reference toxicant, using medium containing EDTA, are shown in
Table 12. The precision (CV) of the 10 EC50s was 10.2%.
i
14.14.1.2 MultiLaboratory Precision
14.14.1.2.1 Data on the multilaboratory precision of this test are not yet
available.
14.14.2 ACCURACY
14.14.2.1 The accuracy of toxicity tests cannot be determined.
225
-------
Cone. ID
Cone. Tested
Response
Response
Response
1
2
3
1
0
1209
1180
1340
2
5
1212
1186
1204
3
10
826
628
816
4
20
493
416
413
5
40
127
147
147
6
80
49.3
40.0
44.0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Cadmium
Test Start Date: Example Test Ending Date:
Test Species: Selenastrum capricornutum
Test Duration: 96 h
DATA FILE: scmanual.icp
OUTPUT FILE: scmanual.i25
Cone.
ID
i
2
3
4
5
6
Number
Replicates
3
3
3
3
3
3
Concentration
ug/i
0.000
5.000
10.000
20.000
40.000
80.000
Response
Means
1243.000
1200.667
756.667
440.667
140.333
44.433
Std.
Dev.
85.247
13.317
111.541
45.347
11.547
4.665
Pooled
Response Means
1243.000
1200.667
756.667
440.667
140.333
44.433
The Linear Interpolation Estimate: 8.0227 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Expanded Confidence Limits:
Resampling time in Seconds:
8.1627 Standard Deviation:
Lower: 7.2541 Upper:
Lower: 6.4087 Upper: I?:0,313
1.65 Random. Seed: -1575623987
ฐ-4733
Figure 6. ICPIN program output for the IC25.
226
-------
1
Cone. ID
Cone. Tested
Response
Response
Response
1
2
3
1
0
1209
1180
1340
2
5
1212
1186
1204
3
10
826
628
816
4
20
493
416
413
5
40
127
147
147
6
80
49.3
40.0
44.0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Cadmium
Test Start Date: Example Test Ending Date:
Test Species: Selenastrum capricornutum
Test Duration: 96 h
DATA FILE: scmanual.icp
OUTPUT FILE: scmanual.iBO
Cone.
ID
1
2
3
4
5
6
Number
Replicates
3
3
3
3
3
3
Concentration
ug/1
0.000
5.000
10.000
20.000
40.000
80.000
Response
Means
1243.000
1200.667
756.667
440.667
140.333
44.433
Std.
Dev.
85.247
13.317
111.541
45.347
11.547
4.665
Pooled
Response Means
1243.000
1200.667
756.667
440.667
140.333
44.433
The Linear Interpolation Estimate: 14.2774 Entered P Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean: 14.2057 Standard Deviation: 1.1926
Original Confidence Limits: Lower: 12.1194 Upper: 16.3078
Expanded Confidence Limits: Lower: 9.7456 Upper: I 18.5413
Resampling time in Seconds: 1.65 Random Seed: -1751550803
Figure 7. ICPIN program output for the IC50.
227
-------
TABLE 12. SINGLE LABORATORY PRECISION OF THE GREEN ALGA, SELENASTRUM
CAPRICORNUTUM, 96-H TOXICITY TESTS, USING THE REFERENCE
TOXICANT CADMIUM CHLORIDE (USEPA, 1991a)
Test Number
1
2
3
4
5
6
7
8
9
10
n
Mean
CV (%)
EC50 (mg/L)
2.3
2.4
2.3
2.8
2.6
2.1
2.1
2.1
2.6
2.4
10.0
2.37
10.2
228
-------
1
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245
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APPENDICES
Page
A. Independence, Randomization, and Outliers . . 248
1. Statistical Independence 248
y/\o
2. Randomization "ฐ
3. Outliers 253
B. Validating Normality and Homogeneity of Variance Assumptions ... 254
1. Introduction 254
2. Tests for Normal Distribution of Data 254
3. Test for Homogeneity of Variance 266
4. Transformations of the Data 268
C. Dunnett's Procedure 271
1. Manual Calculations 271
2. Computer Calculations 277
D. T test with the Bonferroni Adjustment 283
E. Steel's Many-one Rank Test 289
F. Wilcoxon Rank Sum Test 293
G. Fisher's Exact Test with the Bonferroni Adjustment 299
H. Single Concentration Toxicity Test - Comparison of Control with
100% Effluent or Receiving Water 308
I. Probit Analysis ....-.' 312
J. Spearman-Karber Method 315
K. Trimmed Spearman-Karber Method 32ฐ
L. Graphical Method 325
M. Linear Interpolation Method 329
1. General Procedure 329
2. Data Summary and Plots 329
246
-------
APPENDICES (CONTINUED)
Page
3. Monotonicity '....' 32g
4. Linear Interpolation Method ! 330
5. Confidence Intervals 331
6. Manual Calculations 331
7. Computer Calculations , . 335
i
Cited References i 340
247
-------
APPENDIX A
INDEPENDENCE, RANDOMIZATION, AND OUTLIERS
1. STATISTICAL INDEPENDENCE
1 1 Dunnett's Procedure and the t test with Bonferroni's adjustment are
parametric procedures based on the assumptions that (1) the observations
within treatments are independent and normally distributed, and (2) that the
variance of the observations is homogeneous across all toxicant concentrations
and the control. Of the three possible departures from the assumptions, non-
normality, heterogeneity of variance, and lack of independence those caused
by lack of independence are the most difficult to resolve (see Scheffe, 1959).
For toxicity data, statistical independence means that given knowledge of the
true mean for a given concentration or control, knowledge of the error in any
one actual observation would provide no information about the error in any
other observation. Lack of independence is difficult to assess and difficult
to test for statistically. It may also have serious effects on the true alpha
or beta level. Therefore, it is of utmost importance to be aware of the need
for statistical independence between observations and to be constantly
vigilant in avoiding any patterned experimental procedure that might
compromise independence. One of the best ways to help ensure independence is
to follow proper randomization procedures throughout the test.
2. RANDOMIZATION
2 1 Randomization of the distribution of test organisms among test chambers
and the arrangement of treatments and replicate chambers is an important part
of conducting a valid test. The purpose of randomization is to avoid
situations where test organisms are placed serially into test chambers, or
where all replicates for a test concentration are located adjacent to one
another, which could introduce bias into the test results.
2 2 An example of randomization of the distribution of test organisms among
test chambers, and an example of randomization of arrangement of treatments
and replicate chambers are described using the Fathead Minnow Larval Survival
and Growth test. For the purpose of the example, the test design is as
follows- five effluent concentrations are tested in addition to the control.
The effluent concentrations are as follows: 6.25%, 12.5%, 25.0%, 50.0%, and
100.0%. There are four replicate chambers per treatment. Each replicate
chamber contains ten fish.
2.3 RANDOMIZATION OF FISH TO REPLICATE CHAMBERS EXAMPLE
2 3.1 Consider first the random assignment of the fish to the replicate
chambers The first step is to label each of the replicate chambers with the
control or effluent concentration and the replicate number. The next step is
to assign each replicate chamber four double-digit numbers. An example of
this assignment is provided in Table A.I. Note that the double digits 00 and
97 through 99 were not used.
248 '
-------
TABLE A
Assigned
01,
02,
03,
04,
05,
06,
07,
08,
09,
10,
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40,
41,
42,
43,
44,
45,
46,
47,
48,
.1.
RANDOM ASSIGNMENT OF FISH TO REPLICATE CHAMBERS EXAMPLE
ASSIGNED NUMBERS FOR EACH REPLICATE CHAMBER
Numbers
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
6.
6.
6.
6.
12
12
12
12
25
25
25
25
50
50
50
50
100
100
100
100
25%
25%
25%
25%
.5%
.5%
.5%
.5%
.0%
.0%
.0%
.0%
.0%
.0%
.0%
.0%
.0%
.0%
.0%
.0%
Replicate Chamber
Control ,
Control ,
Control ,
Control ,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
2.3.2 The random numbers used to carry out the random assignment of fish to
replicate chambers are provided in Table A.2. The third step is to choose a
starting position in Table A.2, and read the first double digit number. The
first number read identifies the replicate chamber for the first fish taken
from the tank. For the example, the first entry in row 2 was chosen as the
starting position. The first number in this row is 37. According to
Table A.I, this number corresponds to replicate chamber 1 of the 25.0%
effluent concentration. Thus, the first fish taken from the tank is to be
placed in replicate chamber 1 of the 25.0% effluent concentration.
2.3.3 The next step is to read the double digit number to the right of the
first one. The second number identifies the replicate chamber for the second
fish taken from the tank. Continuing the example, the second number read in
row 2 of Table A.2 is 54. According to Table A.I, this number corresponds to
replicate chamber 2 of the 6.25% effluent concentration. Thus, the second
fish taken from the tank is to be placed in replicate chamber 2 of the 6.25%
effluent concentration.
249
-------
TABLE A.2. TABLE OF RANDOM NUMBERS (Dixon and Massey, 1983)
10 09 73
37 54 20
08 42 26
99 01 90
12 80 79
66 06 57
31 06 01
85 26 97
63 57 33
73 79 64
98 52 01
11 80 50
83 45 29
88 68 54
99 59 46
65 48 11
80 12 43
74 35 09
69 91 62
09 89 32
91 49 91
80 33 69
44 10 48
12 55 07
63 60 64
61 19 69
15 47 44
94 55 72
42 48 11
23 52 37
04 49 35
00 54 99
35 96 31
59 80 80
46 05 88
32 17 90
69 23 46
19 56 54
45 15 51
94 86 43
98 08 62
33 18 51
80 95 10
79 75 24
18 63 33
74 02 94
54 17 84
11 66 44
48 32 47
69 07 49
25 33
48 05
89 53
25 29
99 70
47 17
08 05
76 02
21 35
57 53
77 67
54 31
96 34
02 00
73 48
76 74
56 35
98 17
68 03
05 05
45 23
45 98
19 49
37 42
93 29
04 46
52 66
85 73
62 13
83 17
24 94
76 54
53 07
83 91
52 36
05 97
14 06
14 30
49 38
19 94
48 26
62 32
04 06
91 40
25 37
39 02
56 11
98 83
79 28
41 38
76 52 01
64 89 47
19 64 50
09 37 67
80 15 73
34 07 27
45 57 18
02 05 16
05 32 54
03 52 96
14 90 56
39 80 82
06 28 89
86 50 75
87 51 76
17 46 85
17 72 70
77 40 27
66 25 22
14 22 56
68 47 92
26 94 03
85 15 74
11 10 00
16 50 53
26 45 74
95 27 07
67 89 75
97 34 40
73 20 88
75 24 63
64 05 18
26 89 80
45 42 72
01 39 09
87 37 92
20 11 74
01 75 87
19 47 60
36 16 81
45 24 02
41 94 15
96 38 27
71 96 12
98 14 50
77 55 73
80 99 33
52 07 98
31 24 96
87 63 79
35 86
42 96
93 03
07 15
61 47
68 50
24 06
56 92
70 48
47 78
86 07
77 32
80 83
84 01
49 69
09 50
80 15
72 14
91 48
85 14
76 86
68 58
79 54
20 40
44 84
77 74
99 53
43 87
87 21
98 37
38 24
81 59
93 45
68 42
22 86
52 41
52 04
53 79
72 46
08 51
84 04
09 49
07 74
82 96
65 71
22 70
71 43
48 27
47 10
19 76
34
24
23
38
64
36
35
68
90
35
22
50
13
36
91
58
45
43
36
46
46
70
32
12
40
51
59
54
16
68
45
96
33
83
77
05
15
40
43
34
44
89
20
69
31
97
05
59
02
35
67 35
80 52
20 90
31 13
03 23
69 73
30 34
66 57
55 35
80 83
-10 94
72 56
74 67
76 66
82 60
04 77
31 82
23 60
93 68
42 75
16 28
29 73
97 92
86 07
21 95
92 43
36 78
62 24
86 84
93 59
86 25
11 96
35 13
60 94
28 14
56 70
95 66
41 92
66 79
88 88
99 90
43 54
15 12
86 10
01 02
79 01
33 51
38 17
29 53
58 40
43 76
40 37
25 60
11 65
66 53
61 70
26 14
48 18
75 48
42 82
05 58
82 48
00 78
79 51
89 28
69 74
23 74
02 10
72 03
67 88
35 54
41 35
65 75
46 97
25 63
37 29
38 48
44 31
87 67
14 16
10 25
38 96
54 62
97 00
40 77
70 07
00 00
15 85
45 43
15 53
88 96
85 81
33 87
25 91
46 74
71 19
29 69
15 39
68 70
44 01
80 95
20 63
15 95
88 67
98 95
65 81
86 79
73 05
28 46
60 93
60 97
29 40
18 47
90 36
93 78
73 03
21 11
45 52
76 62
96 29
94 75
53 14
57 60
96 64
43 65
65 39
82 39
91 19
03 07
26 25
61 96
54 69
77 97
13 02
93 91
86 74
18 74
66 67
59 04
01 54
39 09
88 69
25 01
74 85
05 45
52 52
56 12
09 97
32 30
10 51
90 91
61 04
33 47
67 43
11 68
33 98
90 74
38 52
82 87
52 03
09 34
52 42
54 06
47 64
56 13
95 71
57 82
16 42
11 39
77 88
08 99
03 33
04 08
48 94
17 70
45 95
61 01
04 25
11 20
22 96
27 93
28 23
45 00
12 48
08 36
31 71
39 24
43 68
79 00
03 54
47 34
54 19
62 52
22 05
56 14
75 80
71 92
33 34
75 75
82 16
17
02
64
97
77
85
39
47
09
44
33
01
10
93
68
86
53
37
90
.22
23
40
81
39
82
93
18
92
59
63
35
91
24
92
47
57
23
06
33
56
07
94
98
39
27
21
55
40
46
15
39 29
00 82
35 08
04 43
12 27
11 19
23 40
18 62
83 49
35 27
50 50
52 77
68 71
29 60
23 47
40 21
14 38
96 28
94 40
54 38
37 08
42 05
22 22
28 70
07 20
42 58
33 21
92 92
25 70
05 52
65 33
23 28
90 10
78 56
70 61
85 39
97 11
84 96
20 82
05 01
35 44
37 54
94 62
00 38
77 93
80 81
36 04
88 46
15 02
01 84
27 49 45
29 16 65
03 36 06
62 76 59
17 68 33
92 91 70
30 97 32
38 85 79
12 56 24
38 84 35
07 39 98
56 78 51
17 78 17
91 10 62
83 41 13
81 65 44
55 37 63
60 26 55
05 64 18
21 45 98
92 00 48
08 23 41
20 64 13
72 58 15
73 17 90
26 05 27
15 94 66
74 59 73
14 66 70
28 25 62
71 24 72
72 95 29
33 93 33
52 01 06
74 29 41
41 18 38
89 63 38
28 52 07
66 95 41
45 11 76
13 18 80
87 30 43
46 11 71
75 95 79
89 19 36
45 17 48
09 03 24
12 33 56
00 99 94
87 69 38
250
-------
2.3.4 Continue in this fashion until all the fish have been randomly assigned
to a replicate chamber. In order to fill each replicate chamber with ten
fish, the assigned numbers will be used more than once. If a number is read
from the table that was not assigned to a replicate chamber, then ignore it
and continue to the next number. If a replicate chamber becomes filled and a
number is read from the table that corresponds to it, then ignore that value
and continue to the next number. The first ten random assignments of fish to
replicate chambers for the example are summarized in Table A.3.
TABLE A.3. EXAMPLE OF RANDOM ASSIGNMENT OF FIRST TEN FISH TO REPLICATE
CHAMBERS
Fish
Assignment
First
Second
Third
Fourth
Fifth
Sixth
Seventh
Eighth
Ninth
Tenth
fish
fish
fish
fish
fish
fish
fish
fish
fish
fish
taken
taken
taken
taken
taken
taken
taken
taken
taken
taken
from
from
from
from
from
from
from
from
from
from
tank
tank
tank
tank
tank
tank
tank
tank
tank
tank
25.
0%
6.25%
50.
100
0%
effluent,
effluent,
effluent
.0% effluent,
6.25%
25.
50.
100
50.
100
0%
0%
.0%
0%
.0%
effluent,
effluent,
effluent,
effluent,
effluent,
effluent,
repl
repl
repl
repl
repl
repl
repl
repl
repl
repl
icate
icate
icate
icate
icate
icate
icate
icate
icate
icate
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
1
2
4
4
1
4
1
3
2
4
2.3.5 Four double-digit numbers were assigned to each replicate chamber
(instead of one, two, or three double-digit numbers) in order to make
efficient use of the random number table (Table A.2). To illustrate, consider
the assignment of only one double-digit number to each replicate chamber: the
first column of assigned numbers in Table A.I. Whenever the numbers 00 and 25
through 99 are read from Table A.2, they will be disregarded and the next
number will be read.
2.4 RANDOMIZATION OF REPLICATE CHAMBERS TO POSITIONS EXAMPLE
2.4.1 Next consider the random assignment of the 24 replicate chambers to
positions within the water bath (or equivalent). Assume that the replicate
chambers are to be positioned in a four row by six column rectangular array.
The first step is to label the positions in the water bath. Table A.4
provides an example layout.
2.4.2 The second step is to assign each of the 24 positions four double-digit
numbers. An example of this assignment is provided in Table A.5. Note that
the double digits 00 and 97 through 99 were not used.
2.4.3 The random numbers used to carry out the random assignment of replicate
chambers to positions are provided in Table A.2. The third step is to choose
a starting position in Table A.2, and read the first double-digit number. The
251
-------
TABLE A.4. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS: EXAMPLE
LABELLING THE POSITIONS WITHIN THE WATER BATH
1
7
13
19
2
8
14
20
3
9
15
21
4
10
16
22
5
11
17
23
6
12
18
24
TABLE A.5. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS: EXAMPLE
ASSIGNED NUMBERS FOR EACH POSITION
Assigned Numbers
01, 25, 49, 73
02, 26, 50, 74
03, 27, 51, 75
04, 28, 52, 76
05, 29, 53, 77
06, 30, 54, 78
07, 31, 55, 79
08, 32, 56, 80
09, 33, 57, 81
10, 34, 58, 82
11, 35, 59, 83
12, 36, 60, 84
13, 37, 61, 85
14, 38, 62, 86
15, 39, 63, 87
16, 40, 64, 88
17, 41, 65, 89
18, 42, 66, 90
19, 43, 67, 91
20, 44, 68, 92
21, 45, 69, 93
22, 46, 70, 94
23, 47, 71, 95
24, 48, 72, 96
Position
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
first number read identifies the position for the first replicate chamber of
the control. For the example, the first entry in row 10 of Table A.2 was
chosen as the starting position. The first number in this row was 73.
According to Table A.5, this number corresponds to position 1. Thus, the
first replicate chamber for the control will be placed in position 1.
252
-------
2.4.4 The next step is to read the double-digit number to the right of the
first one. The second number identifies the position for the second replicate
chamber of the control. Continuing the example, the second number read in row
10 of Table A.2 is 79. According to Table A.5, this number corresponds to
position 7. Thus, the second replicate chamber for the control will be placed
in position 7.
2.4.5 Continue in this fashion until all the replicate chambers have been
assigned to a position. The first four numbers read will identify the
positions for the control replicate chambers, the second four numbers read
will identify the positions for the lowest effluent concentration replicate
chambers, and so on. If a number is read from the table that was not assigned
to a position, then ignore that value and continue to the next number. If a
number is repeated in Table A.2, then ignore the repeats and continue to the
next number. The complete randomization of replicate chambers to positions
for the example is displayed in Table A.6. !
TABLE A.6. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS: EXAMPLE
ASSIGNMENT OF ALL 24 POSITIONS
Control
Control
100.0%
50.0%
100.0%
12.5%
50.0%
50.0%
6.25%
Control
100.0%
25.0%
6.25%
25.0%
Control
50.0%
6.25%
12.5%
100.0%
12.5% .
12.5%
25.0%
25.0%
6.25%
2.4.6 Four double-digit numbers were assigned to each position (instead of
one, two, or three) in order to make efficient use of the random number table
(Table A.2). To illustrate, consider the assignment of only one double-digit
number to each position: the first column of assigned numbers in Table A.5.
Whenever the numbers 00 and 25 through 99 are read from Table A.2, they will
be disregarded and the next number will be read.
3. OUTLIERS
I
3.1 An outlier is an inconsistent or questionable data point that appears
unrepresentative of the general trend exhibited by the majority of the data.
Outliers may be detected by tabulation of the data, plotting, and by an
analysis of the residuals. An explanation should be sought for any
questionable data points. Without an explanation, data points should be
discarded only with extreme caution. If there is no explanation, the analysis
should be performed both with and without the outlier, and the results of both
analyses should be reported.
3.2 Gentleman-Wilk's A statistic gives a test for the condition that the
extreme observation may be considered an outlier. For a discussion of this,
and other techniques for evaluating outliers, see Draper and John (1981).
253
-------
APPENDIX B
VALIDATING NORMALITY AND HOMOGENEITY OF VARIANCE ASSUMPTIONS
1. INTRODUCTION
1.1 Dunnett's Procedure and the t test with Bonferroni's adjustment are
parametric procedures based on the assumptions that the observations within
treatments 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 these tests, 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 nonparametric procedure such as Steel's Many-one Rank
Test may be more appropriate. However, the decision on whether to use
parametric or nonparametric tests may be a judgment call, and a statistician
should be consulted in selecting the analysis.
2. TEST FOR NORMAL DISTRIBUTION OF DATA
2.1 SHAPIRO-WILK'S TEST
2.1.1 One formal test for normality is the Shapiro-Wilk's Test (Conover,
1980). The test statistic is obtained by dividing the square of an
appropriate linear combination of the sample order statistics by the usual
symmetric estimate of variance. The calculated W must be greater than zero
and less than or equal to one. This test is recommended for a sample size of
50 or less. If the sample size is greater than 50, the Kolmogorov "D"
statistic (Stephens, 1974) is recommended. An example of the Shapiro-Wilk's
test is provided below.
2.2 The example uses growth data from the Fathead Minnow Larval Survival and
Growth Test. The same data are used in the discussion of the homogeneity of
variance determination in Paragraph 3 and Dunnett's Procedure in Appendix C.
The data, the mean and variance of the observations at each concentration,
including the control, are listed in Table B.I.
2.3 The first step of the test for normality is to center the observations by
subtracting the mean of all the observations within a concentration from each
observation in that concentration. The centered observations are listed in
Table B.2.
2.4 Calculate the denominator, D, of the test statistic:
A _ ,
D = ฃ (Xd-X)2
Where: X,- - the centered observations and X is the overall mean of
the centered observations. For this set of data, X = 0,
and D = 0.0412.
254
-------
TABLE B.I. FATHEAD LARVAL, PIMEPHALES PROMELAS, LARVAL GROWTH DATA
(WEIGHT IN MG) FOR THE SHAPIRO-MILK'S TEST
NaPCP Concentration (ua/L)
Replicate
A
B
C
D
Mean(Y,-)
Si
i
Control
0.711
0.662
0.718
0.767
0.714
0.0018
.1
32
0.646
0.626
0.723
0.700
0.674
0.0020
2
64
0.669
0.669
0.694
0.676
0.677
0.0001
3
128
0.629
0.680
0.513
0.672
0.624
0.0059
4
256
0.650
0.558
0.606
0.508
0.580
0.0037
5
TABLE B.2. EXAMPLE OF SHAPIRO-WILK'S TEST: CENTERED OBSERVATIONS
NaPCP Concentration
Replicate
A
B
C
D
Control
-0.003
-0.052
0.004
0.053
32
-0.028
-0.048
0.049
0.026
64
-0.008
-0.008
0.017
-0.001
128
0.005
0.056
-0.111
0.048
(ud/L)
256
0.070
-0.022
0.026
-0.072
2.5 Order the centered observations from smallest to largest.
X(1)
-------
TABLE B.3. EXAMPLE OF THE SHAPIRO-MILK'S TEST: ORDERED OBSERVATIONS
1
2
3
4
5
6
7
8
9
10
-0.111
-0.072
-0.052
-0.048
-0.028
-0.022
-0.008
-0.008
-0.003
-0.001
11
12
13
14
15
16
17
18
19
20
0.004
0.005
0.017
0.026
0.026
0.048
0.049
0.053
0.056
0.070
W=
D 1-1 1
2.7 Compute the test statistic, W, as follows:
The differences, X
Yo")
A ,
are listed in Table B.5.
2.8 The decision rule for this test is to compare the critical value from
Table B.6 to the computed W. If the computed value is less than the critical
value, conclude that the data are not normally distributed. For this example,
the critical value at a significance level of 0.01 and 20 observations (n) is
0.868. The calculated value, 0.959, is not less than the critical value.
Therefore, conclude 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 Shapiro-Milk's Test for normality.
2.10 KOLMOGOROV "D" TEST
The
2.10.1 A formal two-sided test for normality is the Kolmogorov "D" Test.
test statistic is calculated by obtaining the difference between the
cumulative distribution function estimated from the data and the standard
normal cumulative distribution function for each standardized observation.
This test is recommended for a sample size greater than 50. If the sample
size is less than or equal to 50, then the Shapiro Milk's Test is recommended.
An example of the Kolmogorov "D11- test is provided below.
2.10.2 The example uses reproduction data from the daphnid, Ceriodaphm'a
dubia, Survival and Reproduction Test. The observed data and the mean of
256
-------
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (Conover, 1980)
V
iV
1
2
3
4
5
2
0.7071
.
3
0.7071
0.0000
4
0.6872
0.1667
5
Number
6
0.6646 0
0.2413 0
0.0000 0
-
-
.
.
-
-
-
-
-
of Observations
.6431
.2806
.0875
7
0.6233
0.3031
0.1401
0.0000
8
0.6052
0.3164
0.1743
0.0561
"
<>
0.5888
0.3244
0.1976
0.0947
0.0000
10
0.5739
0.3291
0.2141
0.1224
0.0399
\ Number of Observations
f\ป
1
2
3
4
5
6
7
8
9
10
\
A-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
11
0.5601
0.3315
0.2260
0.1429
0.0695
0.0000
.
.
.
-
21
0.4643
0.3185
0.2578
0.2119
0.1736
0.1399
0.1092
0.0804
0.0530
0.0263
0.0000
-
-
.
-
12
0.5475
0.3325
0.2347
0.1586
0.0922
0.0303
-
-
-
22
0.4590 '
0.3156
0.2571
0.2131
0.1764
0.1443
0.1150
0.0878
0.0618
0.0368
0.0122
-
.
-
-
13
0.5359
0.3325
0.2412
0.1707
0.1099
0.0539
0.0000
-
.
-
23
0.4542
0.3126
0.2563
0.2139
0.1787
0.1480
0.1201
0.0941
0.0696
0.0459
0.0228
0.0000
-
-
-
14
0.5251
0.3318
0.2460
0.1802
0.1240
0.0727
0.0240
-
-
-
24
0.4493
0.3098
0.2554
0.2145
0.1807
0.1512
0.1245
0.0997
0.0764
0.0539
0.0321
0.0107
-
-
-.
15
0.5150
0.3306
0.2495
0.1878
0.1353
0.0880
0.0433
0.0000
-
-
Number
25
0.4450
0.3069
0.2543
0.2148
0.1822
0.1539
0.1283
0.1046
0.0923
0.0610
0.0403
0.0200
0.0000
-
-
16
0.5056
0.3209
0.2521
0.1939
0.1447
0.1005
0.0593
0.0196
-
17
0.4968
0.3273
0.2540
0.1988
0.1524
0.1109
0.0725
0.0359
0.0000
'
18
0.4886
0.3253
0.2553
0.2027
0.1587
0.1197
0.0837
0.0496
0.0163
~
19
0.4808
0.3232
0.2561
0.2059
0.1641
0.1271
0.0932
ID. 0612
0.0303
0.0000
20
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
0.0711
0.0422
0.0140
of Observations I
26
0.4407
0.3043
0.2533
0.2151
0.1836
0.1563
0.1316
0.1089
0.0876
0.0672
0.0476
0.0284
0.0094
-
-
27
0.4366
0.3018
0.2522
0.2152
0.1848
0.1584
0.1346
0.1128
0.0923
0.0728
0.0540
0.0358
0.0178
0.0000
-
28
0.4328
0.2992
0.2510
0.2151
0.1857
0.1601
0.1372
0.1162
0.0965
0.0778
0.0598
0.0424
0.0253
0.0084
-
29
0.4291
0.2968
0.2499
0.2150
0.1864
0.1616
0.1395
0.1192
0.1002
0.0822
0.0650
0.0483
0.0320
0.0159
0.0000
30
0.4254
0.2944
0.2487
0.2148
0.1870
0.1630
0.1415
0.1219
0.1036
0.0862
0.0697
0.0537
0.0381
0.0227
0.0076
257
-------
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO MILK'S TEST (CONTINUED)
\ Number of Observations
l\"
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
tV
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
31
0.4220
0.2921
0.2475
0.2145
0.1874
0.1641
0.1433
0.1243
0.1066
0.0899
0.0739
0.0585
0.0435
0.0289
0.0144
0.0000
-
-
31
0.3940
0.2719
0.2357
0.2091
0.1876
0.1693
0.1531
0.1384
0.1249
0.1123
0.1004
0.0891
0.0782
0.0677
0.0575
0.0476
0.0379
0.0283
0.0188
0.0094
0.0000
-
-
-
-
32
0.4188
0.2898
0.2462
0.2141
0.1878
0.1651
0.1449
0.1265
0.1093
0.0931
0.0777
0.0629
0.0485
0.0344
0.0206
0.0068
-
-
-
32
0.3917
0.2701
0.2345
0.2085
0.1874
0.1694
0.1535
0.1392
0.1259
0.1136
0.1020
0.0909
0.0804
0.0701
0.0602
0.0506
0.0411
0.0318
0.0227
0.0136
0.0045
-
-
-
-
33
0.4156
0.2876
0.2451
0.2137
0.1880
0.1660
0.1463
0.1284
0.1118
0.0961
0.0812
0.0669
0.0530
0.0395
0.0262
0.0131
0.0000
-
-
33
0.3894
0.2684
0.2334
0.2078
0.1871
0.1695
0.1539
0.1398
0.1269
0.1149
0.1035
0.0927
0.0824
0.0724
0.0628
0.0534
0.0442
0.0352
0.0263
0.017.5
0.0087
0.0000
-
-
-
34
0.4127
0.2854
0.2439
0.2132
0.1882
0.1667
0.1475
0.1301
0.1140
0.0988
0.0844
0.0706
0.0572
0.0441
0.0314
0.0187
0.0062
-
-
34
0.3872
0.2667
0.2323
0.2072
0.1868
0.1695
0.1542
0.1405
9,1278
0.1160
0.1049
0.0943
0.0842
0.0745
0.0651
0.0560
0.0471
0.0383
0.0296
0.0211
0.0126
0.0042
-
-
-
35
0.4096
0.2834
0.2427
0.2127
0.1883
0.1673
0.1487
0.1317
0.1160
0.1013
0.0873
0.0739
0.0610
0.0484
0.0361
0.0239
0.0119
0.0000
-
Number of
35
0.3850
0.2651
0.2313
0.2065
0.1865
0.1695
0.1545
. 0.1410
0.1286
9.1170
0.1062
0.0959
0.0860
0.0765
0.0673
0.0584
0.0497
0.0412
0.0328
0.0245
0.0163
0.0081
0.0000
-
-
36
0.4068
0.2813
0.2415
0.2121
0.1883
0.1678
0.1496
0.1331
0.1179
0.1036
0.0900
0.0770
0.0645
0.0523
0.0404
0.0287
0.0172
0.0057
-
37
0.4040
0.2794
0.2403
0.2116
0.1883
0.1663
0.1505
0.1344
0.1196
0.1056
0.0924
0.0798
0.0677
0.0559
0.0444
0.0331
0.0220
0.0110
0.0000
Observations
36 37
0.3830
0.2635
0.2302
0.2058
0.1862
0.1695
0.1548
0.1415
0.1293
0.1180
0.1073
0.0972
0.0876
0.0783
0.0694
0.0607
0.0522
0.0439
0.0357
0.0277
0.0197
0.0118
0.0039
-
-
0.3808
0.2620
0.2291
0.2052
0.1859
0.1695
0.1550
0.1420
0.1300
0.1189
0.1085
0.0986
0.0892
0.0801
0.0713
0.0628
0.0546
0.0465
0.0385
0.0307
0.0229
0.0153
0.0076
0.0000
-
38
0.4015
0.2774
0.2391
0.2110
0.1881
0.1686
0.1513
0.1356
0.1211
0.1075
0.0947
0.0824
0.0706
0.0592
0.0481
0.0372
0.0264
0.0158
0.0053
38
0.3789
0.2604
0.2281
0.2045
0.1855
0.1693
0.1551
0.-1423
0.1306
0.1197
0.1095
0.0998
0.0906
0.0817
0.0731
0.0648
0.0568
0.0489
0.0411
0.0335
0.0259
0.0185
0.0111
0.0037
-
39
0.3989
0.2755
0.2380
0.2104
0.1880
0.1689
0.1520
0.1366
0.1225
0.1092
0.0967
0.0848
0.0733
0.0622
0.0515
0.0409
0.0305
0.0203
0.0101
0.0000
39
0.3770
0.2589
0.2271
0.2038
0.1851
0.1692
0.1553
0.1427
0.1312
0.1205
0.1105
0.1010
0.0919
0.0832
0.0748
0.0667
0.0588
0.0511
0.0436
0.0361
0.0288
0.0215
0.0143
0.0071
0.0000
40
0.3964
0.2737
0.2368
0.2098
0.1878
0.1691
0.1526
0.1376
0.1237
0.1108
0.0986
0.0870
0.0759
0.0651
0.0546
0.0444
0.0343
0.0244
0.0146
0.0049
40
0.3751
0.2574
0.2260
0.2032
0.1847
0.1691
0.1554
. 0.1430
0.1317
0.1212
0.1113
0.1020
0.0932
0.0846
0.0764
0.0685
' 0.0608
0.0532
0.0459
0.0386
0.0314
0.0244
0.0174
0.0104
0.0035
258
-------
TABLE B.5. EXAMPLE OF THE SHAPIRO-MILK'S TEST:
TABLE OF COEFFICIENTS AND DIFFERENCES
_ w(
1
2
3
4
5
6
7
8
9
10
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
0.0711
0.0422
0.0140
0.181
0.128
0.105
0.097
0.076
0.048
0.034
0.025
0.008
0.005
x<20)
X(19)
X(18>
X(17)
XC16>
x<15)
X(14)
X(13)
X(12)
- ^
- x(2)
- x(3)
- XC4)
y(5)
~ A
y<6)
- A
- >;C7)
y(8)
- A
y(9)
'. ~ A
y(10)
- A
the observations at each concentration, including the control, are listed in
Table B.7.
2.10.3 The first step of the test for normality is to center the observations
by subtracting the mean of all the observations within a concentration from
each observation in that concentration. The centered observations for the
example are listed in Table B.8.
2.10.4 Order the centered observations from smallest to largest:
yd) < y(2) y(n)
where X(1) denotes the ith ordered observation, and n denotes the total number
of centered observations. The ordered observations for the example are listed
in Table B.9.
2.10.5 The next step is to standardize the ordered observations. Let z,-
denote the standardized value of the ith ordered observation. Then,
Zi ~
and
(n-l)
For the example, s = 5.3, and the standardized observations are listed in
Table B.9.
2.10.6 From Table B.10, obtain the value of the standard normal cumulative
distribution function (standard normal CDF) at z*. Denote this value as p{.
Note that negative z are not listed in Table B.10. The value of the standard
normal CDF at a negative number is one minus the value of the standard normal
259
-------
TABLE B.6. QUANTILES OF THE SHAPIRO-WILK'S TEST STATISTIC (Conover, 1980)
n
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
0.01
0.753
0.687
0.686
0.713
0.730
0.749
0.764
0.781
0.792
0.805
0.814
0.825
0.835
0.844
0.851
0.858
0.863
0.868
0.873
0.878
0.881
0.884
0.888
0.891
0.894
0.896
0.898
0.900
0.902
0.904
0.906
0.908
0.910
0.912
0.914
0.916
0.917
0.919
0.920
0.922
0.923
0.924
0.926
0.927
0.928
0.929
0.929
0.930
0.02
0.756
0.707
0.715
0.743
0.760
0.778
0.791
0.806
0.817
0.828
0.837
0.846
0.855
0.863
0.869
0.874
0.879
0.884
0.888
0.892
0.895
0.898
0.901
0.904
0.906
0.908
0.910
0.912
0.914
0.915
0.917
0.919
0.920
0.922
0.924
0.925
0.927
0.928
0.929
0.930
0.932
0.933
0.934
0.935
0.936
0.937
0.937
0.938
0.05
0.767
0.748
0.762
0.788
0.803
0.818
0.829
0.842
0.850
0.859
0.866
0.874
0.881
0.887
0.892
0.897
0.901
0.905
0.908
0.911
0.914
0.916
0.918
0.920
0.923
0.924
0.926
0.927
0.929
0.930
0.931
0.933
0.934
0.935
0.936
0.938
0.939
0.940
0.941
0.942
0.943
0.944
0.945
0.945
0.946
0.947
0.947
0.947
0.10
0.789
0.792
0.806
0.826
0.838
0.851
0.859
0.869
0.876
0.883
0.889
0.895
0.901
0.906
0.910
0.914
0.917
0.920
0.923
0.926
0.928
0.930
0.931
0.933
0.935
0.936
0.937
0.939
0.940
0.941
0.942
0.943
0.944
0.945
0.946
0.947
0.948
0.949
0.950
0.951
0.951
0.952
0.953
0.953
0.954
0.954
0.955
0.955
0.50
0.959
0.935
0.927
0.927
0.928
0.932
0.935
0.938
0.940
0.943
0.945
0.947
0.950
0.952
0.954
0.956
0.957
0.959
0.960
0.961
0.962
0.963
0.964
0.965
0.965
0.966
0.966
0.967
0.967
0.968
0.968
0.969
0.969
0.970
0.970
0.971
0.971
0.972
0.972
0.972
0.973
0.973
0.973
0.974
0.974
0.974
0.974
0.974
0.90
0.998
0.987
0.979
0.974
0.972
0.972
0.972
0.972
0.973
0.973
0.974
0.975
0.975
0.976
0.977
0.978
0.978
0.979
0.980
0.980
0.981
0.981
0.981
0.982
0.982
0.982
0.982
0.983
0.983
0.983
0.983
0.983
0.984
0.984
0.984
0.984
0.984
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.95
0.999
0.992
0.986
0.981
0.979
0.978
0.978
0.978
0.979
0.979
0.979
0.980
0.980
0.981
0.981
0.982
0.982
0.983
0.983
0.984
0.984
0.984
0.985
0.985
0.985
0.985
0.985
0.985
0.986
0.986
0.986
0.986
0.986
0.986
0.987
0.987
0.987
0.987
0.987
0.987
0.987
0.987
0.988
0.988
0.988
0.988
0.988
0.988
0.98
1.000
0.996
0.991
0.986
0.985
0.984
0.984
0.983
0.984
0.984
0.984
0.984
0.984
0.985
0.985
0.986
0.986
0.986
0.987
0.987
0.987
0.987
0.988
0.988
0.988
0.988
0.988
0.988
0.988
0.988
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0.989
0.990
0.990
0.990
0.990'
0.990
0.990
0.990
0.990
0.99
1.000
0.997
0.993
0.989
0.988
0.987 '
0.986
0.986
0.986
0.986
0.986
0.986
0.987
0.987
0.987
0.988
0.988
0.988
0.989
0.989
0.989
0.989
0.989
0.989
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
260
-------
TABLE B.7. CERIODAPHNIA DUBIA REPRODUCTION DATA
FOR THE KOLMOGOROV "D" TEST
Effluent Concentration (%)
Control
1.56
3.12
6.25
12.5
25.0
1
2
3
4
5
6
7
8
9
10
27
30
29
31
16
15
18
17
14
27
32
35
32
26
18
29
27
16
35
13
39
30
33
33
36
33
33
27
38
44
27
34
36
34
31
27
33
31
33
31
19
25
26
17
16
21
23
15
18
10
10
13
7
7
7
10
10
16
12
2
CDF at the absolute value of that number. For example, since the value of the
standard normal CDF at 3.21 is 0.9993, the value of the standard normal CDF at
-3.21 is 1 - 0.9993 = 0.0007. The p1 values for the example data are listed
in Table B.9.
TABLE B.8. CENTERED OBSERVATIONS FOR KOLMOGOROV "D" EXAMPLE
Effluent Concentration (%)
Replicate
Control
1.56
3.12
6.25
12.5
25.0
1
2
3
4
5
6
7
8
9
10
4.6
7.6
6.6
8.6
-6.4
-7.4
-4.4
-5.4
-8.4
4.6
5.7
8.7
5.7
-0.3
-8.3
2.7
0.7
-10.3
8.7
-13.3
4.4
-4.6
-1.6
-1.6
1.4
-1.6
-1.6
-7.6
3.4
9.4
-4.7
2.3
4.3
2.3
-0.7
-4.7
1.3
-0.7 ,
1.3
-0.7
0.0
6.0
7.0
-2.0
-3.0
2.0
4.0
-4.0
-1.0
-9.0
0.6
3.6
-2.4
-2.4
-2.4
0.6
0.6
6.6
2.6
-7.4
261
-------
TABLE B.9. EXAMPLE CALCULATION OF THE KOLMOGOROV "D" STATISTIC
i
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
X(f)
-13.3
-10.3
-9.0
-8.4
-8.3
-7.6
-7.4
-7.4
-6.4
-5.4
-4.7
-4.7
-4.6
-4.4
-4.0
-3.0
-2.4
-2.4
-2.4
-2.0
-1.6
-1.6
-1.6
-1.6
-1.0
-0.7
-0.7
-0.7
-0.3
0.0
0.6
0.6
0.6
0.7
1.3
1.3
1.4
2.0
2.3
2.3
2.6
2.7
3.4
3.6
4.0
4.3
4.4
zi
-2.51
-1.94
-1.70
-1.58
-1.57
-1.43
-1.40
-1.40
-1.21
-1.02.
-0.89
-0.89
-0.87
-0.83
-0.75
-0.57
-0.45
-0.45
-0.45
-0.38
-0.30
-0.30
-0.30
-0.30
-0.19
-0.13
-0.13
-0.13
-0.06
0.00
0.11
0.11
0.11
0.13
0.25
0.25
0.26
0.38
0.43
0.43
0.49
0.51
0.64
0.68
0.75
0.81
0.83
Pi
0.0060
0.0262
0.0446
0.0571
0.0582
0.0764
0.0808
0.0808
0.1131
0.1539
0.1867
0.1867
0.1922
0.2033
0.2266
0.2843
0.3264
0.3264
0.3264
0.3520
0.3821
0.3821
0.3821
0.3821
0.4247
0.4483
0.4483
0.4483
0.4761
0.5000
0.5438
0.5438
0.5438
0.5517
0.5987
0.5987
0.6026
0.6480
0.6664
0.6664
0.6879
0.6950
0.7389
0.7517
0.7734
0.7910
0.7967
Di+
0.0107
0.0071
0.0054
0.0096
0.0251
0.0236
0.0359
0.0525
0.0369
0.0128
-0.0034
0.0133
0.0245
0.0300
0.0234
-0.0176
-0.0431
-0.0264
-0.0097
-0.0187
-0.0321
-0.0154
0.0012
0.0179
-0.0080
-0.0150
0.0017
0.0184
0.0072
0.0000
-0.0271
-0.0105
0.0062
0.0150
-0.0154
0.0013
0.0141
-0.0147
-0.0164
0.0003
-0.0046
0.0050
-0.0222
-0.0184
-0.0234
-0.0243
-0.0134
Dr
0.0060
0.0095
0.0113
0.0071
-0.0085
-0.0069
-0.0192
-0.0359
-0.0202
0.0039
0.0200
0.0034
-0.0078
-0.0134
-0.0067
0.0343
0.0597
0.0431
0.0264
0.0353
0.0488
0.0321
0.0154
-0.0012
0.0247
0.0316
0.0150
-0.0017
0.0094
0.0167
0.0438
0.0271
0.0105
0.0017
0.0320
0.0154
0.0026
0.0313
0.0331
0.0164
0.0212
0.0117
0.0389
0.0350
0.0401
0.0410
0.0300
262
-------
TABLE B.9. EXAMPLE CALCULATION OF THE KOLMOGOROV "D" STATISTIC (CONTINUED)
i X<0 Zi ' p, D,+ Dr
48
49
50
51
52
53
54
55
56
57
58
59
60
4.6
4.6
5.7
5.7
6.0
6.6
6.6
7.0
7.6
8.6
8.7
8.7
9.4
0.87
0.87
1*08
1.08
1.13
1.25
1.25
1.32
1.43
1.62
1.64
1.64
1.77
0.8078
0.8078
0.8599
0.8599
0.8708
0.8944
0.8944
0.9066
0.9236
0.9474
0.9495
0.9495
0.9616
-0.0078
0.0089
-0.0266
-0.0099
-0.0041
-0.0111
0.0056
0.0101
0.0097
0.0026
0.0172
0.0338
0.0384
0.0245
0.0078
0.0432
0.0266
0.0208
0.0277
0.0111
0.0066
0.0069
0.0141
-0.0005
-0.0172
-0.0217
2.10.7 Next, calculate the following differences for each ordered
observation:
D,+ = (i/n) - Pi
Dr = P| - [(i-l)/n]
The differences for the example are listed in Table B.9.
2.10.8 Obtain the maximum of the 0,-+, and denote it as D+. Obtain the
maximum of the D--, and denote it as D-. For the example, D+ = 0.0525, and D-
= 0.0597.
2.10.9 Next, obtain the maximum of D+ and D-, and denote it as D. For the
example, D = 0.0597.
2.10.10 The test statistic, D*, is calculated as follows:
D* = Z>(/n-0.01+0.85/v/Ja)
For the example, D* = 0.4684.
2.10.11 The decision rule for the two tailed test is to compare the critical
value from Table B.ll to the computed D*. If the computed value is greater
than the critical value, conclude that the data are not normally distributed.
For this example, the critical value at a significance level of 0.01 is 1.035.
The calculated value, 0.4684, is not greater than the critical value. Thus,
the conclusion of the test is that the data are normally distributed.
2.10.12 In general, if the data fail the test for normality,, a transformation
such as the log transformation may normalize the data. After transforming the
data, repeat the Kolmogorov "D" test for normality.
263
-------
TABLE B.10. P IS THE VALUE OF THE STANDARD NORMAL CUMULATIVE DISTRIBUTION
AT Z
Z
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
0.20
0.21
0.22
0.23
0.24
0.25
0.26
0.27
0.28
0.29
0.30
0.31
0.32
0.33
0.34
0.35
0.36
0.37
0.38
0.39
0.40
P
0.5000
0.5040
0.5080
0.5120
0.5160
0.5199
0.5239
0.5279
0.5319
0.5359
0.5398
0.5438
0.5478
0.5517
0.5557
0.5596
0.5636
0.5675
0.5714
0.5753
0.5793
0.5832
0.5871
0.5910
0.5948
0.5987
0.6026
0.6064
0.6103
0.6141
0.6179
0.6217
0.6255
0.6293
0.6331
0.6368
0.6406
0.6443
0.6480
0.6517
0.6554
z
0.41
0.42
0.43
0.44
0.45
0.46
0.47
0.48
0.49
0.50
0.51
0.52
0.53
0.54
0.55
0.56
0.57
0.58
0.59
0.60
0.61
0.62
0.63
0.64
0.65
0.66
0.67
0.68
0.69
0.70
0.71
0.72
0.73
0.74
0.75
0.76
0.77
0.78
0.79
0.80
0.81
P
0.6591
0.6628
0.6664
0.6700
0.6736
0.6772
0.6808
0.6844
0.6879
0.6915
0.6950
0.6985
0.7019
0.7054
0.7088
0.7123
0.7157
0.7190
0.7224
0.7257
0.7291
0.7324
0.7357
0.7389
0.7422
0.7454
0.7486
0.7517
0.7549
0.7580
0.7611
0.7642
0.7673
0.7704
0.7734
0.7764
0.7794
0.7823
0.7852
0.7881
0.7910
z
0.82
0.83
0.84
0.85
0.86
0.87
0.88
0.89
0.90
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1.00
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.10
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
1.20
1.21
1.22
P
0.7939
0.7967
0.7995
0.8023
0.8051
0.8078
0.8106
0.8133
0.8159
0.8186
0.8212
0.8238
0.8264-
0.8289
0.8315
0.8340
0.8365
0.8389
0.8413
0.8438
0.8461
0.8485
0.8508
0.8531
0.8554
0.8577
0.8599
0.8621
0.8643
0.8665
0.8686
0.8708
0.8729
0.8749
0.8770
0.8790
0.8810
0.8830
0.8849
0.8869
0.8888
z
1.23
1.24
1.25
1.26
1.27
1.28
1.29
1.30
1.31
1.32
1.33
1.34
1.35
1.36
1.37
1.38
1.39
1.40
1.41
1.42
1.43
1.44
1.45
1.46
1.47
1.48
1.49
1.50
1.51
1.52
1.53
1.54
1.55
1.56
1.57
1.58
1.59
1.60
1.61
1.62
1.63
P
0.8907
0.8925
0.8944
0.8962
0.8980
0.8997
0.9015
0.9032
0.9049
0.9066
0.9082 .
0.9099
0.9115
0.9131
0.9147
0.9162
0.9177
0.9192
0.9207
0.9222
0.9236
0.9251
0.9265
0.9279
0.9292
0.9306
0.9319
0.9332
0.9345
0.9357
0.9370
0.9382
0.9394
0.9406
0.9418
0.9429
0.9441
0.9452
0.9463
0.9474
0.9484
264
-------
TABLE B.10. P IS THE VALUE OF THE STANDARD NORMAL CUMULATIVE DISTRIBUTION
AT Z (CONTINUED)
z
1.64
1.65
1.66
1.67
1.68
1.69
1.70
1.71
1.72
1.73
1.74
1.75
1.76
1.77
1.78
1.79
1.80
1.81
1.82
1.83
1.84
1.85
1.86
1.87
1.88
1.89
1.90
1.91
1.92
1.93
1.94
1.95
1.96
1.97
1.98
1.99
2.00
2.01
2.02
2.03
2.04
P
0.9495
0.9505
0.9515
0.9525
0.9535
0.9545
0.9554
0.9564
0.9573
0.9582
0.9591
0.9599
0.9608
0.9616
0.9625
0.9633
0.9641
0.9649
0.9656
0.9664
0.9671
0.9678
0.9686
0.9693
0.9699
0.9706
0.9713
0.9719
0.9726
0.9732
0.9738
0.9744
0.9750
0.9756
0.9761
0.9767
0.9772
0.9778
0.9783
0.9788
0.9793
z
2.05
2.06
2.07
2.08
2.09
2.10
2.11
2.12
2.13
2.14
2.15
2.16
2.17
2.18
2.19
2.20
2.21
2.22
2.23
2.24
2.25
2.26
2.27
2.28
2.29
2.30
2.31
2.32
2.33
2.34
2.35
2.36
2.37
2.38
2.39
2.40
2.41
2.42
2.43
2.44
2.45
P
0.9798
0.9803
0.9808
0.9812
0.9817
0.9821
0.9826
0.9830
0.9834
0.9838
0.9842
0.9846
0.9850
0.9854
0.9857
0.9861
0.9864
0.9868
0.9871
0.9875
0.9878
0.9881
0.9884
0.9887
0.9890
0.9893
0.9896
0.9898
0.9901
0.9904
0.9906
0.9909
0.9911
0.9913
0.9916
0.9918
0.9920
0.9922
0.9925
0.9927
0.9929
z
2.46
2.47
2.48
2.49
2.50
2.51
2.52
2.53
2.54
2.55
2.56
2.57
2.58
2.59
2.60
2.61
2.62
2.63
2.64
2.65
2.66
2.67
2.68
2.69
2.70
2.71
2.72
2.73
2.74
2.75
2.76
2.77
2.78
2.79
2.80
2.81
2.82
2.83
2.84
2.85
2.86
P
0.9931
0.9932
0.9934
0.9936
0.9938
0.9940
0.9941
0.9943
0.9945
0.9946
0.9948
0.9949
0.9951
0.9952
0.9953
0.9955
0.9956
0.9957
0.9959
0.9960
0.9961
0.9962
0.9963
0.9964
0.9965
0.9966
0.9967
0.9968
0.9969
0.9970
0.9971
0.9972
0.9973
0.9974
0.9974
0.9975
0.9976
0.9977
0.9977
0.9978
0.9979
z
2.87
2.88
2.89
2.90
2.91
2.92
2.93
2.94
2.95
2.96
2.97
2.98
2.99
3.00
3.01
3.02
3.03
3.04
3.05
3.06
3.07
3.08
3.09
3.10
3.11
3.12
3.13
3.14
3.15
3.16
3.17
3.18
3.19
3.20
3.21
3.22
3.23
3.24
3.25
3.26
3.27
P
0.9979
0.9980
0.9981
0.9981
0.9982
0.9982
0.9983
0.9984
0.9984
0.9985
0.9985
0.9986
0.9986
0.9987
0.9987
0.9987
0.9988
0.9988
0.9989
0.9989
0.9989
0.9990
0.9990
0.9990
0.9991
0.9991
0.9991
0.9992
0.9992
0.9992
0.9992
0.9993
0.9993
0.9993
0.9993
0.9994
0.9994
0.9994
0.9994
0.9994
0.9995
265
-------
TABLE B.10. P IS THE VALUE OF THE STANDARD NORMAL CUMULATIVE DISTRIBUTION
AT Z (CONTINUED)
z
3.28
3.29
3.30
3.31
3.32
3.33
3.34
3.35
3.36
3.37
3.38
3.39
3.40
3.41
3.42
3.43
3.44
3.45
P
0.9995
0.9995
0.9995
0.9995
0.9995
0.9996
0.9996
0.9996
0.9996
0.9996
0.9996
0.9997 '
0.9997
0.9997
0.9997
0.9997
0.9997
0.9997
z
3.46
3.47
3.48
3.49
3.50
3.51
3.52
3.53
3.54
3.55
3.56
3.57
3.58
3.59
3.60
3.61
3.62
3.63
P
0.9997
0.9997
0.0997
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9999
0.9999
z
3.64
3.65
3.66
3.67
3.68
3.69
3.70
3.71
3.72
3.73
3.74
3.75
3.76
3.77
3.78
3.79
3.80
3.81
P
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
z
3.82
3.83
3.84
3.85
3.86
3.87
3.88
3.89
3.90
3.91
3.92
3.93
3.94
3.95
3.96
3.97
3.98
3.99
P
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
TABLE B.ll. CRITICAL VALUES FOR THE KOLMOGOROV "D" TEST
Alpha
Level
0.010
0.025
0.050
0.100
0.150
Critical
Value
1.035
0.955
0.895
0.819
0.775
3. TEST FOR HOMOGENEITY OF VARIANCE
3.1 For Dunnett's Procedure and the t test with Bonferroni's adjustment, the
variances of the data obtained from each toxicant concentration 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.
266
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3.2 The data used in this example are growth data from a Fathead Minnow
Larval Survival and Growth Test, and are the same data used in Appendices C
and D. These data are listed in Table B.12, together with the calculated
variance for the control and each toxicant concentration.
TABLE B.12. FATHEAD LARVAL GROWTH DATA (WEIGHT IN MG) USED FOR
BARTLETT'S TEST FOR HOMOGENEITY OF VARIANCE
NaPCP Concentration (uq/L)
Rep! i cate
A
B
C
D
M|an(Y,-)
Si
1
Control
0.711
0.662
0.718
0.767
0.714
0.0018
1
32
0.646
0.626
0.723
0.700
0.674
0.0020
2
64
0.669
0.669
0.694
0.676
0.677
0.0001
3
128
0.629
0.680
0.513
0.672
0.624
0.0059
4
256
0.650
0.558
0.606
0.508
0.580
0.0037
5
3.3 The test statistic for Bartlett's Test (Snedecor and Cochran, 1980) is as
follows:
rj) InS2- EVjlnS'f]
S ฃ
Where: V,. = degrees of freedom for each toxicant concentration and control
p = number of levels of toxicant concentration including the
control
In = loge
i = 1, 2, ..., p where p is the number of concentrations
n, = .the number of replicates for concentration i.
267
-------
C - 1+ (3 (p-1) ) -1 [ ฃ 1/Vฑ - (
3.4 Since B is approximately distributed as chi -square with p - 1 degrees of
freedom when the variances are equal, the appropriate critical value is
obtained from a table of the chi-square distribution for p-1 degrees of
freedom and a significance level of 0.01. If B is less than the critical
value then the variances are assumed to be equal.
3.5 For the data in this example, V,- = 3, p = 5, 32 = 0.0027, and
C ซ 1.133. The calculated B value is:
(15) [In (0.0027 )] -3 fin (Si)
B =
1.133 " '
15 (-5.9145) -3 (-32.4771)
1.133
= 7.691
3.6 Since B is approximately distributed as chi-square with p-1 degrees of
freedom when the variances are equal, the appropriate critical value for the
test is 13.277 for a significance level of 0.01. Since B = 7.691 is less than
the critical value of 13.277, conclude that the variances are not different.
4. TRANSFORMATIONS OF THE DATA
4.1 When the assumptions of normality and/or homogeneity of variance are not
met, transformations of the data may remedy the problem, so that the data can
be analyzed by parametric procedures, rather than by nonparametric technique
such as Steel's Many-one Rank Test or Wilcoxon's Rank Sum Test. Examples of
transformations include log, square root, arc sine square root, and
reciprocals. After the data have been transformed, Shapiro-Milk's and
Bartlett's tests should be performed on the transformed observations to
determine whether the assumptions of normality and/or homogeneity of variance
are met.
4.2 ARC SINE SQUARE ROOT TRANSFORMATION (USEPA, 1993)
4.2.1 For data consisting of proportions from a binomial (response/no
response; live/dead) response variable, the variance within the ith treatment
is proportional to P,- (1 - P.), where P,- is the expected proportion for the
treatment. This clearly violates the homogeneity of variance assumption
required by parametric procedures such as Dunnett's Procedure or the t test
with Bonferroni's adjustment, since the existence of a treatment effect
implies different values of Pi for different treatments, i. Also, when the
observed proportions are based on small samples, or when P,- is close to zero
268
-------
or one, the normality assumption may be invalid. The arc sine square root
(arc sine J P ) transformation is commonly used for such data to stabilize the
variance and satisfy the normality requirement.
i
4.2.2 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
calculated, 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 aind the modification is
provided below.
4.2.3 Calculate the response proportion (RP) at each effluent concentration,
where: !
RP = (number of surviving or "unaffected" organisms)/(number exposed)
Example; If 12 of 20 animals in a given treatment replicate survive:
i
RP = 12/20
= 0.60
4.2.4 Transform each RP to its arc sine square root, as follows:
4.2.4.1 For RPs greater than zero or less than one:
Angle (radians) = arc sine
Example: If RP = 0.60:
Angle = arc sine
i
= arc sine 0.7746
= 0.8861 radians
4.2.4.2 Modification of the arc sine square root when RP = 0:
Angle (in radians) = arc sine ^1/4 #
Where: N = Number of animals/treatment replicate
269
-------
Example: If 20 animals are used:
Angle = arc sine /i/so
= arc sine 0.1118
= 0.1120 radians
4.2.4.3 Modification of the arc sine square root when RP
Angle - 1.5708 radians - (radians for RP = 0)
Example: Using above value:
Angle = 1.5708 - 0.1120
= 1.4588 radians
= 1.0:
270
-------
APPENDIX C
DUNNETT'S PROCEDURE
1. MANUAL CALCULATIONS
j
1.1 Dunnett's Procedure (Dunnett, 1955; Dunnett, 1964) 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 concentrations and control (see Appendix B for a discussion on validating
the assumptions). Dunnett's Procedure uses a pooled estimate of the variance,
which is equal to the error value calculated in an analysis of variance.
Dunnett's Procedure can only be used when the same number of replicate test
vessels have been used at each concentration and the control. When this
condition is not met, a t test with Bonferroni's adjustment is used (see
Appendix D).
1.2 The data used in this example are growth data from a Fathead Minnow
Larval Survival and Growth Test, and are the same data used in Appendices B
and D. These data are listed in Table C.I.
TABLE C.I. FATHEAD MINNOW, PlMEPHALES PROMELAS, LARVAL GROWTH DATA
(WEIGHT IN MG) USED FOR DUNNETT'S PROCEDURE
Replicate
Control
NaPCP Concentration fyq/L)
32
64
128
256
A
B
C
D
Mean (7,-)
Total (Tf)
0.711
0.662
0.646
0.690
0.677
2.709
0.517
0.501
0.723
0.560
0.575
2.301
0.602
0.669
0.694
0.676
0.660
2.641
0.566
0.612
0.410
0.672
0.565
2.260
0.455
0.502
0.606
0.254
0.454
1.817
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:
271
-------
Where: p - number of effluent concentrations including:
SST = E Yi:j2-G2/N Total Sum of Squares
3SB = ETj2/n.i-G2/N Between Sum of Squares
SSW = SST-SSB
Within Sum of Squares
G - the grand total of all sample observations; c? = E r^
T5 - the total of the replicate measurements for concentration i
N = the total sample size; N = ~
n,- - the number of replicates for concentration i
Y,j = the jth observation for concentration i
1.4 For the data in this example:
n1 = n2 - n3 = n4 = n5 = 4
n = 20
TI s Yn + Y12 + Y13 + YM = 2.709
%: fe:fe:fe:fe:l:Si
T4 = Y41 + Y42 + Y43 + Y44 = 2 26ฐ
T V _i_ V j- V j. V 1 Q17
5 Y51 + Y52 + Y53 + Y54 " 1'bl/
G = T, -J- T2 + T3 + T4 + T5 = 11.728
SST = E Yi:j2-Gz/N
= 7.146 - (11.728)2/20
= 0.2687
272
-------
SSB = ETi2/ni-G2/N
1/4 (28.017 - 11.728)2/20
0.1270
SSW = SST-SSB
0.2687 - 0..1270
0.1417
1.5 Summarize these data in the ANOVA table (Table C.2).
TABLE C.2. ANOVA TABLE FOR DUNNETT'S PROCEDURE
Source df
Between p - 1
Within N - p
Total N - 1
Sum of
Squares (SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
i
s2 ;- SSB/(P-D
S2 = SSW/(N-p)
1 '
1
i
1.6 Summarize data for ANOVA (Table C.3).
TABLE C.3. COMPLETED ANOVA TABLE FOR DUNNETT'S PROCEDURE
Source
Between
Within
df
5-1=4
20 - 5 = 15
SS
0.1270
0.1417
Mean Square
0.0318
i
0.0094
Total
19
0.2687
273
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1.7 To perform the individual comparisons, calculate the t statistic for each
concentration and control combination, as follows:
Where: Y{ ซ= mean for concentration i
Y, = mean for the control
S = square root of the within mean square
n., - number of replicates in the control
n- = number of replicates for concentration i.
1.8 Table C.4 includes the calculated t values for each concentration and
control combination.
TABLE C.4. CALCULATED T VALUES.
NaPCP
Concentration
32
64
128
256
2
3
4
5
1.487
0.248
1.633
3.251
1.9 Since the purpose of the test is only to detect a decrease in growth from
the control, a one-sided test is appropriate. The critical value for the
one-sided comparison (2.36), with an overall alpha level of 0.05, 15 degrees
of freedom and four concentrations excluding the control is read from the
table of Dunnett's "T" values (Table C.5; this table assumes an equal number
of replicates in all treatment concentrations and the control). The mean
weight for concentration i is considered significantly less than the mean
weight for the control if t,. is greater than the critical value. Since T5 is
greater than 2.36, the 256 M9/L concentration has significantly lower growth
than the control. Hence the NOEC and LOEC for growth are 128 ng/L and 256
/*g/L, respectively.
274
-------
CO
S-
O)
=
00
CO
-------
1.10 To quantify the sensitivity of the test, the minimum significant
difference (MSD) may be calculated. The formula is as follows:
MSD = d StfjCL/nJ + (1/23)
Where: d = critical value for the Dunnett's Procedure
Sw = the square root of the within mean square
n - the number of replicates at each concentration,
assuming an equal number of replicates at all treatment
concentrations
n., = number of replicates in the control
For example:
MSD = 2.36 (0.097) [3^(1/4)+(1/4)] = 2.36 (0.097) (^2/4)
= 2.36 (0.097)(0.707)
- 0.162
1.11 For this set of data, the minimum difference between the control mean
and a concentration mean that can be detected as statistically significant is
0.087 mg. This represents a decrease in growth of 24% from the control.
1.11.1 If the data have not been transformed, the MSD (and the percent
decrease from the control mean that it represents) can be reported as is.
1.11.2 In the case where the data have been transformed, the MSD would be in
transformed units. In this case carry out the following conversion to
determine the MSD in untransformed units.
1.11.2.1 Subtract the MSD from the transformed control mean. Call this
difference D. Next, obtain untransformed values for the control mean and the
difference, D.
MSD,
Where: MSDt
Control,
controlu - Du
the minimum significant difference for untransformed data
the untransformed control mean
the untransformed difference
276
-------
I
1.11.2.2 Calculate the percent reduction from the control that MSD
rpnyp^pntc ac- , u
represents as:
MSDU
Percent Reduction = X 100
Control,
u
1.11.3 An example of a conversion of the MSD to untransformed units, when the
arc sine square root transformation was used on the data, follows:
Step 1. Subtract the MSD from the transformed control mean. As an
example, assume the data in Table C.I were transformed by the arc
sine square root transformation. Thus:
.
0.677 - 0.162 = 0.515
Step 2. Obtain untransformed values for the control mean (0.677) and the
difference (0.515) obtained in Step 1 above. [
2
[ Sine (0.677)]; = 0.392
[ Sine (0.515)]2 = 0.243
I
Step 3. The untransformed MSD (MSDJ is determined by subtracting the
untransformed values obtained in Step 2.
MSDU = 0.392 - 0.243 = 0.149
i
In this case, the MSD would represent a 38.0% decrease in survival
from the control [ (0.149/0. 392) ( 100) ]. i
2. COMPUTER CALCULATIONS
2.1 This computer program incorporates two analyses: an analysis of variance
(ANOVA), and a multiple comparison of treatment means with the control mean
(Dunnett's Procedure). The ANOVA is used to obtain the error value.
Dunnett's Procedure indicates which toxicant concentration means (if any) are
statistically different from the control mean at the 5% level of significance.
The program also provides the minimum difference between the control and
treatment means that could be detected as statistically significant, and tests
the validity of the homogeneity of variance assumption by Bartlett's Test.
The multiple comparison is performed based on procedures described by Dunnett
2.2 The source code for the Dunnett's program is structured into a series of
subroutines, controlled by a driver routine. Each subroutine has a specific
function in the Dunnett's Procedure, such as data input, transforming the
data, testing for equality of variances, computing p values, and calculating
the one-way analysis of variance.
2.3 The program compares up to seven toxicant concentrations against the
control, and can accommodate up to 50 replicates per concentration.
277
-------
2 4 If the number of replicates at each toxicant concentration and control
are not equal, a t test with Bonferroni's adjustment is performed instead of
Dunnett's Procedure (see Appendix D).
2 5 The program was written in IBM-PC FORTRAN by Computer Sciences
Corporation, 26 W. Martin Luther King Drive, Cincinnati, OH 45268. A compiled
executable version of the program can be obtained from EMSL-Cincinnati by
sending a written request to EMSL at 3411 Church Street, Cincinnati, OH
45244.
2.6 DATA INPUT AND OUTPUT
261 Reproduction data from a daphnid, Ceriodaphnia dubia, survival and
reproduction test (Table C.6) are used to illustrate the data input and output
for this program.
TABLE C.6. SAMPLE DATA FOR DUNNETT'S PROGRAM
CERIODAPHNIA DUBIA REPRODUCTION DATA
Replicate Control
Effluent Concentration (%)
1.56
3.12
6.25
12.5
1
2
3
4
5
6
7
8
9
10
27
30
29
31
16
15
18
17
14
27
32
35
32
26
18
29
27
16
35
13
39
30
33
33
36
33
33
27
38
44
27
34
36
34
31
27
33
31
33
31
10
13
7
7
7
10
10
16
12
2
2.6.2 Data Input
2.6.2.1 When the program is entered, the user is asked to select the type of
data to be entered:
1. Response proportions, like survival or fertilization proportions.
2. Counts and measurements, like offspring counts, cystocarp counts'or
weights.
278
-------
2.6.2.2 After the type of data is chosen, the user has the following options:
1. Create a data file
2. Edit a data file
3. Perform analysis on existing data set !
4. Stop
2.6.2.3 When Option 1 (Create a data file) is selected for counts and
measurements, the program prompts the user for the follov/ing information:
1. Number of concentrations, including control
2. For each concentration:
- number of observations
- data for each observation
i
2.6.2.4 After the data have been entered, the user may save the file on a
disk, and the program returns to the menu (see below).
t
2.6.2.5 Sample data input is shown in Figure C.I.
2.6.3 Program Output
2.6.3.1 When Option 3 (Perform analysis on existing data set) is selected
from the menu, the user is asked to select the transformation desired, and
indicate whether they expect the means of the test groups to be less or
greater than the mean for the control group (see Figure 0.2).
2.6.3.2 Summary statistics (Figure C.3) for the raw and transformed data, if
applicable, the ANOVA table, results of Bartlett's Test, the results of the
multiple comparison procedure and the minimum detectable difference are
included in the program output.
279
-------
EMSL Cincinnati Dunnett Software
Version 1.5
1) Create a data file
2) Edit a data file
3) Perform ANOVA on existing data
4) Stop
Your choice ? 1
Number of groups, including control ? 5
Number of observations for group 1 ? 10
Enter the data for group 1 one observation at a time,
NO. 1? 27
NO. 2? 30
NO. 3? 29
NO. 4? 31
NO. 5? 16
NO. 6? 15
NO. 7? 18
NO. 8? 17
NO. 9? 14
NO. 10? 27
Number of observations for group 2 ? 10
Do you wish to save the data on disk ?y
Disk file for output ? cerio
Figure C.I. Sample Data Input for Dunnett's Program for Reproduction
Data from Table C.6.
280
-------
EMSL Cincinnati Dunnett Software
Version 1.5
1) Create a data file
2) Edit a data file
3) Perform analysis on existing data set
4) Stop
Your choice ? 3
File name ? cerio
Available Transformations
1) no transform
2) square root
3) loglO
Your choice ? 1
Dunnett's test as implemented in this program is i
a one-sided test. You must specify the direction
the test is to be run; that is, do you expect the
means for the test groups to be less than or
greater than the mean for the control group mean.
Direction for Dunnett's test : L=less than, G=greater than ? L
Figure C.2. Example of Choosing Option 3 from the Menu of the Dunnett
Program.
281
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Group
Ceriodaphnia Reproduction Data from Table C.6
Summary Statistics and ANOVA
Transformation = None
n Mean s.d. CV%
1
- control
2
3
4
5*
10
10
10
10
10
22.4000
26.3000
34.6000
31.7000
9.4000
6.9314
8.0007
4.8351
2.9458
3.8930
30.9
30.4
14.0
9.3
41.4
*) the mean for this group is significantly less than the control
mean at alpha - 0.05 (1-sided) by Dunnett's test
Minimum detectable difference for Dunnett's test = -5.628560
This difference corresponds to -25.13 percent of control
Between concentrations
Sum of squares - 3887.880000 with 4 degrees of freedom.
Error mean square = 31.853333 with 45 degrees of freedom.
Bartlett's test p-value for equality of variances = .029
Do you wish to restart the program ?
Figure C.3. Example of Program Output for the Dunnett's Program Using the
Reproduction Data from Table C.6.
282
-------
APPENDIX D
T TEST WITH BONFERRONI'S ADJUSTMENT
1. The t test with Bonferroni's adjustment is used as an alternative to
Dunnett's Procedure when the number of replicates is not the same for all
concentrations. This test sets an upper bound of alpha on the overall error
rate, in contrast to Dunnett's Procedure, for which the overall error rate is
fixed at alpha. Thus, Dunnett's Procedure is a more powerful test.
2. The t test with Bonferroni's adjustment is based on the same assumptions of
normality and homogeneity of variance as Dunnett's Procedure (see Appendix B
for testing these assumptions), and, like Dunnett's Procedure, uses a pooled
estimate of the variance, which is equal to the error value calculated in an
analysis of variance.
3. An example of the use of the t test with Bonferroni's adjustment is
provided below. The data used in the example are the same as in Appendix C,
except that the third replicate from the 256 fj.g/1 concentration is presumed to
have been lost. Thus, Dunnett's Procedure cannot be used. The weight data
are presented in Table D.I.
TABLE D.I. FATHEAD MINNOW, PIMEPHALES PROMELAS, LARVAL GROWTH DATA
(WEIGHT IN MG) USED FOR THE T-TEST WITH BONFERRONI'S
ADJUSTMENT
Replicate
Control
NaPCP Concentration f,itq/L)
32
64
128
256
A
B
C
D
Mean(Y,.)
Total (Tf)
0.711
0.662
0.646
0.690
0.677
2.709
0.517
0.501
0.723
0.560
0.575
2..301
0.602
0.669
0.694
0.676
0.660
2.641
0.566
0.612
0.410
0.672
0.565
2.260
i
0.455
0.502
(LOST)
0.254
0.404 .
1.211
283
-------
3.1 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:
Where: p = number of effluent concentrations including the control
N = the total sample size; N =
n.- = the number of replicates for concentration i
SST = ฃ Yi12-G2/N
ij
Total Sum of Squares
SSB = T,Ti2/ni-G2/N
Between Sum of Squares
SSW = SST-SSB
Within Sum of Squares
Where: G - The grand total of all sample observations; G = ฃ Tฑ
1=1
J. - The total of the replicate measurements for concentration i
Yj- - The jth observation for concentration i
3.2 For the data in this example:
'1
n2 =
ฃ: $;
ฃ:ฃ:
12
'.32
= 4
13
23
T3 + T4
Y14 =2.709
Y24 = 2.301
YM = 2.641
= 2.260
Y54= 1.211
+ T5 = 11
SSB = ^Ti2/ni-G2/N
= 6.668 - (11.122)2 /19
= 0.158
284
-------
SST = E YฑJ2-G2/N
= 6.779 - (11.122)2/19
= 0.269
SSW = SST-SSB
= 0.269 - 0.158 i
= 0.111
3.3 Summarize these data in the ANOVA table (Table D.2):
TABLE D.2. ANOVA TABLE FOR BONFERRONI'S ADJUSTMENT
Source df Sum of Mean Square (MS)
Squares (SS) (SS/df)
Between p - 1 SSB S* = SSB/(p-l)
Within N - p SSW SJ; = SSW/(N-p)
Total N - 1 SST
3.4 Summarize these data in the ANOVA table (Table D.3):
3.5 To perform the individual comparisons, calculate the t statistic for each
concentration and control combination, as follows: i
Where: Y,- = mean for each concentration
i
Y1 = mean for the control <
Su = square root of the within mean square
" i
n, = number of replicates in the control.
n,- = number of replicates for concentration i.
i
285
-------
TABLE D.3. COMPLETED ANOVA TABLE FOR THE T-TEST WITH
BONFERRONI'S ADJUSTMENT
Source df SS Mean Square
Between
Within
5-1=4
19 - 5 = 14
0.158
0.111
0.0395
0.0029
Total 18 0.269
3.6 Table D.4 includes the calculated t values for each concentration and
control combination.
TABLE D.4. CALCULATED T VALUES
NaPCP
Concentration i t-
32
64
128
256
2
3
4
5
1.623
0.220
1.782
4.022
3.7 Since the purpose of the test is only to detect a decrease in growth from
the control, a one-sided test is appropriate. The critical value for the
one-sided comparison (2.510), with an overall alpha level of 0.05, fourteen
degrees of freedom and four concentrations excluding the control, was obtained
from Table D.5. The mean weight for concentration "i" is considered
significantly less than the mean weight for the control if t,- is greater than
the critical value. Since t5 is greater than 2.510, the 256 jug/L
concentration has significantly lower growth than the control. Hence the NOEC
and LOEC for growth are 128 /jg/L and 256 ng/L, respectively.
286
-------
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APPENDIX E
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 Dunnett's
Procedure, and may be applied to 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, use Wilcoxon's
Rank Sum Test, with Bonferroni's adjustment (see Appendix F).
2. For an analysis using Steel's Test, for each control and concentration
combination, combine the data and arrange the observations in order of size
from smallest to largest. Assign the ranks 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. (Extensive ties would invalidate
this procedure). The sum of the ranks within each concentration and within
the control is then calculated. To determine if the response in a
concentration is significantly different from the response in the control, the
minimum rank sum for each concentration and control combination is compared to
the significant values of rank sums given later in this section. In this
table, k equals the number of treatments excluding the control and n equals
the number of replicates for each concentration and the control.
i
3. An example of the use of this test is provided below., The test employs
reproduction data from a Ceriodaphnia dubia 7-day, chronic test. The data are
listed in Table E.I. Significant mortality was detected via Fisher's Exact
Test in the 50% effluent concentration. The data for this concentration is
not included in the reproduction analysis.
TABLE E.I. EXAMPLE OF STEEL'S MANY-ONE RANK TEST: DATA FOR THE
.DAPHNID, CERIODAPHNIA DUBIA, 7-DAY CHRONIC TEST
Effluent
Concentration
Control
3%
6%
12%
25%
50%
i
Replicate
1
20
13
18
14
9
0
2
26
15
22
22
0
0
3
26
14
13
20
9
0
4
23
13
13
23
7
0
5
24
23
23
20
6
0
6
27
26
22
23
10
0
7
26
0
20
25
12
0
8
I
23
25
22
24
14
0
9
27
26
23
25
9
0
10
24
27
22
21
13
0
No.
Live
Adults
10
9
10
10
8
0
289
-------
4. For each control and concentration combination, combine the data and
arrange the observations in order of size from smallest to largest. Assign
ranks (1, 2, 3,..., 16) 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 each tied observation.
5. An example of assigning ranks to the combined data for the control and 3%
effluent concentration is given in Table E.2. This ranking procedure is
repeated for each control and concentration combination. The complete set of
rankings is listed in Table E.3. The ranks are then summed for each effluent
concentration, as shown in Table E.4.
TABLE E.2. EXAMPLE OF STEEL'S MANY-ONE RANK TEST: ASSIGNING
RANKS TO THE CONTROL AND 3% EFFLUENT CONCENTRATION
Rank
Number of Young
Produced
Control or % Effluent
1
2.5
2.5
4
5
6
8
8
8
10.5
10.5
12
15
15
15
15
15
19
19
19
0
13
13
14
15
20
23
23
23
24
24
25
26
26
26
26
26
27
27
27
3
3
3
3
3
Control
Control
Control
3
Control
Control
3
Control
Control
Control
3
3
Control
Control
3
6. For this set of data, 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, compare 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
290
-------
TABLE E.
Repl icate
(Organism)
1
2
3
4
5
6
7
8
9
10
20
26
26
23
?4
27
26
23
27
24
3.
Control1
(6,4.5
(15,17
(15,17
(8,11.
(10.5,
(19,19
(15,17
(8,11.
(19,19
(10.5,
,3,11)
,17,17)
,17,17)
5,8.5,12
14.5,12,
.5,19.5,
,17,17)
5,8.5,12
.5,19.5,
14.5,12,
.5)
14.5)
19.5)
.5)
19.5)
14.5)
13
15
14
13
23
26
0
25
26
27
TABLE OF RANKS
Effluent Concentration (%)
3
(2.5)
(5)
(4)
(2.5)
(8)
(15)
(1)
(12)
(15)
(19)
18
22
13
13
23
22
20
22
23
22
6
(3)
(7.5)
(1.5)
(1.5)
(11.5)
(7.5)
(4.5)
(7.5)
(11.5)
(7.5)
14
22
20
23
20
23
25
24
25
21
12
(1)
(6)
(3)
(8.5)
(3)
(8.5)
(14.5)
(12)
(14.5)
(5)
25
9 (5)
0 (1)
9 (5)
7 (3)
6 (2)
10 (7)
12 (8)
14 (10)
9 (5)
13 (9)
1 Control ranks are given in the order of the concentration with which they
were ranked.
TABLE E.4. RANK SUMS
Effluent Rank Sum
Concentration
3 84
6 64
12 76
25 55
level of 0.05, the critical rank in a test with four concentrations and ten
replicates is 76 (see Table E.5, forR=4).
7. Comparing the rank sums in Table E.4 to the appropriate critical rank, the
6%, 12% and 25% effluent concentrations are found to be significantly
different from the control. Thus the NOEC and LOEC for reproduction are 3%
and 6%, respectively.
291
-------
TABLE E.5. SIGNIFICANT VALUES OF RANK SUMS: JOINT CONFIDENCE
COEFFICIENTS OF 0.95 (UPPER) and 0.99 (LOWER) FOR
ONE-SIDED ALTERNATIVES (Steel, 1959)
k = number of treatments (excludina
'
I 2
4 1 11
5 18
6
7
8
9
10
11
12
13
15
27
23
37
32
49
43
63
56
79
71
97
87
116
105
138
125
14 161
1 147
15 1 186
1 170
16 1 213
1
17
18
19
20
196
241
223
272
252
304
282
339
315
3
10
17
--
26
22
36
31
48
42
62
55
77
69
95
85
114
103
135
123
158
144
182
167
209
192
237
219
267
248
299
278
333
310
4
10
17
25
21
35
30
47
41
61
54
76
68
93
84
112
102
133
121
155
142
180
165
206
190
234
217
264
245
296
275
330
307
5
10
16
25
21
35
30
46
40
60
53
75
67
92
83
111
100
132
120
154
141
178
164
204
188
232
215
262
243
294
273
327
305
6
10
16
24
34
29
46
40
59
52
74
66
91
82
110
99
130
119
153
140
177
162
203
187
231
213
260
241
292
272
325
303
7
--
16
_
24
34
29
45
40
59
52
74
66
90
81
109
99
129
118
152
139
176
161
201
186
229
212
259
240
290
270
323
301
control )
8
--
16
_
24
- -
33
29
45
39
58
51
73
65
90
81
108
98
129
117
151
138
175
160
200
185
228
211
257
239
288
268
322
300
9
--
15
__
23
33
29
44
39
58
51
72
65
89
80
108
98
128
117
150
137
174
160
199
184
227
210
256
238
287
267
320
299
292
-------
APPENDIX F
WILCOXON RANK SUM TEST
1. Wilcoxon's Rank Sum Test is a nonparametric test, to be used as an
alternative to Steel's Many-one Rank Test when the number of replicates are
not the same at each concentration. A Bonferroni's adjustment of the pairwise
error rate for comparison of each concentration versus the control is used to
set an upper bound of alpha on the overall error rate, in contrast to Steel's
Many-one Rank Test, for which the overall error rate is fixed at alpha. Thus,
Steel's Test is a more powerful test.
2. An example of the use of the Wilcoxon Rank Sum Test is provided in Table
F.I. The data used in the example are the same as in Appendix E, except that
two males are presumed to have occurred, one in the control and one in the 25%
effluent concentration. Thus, there is unequal replication for the
reproduction analysis.
3. For each concentration and control combination, combine the data and
arrange the values in order of size, from smallest to largest. Assign ranks
to the ordered observations (a rank of 1 to the smallest, 2 to the next
smallest, etc.). If ties in rank occur, assign the average rank to each tied
observation.
TABLE F.I. EXAMPLE OF WILCOXON'S RANK SUM TEST: DATA FOR THE
DAPHNID, CERIODAPHNIA DUBIA, 7-DAY CHRONIC TEST
Effluent
Concentration
Cont
3%
6%
12%
25%
50%
l
Replicate
1
M
13
18
14
9
0
2
26
15
22
22
0
0
3
26
14
13
20
9
0
4
23
13
13
23
7
0
5
24
23
23
M
6
0
6
27
26
22
23
10
0
7
26
0
20
25
12
0
8
23
25
22
24
14
0
9
27
26
23
25
i 9
0
10
24
27
22
21
13
0
No.
Live
Adults
10
9
10
10
8
0
4. An example of assigning ranks to the combined data for the control and
3% effluent concentration is given in Table F.2. This ranking procedure is
repeated for each of the three remaining control versus test concentration
combinations. The complete set of ranks is listed in Table F.3. The ranks
are then summed for each effluent concentration, as shown in Table F.4.
5. For this set of data, determine if the reproduction in any of the effluent
concentrations is significantly lower than the reproduction by the control
293
-------
TABLE F.2. EXAMPLE OF WILCOXON'S RANK SUM TEST: ASSIGNING
RANKS TO THE CONTROL AND EFFLUENT CONCENTRATIONS
Rank Number of Young Control or % Effluent
Produced
1
2.5
2.5
4
5
7
7
7
9.5
9.5
11
14
14
14
14
14
18
18
18
0
13
13
14
15
23
23
23
24
24
25
26
26
26
26
26
27
27
27
3
3
3
3
3
Control
Control
3
Control
Control
3
Control
Control
Control
3
3
Control
Control
3
organisms. If this occurs, the rank sum at that concentration would be
significantly lower than the rank sum for the control. Thus, compare 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 critical rank in a test with four concentrations and nine
replicates in the control is 72 for those concentrations with ten replicates,
and 60 for those concentrations with nine replicates (see Table F.5, for K =
4).
6. Comparing the rank sums in Table F.4 to the appropriate critical rank, the
6%, 12% and 25% effluent concentrations are found to be significantly
different from the control. Thus, the NOEC and LOEC for reproduction are 3%
and 6%, respectively.
294
-------
TABLE F.3. TABLE OF RANKS
Replicate Control1
(Organism)
1
2
3
4
5
6
7
8
9
10
M
26 (14,16,15,16)
26 (14,16,15,16)
23 (7,10.5,6.5,11.5)
24 (9.5,13.5,10,13.5)
27 (18,18.5,17.5,18.5)
26 (14,16,15,16)
23 (7,10.5,6.5,11.5)
27 (18,18.5,17.5,18.5)
24 (9.5,13.5,10,13.5)
13
15
14
13
23
26
0
25
26
27
Effluent
3
(2.5)
(5)
(4)
(2.5)
(7)
(14)
(1)
(11)
(14)
(18)
18
22
13
13
23
22
20
22
23
22
6
(3)
(6.
(1.
(1.
(10
(6.
(4)
(6.
(10
(6.
Concentration (%)
5)
5)
5)
.5)
5)
5)
.5)
5)
1
14
22
20
23
M
23
25
24
25
:21
12
(1)
(4)
(2)
(6.
(6.
(12
5)
5)
.5)
(10)
(12
(3)
.5)
9
0
9
7
6
10
12
14
9
13
25
(b)
(1)
(5)
(3)
(2)
(7)
(8)
(10)
(5)
(9)
Control ranks are given in the order of the concentration! with which they
were ranked.
TABLE F.4. RANK SUMS
Eff1uent
Concentration
Rank Sum
No. of
Replicates
Critical
Rank Sum
3
6
12
25
79
57
58
55
10
10
9
10
72
72
60
72
295
-------
TABLE F.5. CRITICAL VALUES FOR WILCOXON'S RANK SUM TEST WITH
BONFERRONI'S ADJUSTMENT OF ERROR RATE FOR COMPARISON
OF "K" TREATMENTS VERSUS A CONTROL FIVE PERCENT CRITICAL
LEVEL (ONE-SIDED ALTERNATIVE: TREATMENT CONTROL)
K No. Reolicates No. of Reolicates Per Effluent
in Control
1 3
4
5
6
7
8
9
10
2 3
4
5
6
7
8
9
10
3 3
4
5
6
7
8
9
10
3
6
6
7
8
8
9
10
10
_
--
6
7
7
8
8
9
--
--
6
7
7
7
8
4
10
11
12
13
14
15
16
17
..
10
11
12
13
14
14
15
10
11
11
12
13
13
14
5
16
17
19
20
21
23
24
26
15
16
17
18
20
21
22
23
16
17
18
19
20
21
22
6
23
24
26
28
29
31
33
35
22
23
24
26
27
29
31
32
21
22
24
25
26
28
29
31
7
30
32
34
36
39
41
43
45
29
31
33
34
36
38
40
42
29
30
32
33
35
37
39
41
8
39
41
44
46
49
51
54
56
38
40
42
44
46
49
51
53
37
39
41
43
45
47
49
51
Concentration
9
49
51
54
57
60
63
66
69
47
49
52
55
57
60
62
65
46
48
51
53
56
58
61
63
10
59
62
66
69
72
72
79
82
58
60
63
66
69
72
75
78
57
59
62
65
68
70
73
76
296
-------
TABLE F.5. CRITICAL VALUES FOR WILCOXON'S RANK SUM TEST WITH BONFERRONI'S
ADJUSTMENT OF ERROR RATE FOR COMPARISON OF '"K" TREATMENTS
VERSUS A CONTROL FIVE PERCENT CRITICAL LEVEL (ONE-SIDED
ALTERNATIVE: TREATMENT CONTROL) (CONTINUED)
K No. Replicates No. of Reol
in Control
4 3
4
5
6
7
8
9
10
5 3
4
5
6
7
8
9
10
6 3
4
5
6
7
8
9
10
7 3
4
5
6
7
8
9
10
3
...
--
--
6
6
7
7
7
..
_-
--
--
6
6
7
7
..
--
--
_-
6
6
6
7
._
.--
--
--
--.
6
6
7
4
...
--
10
11
12
12
13
14
..
--
10
11
11
12
13
13
_
--
10
11
11
12
12
13
..
--
--
10
11
11
12
13
icates Per Effl
5
..
15
16
17
18
19
20
21
15
16
17
18
19
20
21
_
15
16
16
17
18
19
20
_
--
15
16
17
18.
19
20
6
21
22
23
24
26
27
28
30
_ _
22
23
24
25
27
28
29
_ _
21
22
24
25
26
27
29
..
21
22
23
25
26
27
28
7
28
30
31
33
34
36
38
40
28
29
31
32
34
35
37
39
28
29 ,
30 '
32
33
35
37
38
..
29
30
32
33
35
36
38
uent
8
37
38
40
42
44
46
48
50
36
38
40
42
43
45
47
49
36
38
39
41
43
:45
47
:49
36
37
39
41
43
44
46
48
Concentration
9
46
48
50
52
55
57
60
62
46
48
50
52
54
56
59
61
45
47
49
51
54
56
58
60
45
47
49
51
53
55
58
60
10
56
59
61
64
67
69
72
75
56
58
61
63
66
68
71
74
56
58
60
63
65
68
70
73
56
58
60
62
65
67
70
72
297
-------
TABLE F.5. CRITICAL VALUES FOR WILCOXON'S RANK SUM TEST WITH BONFERRONI'S
ADJUSTMENT OF ERROR RATE FOR COMPARISON OF "K" TREATMENTS
VERSUS A CONTROL FIVE PERCENT CRITICAL LEVEL (ONE-SIDED
ALTERNATIVE: TREATMENT CONTROL) (CONTINUED)
K No. Replicates No. of Reolicate Per
in Control
8 3
4
5
6
7
8
9
10
9 3
4
5
6
7
8
9
10
10 3
4
5
6
7
8
9
10
3
--
--
--
--
6
6
6
..
--
--
--
--
--
6
6
.
--
--
--
--
--
6
6
4
--
--
10
11
11
12
12
--
--
10
10
11
11
12
..
--
--
10
10
11
11
12
5
--
15
16
17
18
19
19
--
15
16
17
18
18
19
..
--
15
16
16
17
18
19
6
21
22
23
24
25
27
28
21
22
23
24
25
26
28
..
21
22
23
24
25
26
27
Effluent Concentration
7
29
30
31
33
34
36
37
28
30
31
33
34
35
37
..
28
29
31
32
34
35
37
8
36
37
39
40
42
44
46
48
37
39
40
42
44
46
47
37
38
40
42
43
45
47
9
45
47
49
51
53
55
57
59
45
46
48
50
52
55
57
59
45
46
48
50
52
54
56
58
10
55
57
59
62
64
67
69
72
55
57
59
62
64
66
69
71
55
57
59
61
64
66
68
71
298
-------
APPENDIX G
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 dubia data,
it provides a conservative test of the equality of any two survival
proportions assuming only the independence of responses from a Bernoulli
population. Additionally, since it is a conservative test, a pair-wise
comparison error rate of 0.05 is suggested rather that ati experiment-wise
error rate.
2. The basis for Fisher's Exact Test is a 2x2 contingency table. However, in
order to use this table the contingency table must be arranged in the format
shown in Table G.I. From the 2x2 table, set up for the control and the
concentration you wish to compare, you can determine statistical significance
by looking up a value in the table provided later in this section.
TABLE G.I. FORMAT FOR CONTINGENCY TABLE
Number of
Successes
Failures
Number of
Observations
Row 1
Row 2
Total
a
b
A - a
B - b
A
B
a + b [(A + B) - a - b]
A + B
3. Arrange the table so that the total number of observations for row one is
greater than or equal to the total for row two (A > B). Categorize a success
such that the proportion of successes for row one is greater than or equal to
the proportion of successes for row two (a/A > b/B). For the Ceriodaphnia
dubia survival data, a success may be 'alive' or 'dead', 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 the
contingency table. The table of significance levels of b is Table G.5. Enter
Table G.5 in the section for A, subsection for B, and the line for a. If the
b value of the contingency table is equal to or less than the integer in the
column headed 0.05 in Table G.5, then the survival proportion for the effluent
concentration is significantly different from the survival proportion for the
control. A dash or absence of entry in Table G.5 indicates that no
contingency table in that class is significant.
299
-------
4. To illustrate Fisher's Exact Test, a set of survival data (Table G.2) from
the daphnid, Ceriodaphnia dubia, survival and reproduction test will be used.
TABLE G.2. EXAMPLE OF FISHER'S EXACT TEST:
CERIODAPHNIA DUBIA MORTALITY DATA
Effluent
Concentration (%)
No. Dead
Total1
Control
1
3
6
12
25
1
0
0
0
0
10
9
10
10
10
10
10
1 Total number of live adults at the beginning of the test.
5. For each control and effluent concentration construct a 2x2 contingency
table.
6. For the control and effluent concentration of 1% the appropriate
contingency table for the test is given in Table G.3.
7. Since 10/10 > 8/9, the category 'alive' is regarded as a success. For A =
10, B = 9 and, a = 10, under the column headed 0.05, the value from Table G.5
is b = 5. Since the value of b (b = 8) from the contingency table
(Table G.3), is greater than the value of b (b = 5) from Table G.5, the test
concludes that the proportion of survival is not significantly different for
the control and 1% effluent.
8. The contingency tables for the combinations of control and effluent
concentrations of 3%, 6%, 12% are identical to Table G.3. The conclusion of
no significant difference in the proportion of survival for the control and
the level of effluent would also remain the same.
9. For the combination of control and 25% effluent, the contingency table
would be constructed as Table G.4. The category 'dead' is regarded as a
success, since 10/10 > 1/9. The b value (b = 1) from the contingency table
(Table G.4) is less than the b value (b = 5) from the table of significance
levels of b (Table G.5).
300.
-------
Thus, the percent mortality for 25% effluent is significantly greater than the
percent mortality for the control. Thus, the NOEC and LOEC for survival are
12% and 25%, respectively.
TABLE G.3. 2x2 CONTINGENCY TABLE FOR CONTROL AND 1% EFFLUENT
1% Effluent
Control
Total
Number
Alive
10
8
18
of
Dead
0
1
1
i
Number of
Observations
10
9
19
'
Table G.4. 2x2 CONTINGENCY TABLE FOR CONTROL AND 25% EFFLUENT
25% Effluent
Control
Total
Number
Dead
10
1
11
of
Alive
0
8
8
Number of
Observations
10
9
| 19
301
-------
TABLE 6.5. SIGNIFICANT LEVELS OF B: VALUES OF B (LARGE TYPE)
AND CORRESPONDING PROBABILITIES (SMALL TYPE)1
A*3 B=3
A=4 B=4
3
A*5 B-5
4
3
2
Ar6 B=6
5
4
3
2
A=7 B*7
6
5
a
3
4
4
5
4
5
4
5
5
6
5
4
6
5
4
6
5
6
5
6
7
6
5
4
7
6
5
4
7
6
5
7
6
5
7
6
7
Probability
0-05
0 -050
1 -014
0 429
1 -02*
0 424.
1 -040
0 440
0 418
0 -049
2 430
1 440
0 430
1 416 +
0 413
0 445 +
1 433
0 424
0 412
0 448
0 438
3 435-
1 415-
1 410+
0 435
2 421
1 425+
0 -016
0 449
2 445+
1 445+
0 427
1 424
0 415+
0 446+
0 408
0 433
0 428
0-025
1 414
1 424
1 424
0 408
0 418
1 408
0 408
0 415 +
0 413
0 -005-
0 424
0 412
2 410+
1 415"
0 410+
2 421
0 404
0 418
1 410+
0 408
1 424
0 415+
0 408
0-01
0 -004
0 408
1 -008
0 -COB
0 402
0 405-
1 -002
0 -002
1 405-
0 -004
0 -001
0 -008
0 403
0 -008
0-005
0 -004
0 401
0 402
0 -006-
1 -002
0 402
1 -005"
0 -004
0 401
0 403
A=8 B=8
7
6
5
4
3
2
A=9 B=9
8
7
6
a
8
7
6
5
4
8
7
6
5
8
7
6
5
8
7
6
5
8
7
6
8
7
8
9
8
7
6
5
4
9
8
7
6
5
9
8
7.
6
5
9
8
7
6
5
Probability
0-05
4 -038
2 420
1 -020
0 -013
0 438
3 426
2 -035"
1 -032
0 419
2 -015'
1 -016
0 -009
0 428
2 435'
1 -032
0 416
0 -044
1 -018
0 410 +
0 -030
0 -006
0 424
0 -022
5 -041
3 425-
2 -028
1 -025-
0 -015-
0 -041
4 -029
3 443
2 444
1 436
0 -020
3 -019
2 -024
1 -020
0 -010+
0 429
3 -044
2 -047
1 -035-
0 417
0 442
0-025
3 -013 '
2 -020
1 -020
0 -013
2 -007
1 -oos
0 -006
0 -019
2 -015-
1 -016
0 409
1 -007
0 -005"
0 -016
1 -018
0 -010 +
0 -006
0 -024
0 -022
4 415-
3 -025-
1 -008
1 -025-
0 -015-
3 -009
2 413
1 412
0 -007
0 420
3 -019
2 -024
1 -020
0 -010+
2 -011
1 411
0 -006
0 417
0-01
2 -003
1 -005 +
0 -003
2 -007
1 -009
0 -006
1 -003
0 -002
0 -009
1 -007
0 -DOS"
0 -002
0 -006
3 -005"
2 -008
1 -008
0 -005-
3 -009
1 -003
0 -002
0 -007
2 -005
1 -006
0 403
1 -002
0 -001
0 -006
0-005
2 -003
0 -001
0 403
1 -001
0 -001
1 -003
0 -002
0 -001
0 -oos'
0 -002
3 -005"
1 -002
0 -001
0 -005"
2 -002
1 -003
0 -002
2 -005-
0 -001
0 -003
1 -002
0 -001
The table shows:(1) In bold type, for given a, A and B, the value of b (Ca) which is just significant
at the probability level quoted (one-tailed test); and (2) In small type, for given A, B and r = a +
b, the exact probability (if there is independence) that b is equal to or less than the integer shown
in bold type. From Pearson and Hartley (1962).
302
-------
TABLE G.5.
SIGNIFICANT LEVELS OF B: VALUES OF B (LARGE TYPE) AND
CORRESPONDING PROBABILITIES (SMALL TYPE)1 (CONTINUED)
A=9 B=5
4
3
2
A=10 B=10
9
8
7
6
5
a
9
8
7
6
9
8
7
6
9
8
7
9
10
9
8
7
6
5
4
10
9
8
7
6
5
10
9
8
7
6
5
10
9
8
7
6
5
10
9
8
7
6
10
9
8
7
6
Probability
0-05
2 -027
1 -023
0 -010+
0 -028
1 -014
0 -007
0 -021
0 -049
1 -045+
0 -018
0 -045+
0 -018
6 -043
4 -029
3 -035-
2 -035-
1 -029
0 -016
0 -043
5 -033
4 -050-
2 -019
1 -015-
1 -040
0 -022
4 -023
3 -032
2 -O31
1 -023
0 -Oil
0 -029
3 -015-
2 -018
1 -013
1 -038
0 -017
0 -041
3 -036
2 -033
1 -024
0 -010+
0 -026 '
2 -022
1 -017
1 -047
0 -019
0 -042-
0-025
1 -005-
1 -023
0 -010+
1 -014
0 -007
0 -021
0 -005-
0 -018
0 -018
5 -016
3 -oio-
2 -012
1 -010"
0 -006+
0 -016
4 -011
3 -017
2 -019
1 -015-
0 -008
0 -022
4 -023
2 -009
1 -O08
1 -023
0 -Oil
3 -015-
2 -018
1 -013
0 -006
0 -017
2 -008
1 -008
1 -024
0 -010+
2 -022
1 -017
0 -007
0 -019
0-01
1 -005-
0 -003
0 -001
0 -007
0 -005
0 -005-
4 -005+
3 -010
1 -003
1 -010-
0 -005+
3 -003
2 -oos-
1 -004
0 -002
. 0 -008
3 -007
2 -009
1 -008
0 -004
2 -003
1 -004
0 -002
0 -003
2 -003
1 -oos
0 -003 .
-
1 -004
0 -ooz
0 -007
-
0-005
1 -006-
0 -003
0 -001
0 -005-
_
3 -002
2 -003
1 -003
0 -002
3 -003
2 -005-
1 -004
0 -002 .
2 -002
1 -002
0 -001
0 -004
2 -003
1 -004
0 -002
.
1 -001
0 -001
0 -003
1 -004
0 -002
.
A=10 B=4
3
2
A=11 B=11
10
9
8
7
6
a.
10
9
8
7
10
9
8
10
9
11
10
9
8
7
6
5
4
11
10
9
8
7
6
5
11
10
9
8
7
6
5
11
10
9
8
7
6
5
11
10
9
8
7
6
11
10
9
Probability
0-05
1 -Oil
1 -041
,0 -016-
0 -035-
1 -038
0 -014
0 -035-
0 -015+
0 -045+
7 -04S+
5 -032
4 -040
3 -043
2 -040
1 -032
0 -018
0 -045+
6 -035+
4 -021
3 -024
2 -023
1 -017
1 -043
0 -023
5 -026
4 -038
3 -040
ซ 2 -035-
1 -025 "
0 -012
0 -030
4 -018
3 -024
2 -022
1 -015-
1 -037
0 -017
0 -040
4 -043
3 -047
2 -039
1 -026-
0 -010+
0 -025-
3 -029
2 -028
1 -018
0-025
1 -011
0 -005-
0 -015-
0 -003
0 -014
0 -015+
6 -018
4 -012
3 -015"
2 -015-
1 -012
0 -006
0 -018
5 -012
4 -021
3 -024
2 -023
1 -017
0 -009
0 -023
4 -008
3 -012
2-012
1 -OO9
1 -02S-
0 -012
'
4 -018
3 -024
2 -022
1 -015-
0 -007
0 -017
3 -011
2 -013
1 -009
1 -025-
0 -010+
0 -025-
2 006
1 -005+
1 -018
0-01
0 -001
0 -005"
0 -003
5 -006
3 -004
2 -004
1 -004
0 -002
0 -006
4 -004
3 -007
2 -007
1 -006
0 -003
0 -009
4 -008
2 -003
1 -003
1 -009
0 -004
3 -005"
2 -009
1 -005-
0 -002
0 -007
2 -002
1 002
1 -009
0 -004
'.
2 -006
1 -005+
0 -002
0-005
0 -001
0 -005-
0 -003
4 -002
3 -004
2 -004
1 -004 .
0 -002
4 -004
2 -002
1 -002
0 -001
0 -003
'.
3 -002
2 -003
1 -003
0 , -001
0 -004
3 -005"
1 -001
1 -005"
0 -002
2 -002
1 -002
0 -001
0 -004
1 -001
0 -001
0 -002
303
-------
TABLE G.5. SIGNIFICANT LEVELS OF B: VALUES OF B (LARGE TYPE)
AND CORRESPONDING PROBABILITIES (SMALL TYPE)1
(CONTINUED)
.
As11 B=6
5
4
3
2
A*12 B=12
11
10
9
o:
8
7
6
11
10
9
8
7
11
10
9
8
11
10
9
11
10
12
11
10
9
8
7
6
5
4
12
11
10
9
8
7
6
5
12
11
10
9
8
7
6
5
12
11
10
9
8
Probability
0-05
1 -043
0 417
0 -O37
2 -018
1 -013
1 -038
0 -013
0 428
1 -009
1 -033
0 -on
0 428
1 433
0 .011
0 -027
0 .013
0 .038
8 .047
6 -034
5 445-
4 450-
3 450"
2 445-
1 -034
0 .018
0 447
7 437
5 -024
4 429
3 -030
2 -029
1 419
1 445-
0 424
6 429
5 443
4 448
3 -046
2 438
it
0 412
0 430
5 421
4 429
3 428
2 424
1 418
0-025
0 407
0 417
2 418
1 413
0 405-
0 413
1 409
0 404
0 -011
0 .003
0 411
0 413
7 419
5 414
4 -018
3 -020
2 418
1 -014
0 -007
0 419
6 414
5 424
3 410+
2 -009
1 407
1 419
0 409
0 424
5 410
4 -01 6+
3 417
2 415"
1 410+
0 405*
0 412
5 421
3 -009
2 408
2 424
1 -016
0-01
0 407
1 403
0 -001
0 -006-
1 409
0 404
0 403
6 407
4 -DOS"
3 -006
2 -008
1 40B-
0 402
0 -007
5 -cos-
4 -008
2 -003
2 -009
1 407
0 -003
0 -009
5 -010"
3 405-
2 -005-
1 -004
0 -002
0 405-
4 -008
3 -009
2 408
1 -006
0 402
0-005
_
1 -003
0 401
0 405-
0 401
0 -004
0 403
5 402
4 -oos-
2 -002
1 -001
1 405-
0 -002
5 405-
3 -002
2 -003
1 -002
0 -001
0 -003
4 -003
3 -005-
2 -005-
1 -004
0 -002
0 40S-
3 -002
2 -002
1 -002
0 -001
0 -002
A=12 B=9
8
7
6
5
4
3
2
A=13 B=13
a
7
6
5
12
11
10
9
.8
7
6
12
11
10
9
8
7
6
12
11
10
9
8
7
6
12
11
10
9
8
7
12
11
10
9
8
12
11
10
9
12
11
13
12
11
10
9
8
Probability
0-05
1 -037
0 -017
0 439
5 449
3 418
2 -015+
2 -040
1 -025 "
0 410+
0 424
4 438
3 -038
2 429
1 -017
1 -040
0 -018
0 -034
3 -025"
2 -022
1 -013
1 -032
0 411
0 426-
0 4SO-
2 -oi$-
1 -010-
1 428
0 -009
0 420
0 441
2 -050
1 427
0 -008
0 -019
0 -038
1 -029
0 -009
0 422
0 -044
0 411
0 433
9 448
7 -037
6 -048
4 -024
3 424
2 -021
0-025
0 407
0 -017
4 414
3 418
2 415+
1 410-
1 -025-
0 -010+
0 424
3 -009
2 -010-
1 406
1 -017
0 -007
0 -016
3 425-
2 422
1 -013
0 405"
0 -Oil
0 -02B"
2 -015
1 410-
0 -003
0 -009
0 -020
1 407
0 -003
0 -008
0 419
0 402
0 -009
0 -022
0 411
8 420
6 -015+
5 -021
4 -024
3 -024
2 -021
0-01
0 -007
.3 -004
2 404
1 -003
1 -010-
0 -004
3 -009
2 -010-
1 -006
0 -002
0 -007
2 405-
1 -004
0 -002
0 -005-
1 -002
1 -010"
0 -003
0 -009
1 -007
0 403
0 -008
0 -002
0 -009
7 -007
5 -006
4 408
3 -008
2 -008
1 -006
0-005
3 -004
2 -004
1 -003
0 -001
0 -004
2 -002
1 -002
0 401
0 -002
2 -005-
1 -004
0 -002
0 405"
1 -002
0 -001
0 -003
0 -001
0 -003
'
0 -002
6 -003
4 -002
3 -002
2 -002
1 -002
0 -001
304
-------
TABLE 6.5. SIGNIFICANT LEVELS OF B: VALUES OF B (LARGE TYPE)
CORRESPONDING PROBABILITIES (SMALL TYPE)r
(CONTINUED)
AND
A=13 B=13
12
11
10
9
8
7
at
7
6
5
4
13
12
11
10
9
8
7
6
5
13
12
11
10
9
8
7
6
5
13
12
11
10
9
8
7
6
5
13
12
11
10
9
8
7
6
5
13
12
11
10
9
8
7
6
13
12
Probability
0-05
2 -048
1 -037
0 -020
0 -048
8 -039
6 -027
5 -033
4 -036
3 -034
2 -029
1 -020
1 -048
0 -024
7 -031
6 -048
4 -021
3 -021
3 -060-
2 -040
1 -027
0 -O13
0 -030
6 -024
5 -03S-
4 -037
3 -033
2 -028
1 -017
1 -038
0 -017
0 -038
5 -017
4 -023
3 -022
2 -017
2 -040
1 -02B-
0'-010+
0 -023
0 -049
5 -042
4 -047
3 -041
2 -029
1 -017
1 -037
0 -015-
0 -032
4 -031
3 -031
0-025
1 -016 +
0 -007
0 -020
7 -01 6-
5 -DID-
4 -013
3 -013
2 -011
1 -008
1 -020
0 -010-
0 -024
6 -011
5 -018
4 -021
3 -021
2 -017
1 -011
0 -006-
0 -013
6 -024
4 -012
3 -012
2 -010+
1 -006
1 -017
0 -007
0 -017
5 -017
4 -023
3 -022
2 -017
1 -010 +
1 -02S-
0 -010 +
0 -023
4 -012
3 -014
2 -011
1 -007
1 -017
0 -006
0 -P1B-
'
3 .-007
2 -007
0-01
0 -003
0 -007
6 -005 -f
5 -010-
3 -004
2 -004
1 -003
1 -008
0 -004
0 -010-
5 -003
4 -008
3 -007
2 -008
1 -004
0 -002
0 -DOS-
5 -007
3 -003
2 -003
1 -002
1 -006
0 -003
0 -007
4 -008"
3 -007
2 -006
1 -004
0 -001
0 -004
3 -003
2 -003
1 -002
1 -007
0 -002
0 -006
'
.
3 -007
2 -007
0-005
0 -003
5 -002
4 -003
3 -004
2 -004
1 -003
0 -001
0 -004
5 -003
3 -002
2 -002
1 -001
1 -004
0 -002
0 -006-
4 -002
3 -003
2 -003
1 -002
0 -001
0 -003
4 -OOB-
2 -001
1 -001
1 -004
0 -001
0 -004
3 -003
2 -003
1 -002
0 -001
0 -002
'
2 -001
1 -001
A=13 B=7
6
5
4
3
2
A=14 B=14
13
a
11
10
9
8
7
6
13
12
11
10
9
8
7
13
12
11
10
9
8
13
12
11
10
9
13
12
11
10
13
12
14
13
12
11
10
9
8
7
6
5
4
14
13
12
11
10
9
8
; Probability
0-05
2 -022
1 -012
1 -029
0 -010 +
0 -022
0 -044
3 -021
2 -017
2 -046
1 -024
1 -060-
0 -017
0 -034
2 -012
2 -044
1 -022
1 -047
0 -01B-
0 -029
2 -044
1 -022
0 -006
0 -01 S-
0 -029
1 -02B
0 -007
0 -018
0 -036
0 -010-
0 -029
10 -049
8 -038
6 -023
5 -027
4 -028
3 -027
2 -023
1 -018
1 -038
0 -020
0 -049
9 -041
7 -029
6 -037
5 -041
4 -041
3 -038
2 -031
0-025
2 -022
1 -012
0 -004
O1 -010 +
0 -022
3 -021
2 -017
1 -010-
1 -024
0 -008
0 -017
2 -012
1 -008
1 -022
0 -007
0 -01 6-
1 -006
1 -022
0 -006
0 -01 B-
1 -02B
0 -007
0 -018
0 -010-
9 -020
7 -016
6 -023
4 -011
3 -011
2 -009
2 -023
1 -018
0 -008
0 -020
8 -018
6 -011
5 -015 +
4 -017
3 -016
2 -013
1 -009
0-01
1 -004
0 -002
0 -004
__
2 -004
1 -003
1 -010-
0 -003
0 -008
1 -002
1 -008
0 -002
0 -007
__
1 -006
0 -002
0 -006
__
0 -002
0 -007
__
0 -010-
8 -008
6 -006
5 -009
3 -004
2 -003
2 -009
1 -006
0 -003
0 -008
__
7 -006
5 -004
4 -005 +
3 -006
2 -005-
1 -003
1 -009
0-005
1 -004
0 -002
0 -004
___
2 -004
1 -003
0 -001
0 -003
1 -002
0 -001
0 -002
_
__
0 -000
0 -002
___
0 -002
,
, ,_
7 -003
5 -002
4 -003
3 -004
2 -003
1 -002
0 -001
0 -003
6 -002
5 -004
3 -002
2 -001
2 -005-
1 -003
305
-------
TABLE G.5. SIGNIFICANT LEVELS OF B: VALUES OF B (LARGE TYPE)
AND CORRESPONDING PROBABILITIES (SMALL TYPE)1
(CONTINUED)
A*14 8=13
12
11
10
9
8
7
6
5
14
13
12
11
10
9
8
7
6
14
13
12
11
10
9
8
7
6
5
14
13
12
11
10
9
8
7
6
5
14
13
12
11
m
1U
9
8
7
6
14
13
12
11
10
9
8
7
A
Probability
0-05
1 -021
1 -048
0 426-
8 -033
6 -021
5 -025+
4 -028
3 424
2 -oia
2 -042
1 -028
0. 413
0__n
430
7 -028
6 -039
5 -043
4 -042
3 -038
2 -027
1 -O17
1 -038
0 -017
0 -038
6 -020
5 428
4 -O28
3 -O24
2 -018
2 -O40
1 -024
0 -010-
0 -022
0 -047
6 .047
4 418
3 417
3 442
2___
429
1 417
1 438
0 414
0 430
5 438
4 -039
3 432
2 422
2 448
1 428
0 409
0 420
0-025
1 421
0 410 +
0 425-
7 412
6 421
4 409
3 409
3 424
2 410
1 412
0 405+
0 413
6 409
5 414
4 418
3 418-
2 411
1 407
1 417
0 407
0 417
6 420
4 409
3 409
3 424
2 418
1 411
1 424
0 410-
0 422
5 414
4 418
3 417
2 412
1 -nn-7
1 407
1 417
0 -000
0 414
4 410-
, 3 411
2 -ooo
2 422
1 412
0 404
0 409
0 420
0-01
0 404
6 404
5 -007
4 -000
3 -009
H -007
1 -006-
0 -002
0 405 +
__
6 409
4 404
3 405-
2 404
1 -003
1 407
0 403
0 407
5 -008
4 -009
3 409
2 -O07
1 -004
0 -002
0 -004
0 410-
_
4 404
3 -005-
2 -004
1 402
Innl
OO7
0 -002
0 -008
4 -010-
2 402
2 -009
1 405-
0 -002
0 404
0 409
0-005
0 -004
6 -004
4 -002
3 -003
2 -002
1 -002
1 -005-
0 -002
___
5 -003
4 -004
3 405-
2 -004
1 -003
0 -001
0 403
4 -002
3 -002
2 402
1 -001
1 -004
0 -002
0 -004
4 -004
3 -005-
2 -004
1 -002
fi nni
0 -002
3 -002
2 -002
1 -001
1 405-
0 -002
0 -004
A=14 B=7
6
5
4
3
2
A=15 8=15
14
13
12
11
10
9
8
7
14
13
12
11
10
9
8
7
14
13
12
11
10
9
8
14
13
12
11
10
9
14
13
12
11
14
13
12
15
14
13
12
11
10
9
8
7
6
5
4
Probability
0-05
4 -026
3 -025
2 417
2 441
1 -021
1 -043
0 -015-
0 -030
3 -018
2 414
2 -037
1 -018
1 438
0 412
0 -024
0 444
2 410 +
2 -037
1 -017
1 -038
0 -011
0 -022
0 -040
2 -039
1 419
1 -044
0 -011
0 -023
0 441
1 -022
0 408
0 -015-
0 429
0 408
0 425
0 450
11 450-
9 -040
7 425+
6 -030
5 -033
4 -033
3 430
2 425+
1 -018
1 440
0 421
0 -050-
0-025
3 -006
2 -006
2 417
1 -009
1 -021
0 -007
0 415-
3 -018
2 414
1 -007
1 418
0 405 +
0 -012
0 -024
*^~
2 410 +
1 406
1 -017
0 406-
0 -011
0 -022
1 -005-
1 419
0 -005-
0 -011
0 423
1 -022
0 -008
0 -015-
0 408
0 -025
10 421
8 -018
6 410+
5 -013
4 -013
3 -013
2 410+
1 -007
1 418
0 -008
0 -012
~
0-01
3 -006
2 -006
1 -003
1 -009
0 -003
0 -007
~~
:
2 -003
1 -002
1 -007
0 -002
0 -005 +
~~
~~-
~~
1 -001
1 -008
0 -002
0 -005-
"
1 -005-
0 -002
0 -005- '
_
^~
0 -001
0 -006
"""
1
0 -008
_
9 -008
7 -007
5 -004
4 -005-
3 -005"
2 404
1 -003
1 -007
0 -003
0 -008
^^
0-005
2 -001
1 -001
1 -003
0 -001
0 -003
"
1 "
* " '
2 -003
1 -002
0 -001
0 -002
-
~~
1 -001
0 -001
0 -002
0 -005-
"~~
1 -005-
0 -002
0 -005-
-
0 -001
-
_
"~~
8 -003
6 -003
5 -004
4 -006-
3 -005-
2 -004
1 -003
0 -001
0 -003
~
306
-------
TABLE G.5.
SIGNIFICANT LEVELS OF B: VALUES OF B (LARGE TYPE)
AND CORRESPONDING PROBABILITIES (SMALL TYPE)1
(CONTINUED)
A=15 B=14
13
12
11
10
9
0!
15
14
13
12
11
10
8
7
5
15
14
13
12
11
10
9
8
7
6
15
14
13
12
11
10
9
8
7
15
14
13
12
11
10
9
8
7
6
15
14
13
12
11
10 .
9
8
7
6
15
14
Probability
0-05
10 -042
8 -031
7 -041
6 -O4S
5 -048
4 -048
3 -041
2 -033
1 -022
-049
0 -025+
9 -03B-
7 -023
6 -029
5 -031
4 -030
3 -026
2 -020
2 -043
1 -029
0 -013
0 -031
8 -028
7-043
6 -049
5 -049
4 -045+
3 -038
2 -028
1 -018
1 -038
017
-037
7 -022
6 -032
5 -034
4 -032
3 -028
2 -019
2 -040
1 -024
1 -049
0 -022
0 -046
6 -017
.5-023
4 ' -022
3 -018
3 -042
2 -029
1 -016
1 -034
0 -013
0 -028
6 -042
5 -047
0-025
9 -017
7 -013
6 -O17
5 -020
4 -020
3 -018
2 -014
1 -009
1 -022
0 -011
8 -013
7 ;023
5 -011
4 -012
3 -011
2 -008
2 -020
1 -013
0 -005+
0 -O13
7 -010-
6 -016
5 -019
4 -019
3' -017
2 -012
1 -007
1 -018
0 -007
0 -017
7 -022
5 -011
4 -012
3 -010+
2 -008
2 -O19
1 -011
1 -024
0 -010-
0 -022
6 -O1 7
5 -023
4 -022
3 -018
2 -013
1 -007
0 -016
0 -006
1 -013
5 -012
4 -015-
0-01
8 -O08
6 -oos"
5 -007
4 -007
3 -007
2 -006
1 -004
,1 -oos
0 -004
7 -005"
6 -009
4 -004
3 -004
2 -003
2 -008
1 -005+
0 -002
0 -006+
7 -010 -
5 -006
4 -007
3 -006
2 -006"
1 -003
1 -007
0 -003
0 -007
"^
6 -007
4 -003
3 -003
2 -003
2 -008
1 -004
0 -002
0 -004
0 -010-
5 -oos-
4 -007
3 -007
2 -oos-
1 ' -003
1 -007
0 -002
0 -006
4 -003
3 -004
0-005
7 -002
6 -005-
4 -002
3 -002
2 -002
1 -001
1 -004
0 -001
0 -004
7 -005-
5 -003
4 -004
3 -004
2 -003
1 -002
0 -001
0 -002
6 -003
4 -002
3 -002
2 -002
2 -006-
1 -003
0 -001
0 -O03
5 -002
4 -003
3 -003
2 -003
1 002
1 -004
0 -002
0 -004
5 -005-
3 -002
2 -001
2 -oos-
1 -003
0 .001
0 -002
4 -003
A=15 B=9
8
7
6
5
4
3
2
a
13
12
11
10
9
8
7
6
15
14
13
12
11
10
9
8
7
6
15
14
13
12
11
10
9
8
7
15
14
13
12
11
10
9
8
15
14
13
12
11
10
9
15
14
13
12
11
10
15
14
13
12
11
15
14
13
Probability
0-05
4 -042
3 -032
2 -021
2 -045-
1 -024
1 -048
0 -019
0 -037
5 -032
4 -033
3 -026
2 -017
2 -037
1 -019
1 -038
0 -013
0 .026
0 -oso-
4 -023
3 -021
2 -O14
2 -032
1 -015+
1 -032
0 -010+
0 -020
0 -038
3 -01 6+
2 -011
2 -031
1 -014
1 -029
0 -009
0 -017
0 -032
2 -coo
2 -032
1 -014
1 -031
0 -008
0 -016
0 -030
2 -035+
1 -016
1 -03'7
0 -009
0 -018
0 -033
1 -020
0 -005-
0 -012
0 -025-
0 -043
0 -007
0 -022
0 -044
0-025
3 -013
2 -009
2 -021
, 1 -01 1
1 -O24
0 -009
0 -019
4 -008
3 -009
2 -006
2 -017
1 -008
1 -019
0 -006
0 -013
__
4 -023
3 -021
2 -014
1 -007
1 -015+
0 -006-
0 -010+
0 -O20
3 -016+
2 -Oil
1 -008
1 -014
0 -004
0 -009
0 -017
; 2 -009
1 -005 "
1 -014
0 -004
0 -008
0 -cue
1 -004
1 -018
0 -004
0 -009
0 -018
1 -020
0 -oos-
0 -012
0 -025-
0 -007
0 -022
0-01
2 -003
2 -009
1 -oos-
0 -002
0 -004
0 -009
4 -008
3 -009
2 -006
1 -003
1 -008
0 -003
0 -006
^_
3 -006-
2 -004
1 -002
1 -007
0 -002
0 -005-
2 -003
1 -002
1 -006
0 -002
0 -004
0 -009
2 -009
1 -oos-
0 -001
0 -004
0 -008
1 -004
0 -001
0 -004
0 -009
0 -001
0 -oos-
__
,^_
0 -007
0-005
2 -003
1 -002
1 -005 -
0 -002
0 -004
3 -002
2 -002
1 -001
1 -003
0 -001
0 -003
3 -005-
2 -004
1 -002
0 -001
0 -002
0 -COS'
2 -003
1 -002
0 -001
0 -002
0 -004
1 -001
1 -005-
0 -001
0 -004
1 -004
0 -001
0 -004
0 -001
0 -005"
307
-------
APPENDIX H
SINGLE CONCENTRATION TOXICITY TEST - COMPARISON OF CONTROL
WITH 100% EFFLUENT OR RECEIVING WATER
1. To statistically compare a control with one concentration, such as 100%
effluent or the instream waste concentration, a t-test is the recommended
analysis. The t-test is based on the assumptions that the observations are
independent and normally distributed and that the variances of the
observations are equal between the two groups.
2 Shapiro Wilk's test may be used to test the normality assumption (see
Appendix B for details). If the data do not meet the normality assumption,
the nonparametric test, Wilcoxon's Rank Sum Test, may be used to analyze the
data An example of this test is given in Appendix F. Since a control and
one concentration are being compared, the K = 1 section of Table F.5 contains
the needed critical values.
3 The F test for equality of variances is used to test the homogeneity of
variance assumption. When conducting the F test, the alternative hypothesis
of interest is that the variances are not equal.
4. To make the two-tailed F test at the 0.01 level of significance, put the
larger of the two variances in the numerator of F.
F =
where Sฑ2 > S22
5. Compare F with the 0.005 level of a tabled F value with n, - 1 and n2 - 1
degrees of freedom, where n., and n2 are the number of replicates for each of
the two groups.
6 A set of Cen'odaphm'a dubia reproduction data from an effluent screening
test will be used to illustrate the F test. The raw data, mean and variance
for the control and 100% effluent are given in Table H.I.
7. Since the variability of the 100% effluent is greater than the variability
of the control, S for the 100% effluent concentration is placed in the
numerator of the F statistic and S for the control is placed in the
denominator.
F =
36.61
14.55
8. There are 9 replicates for the effluent concentration and 8 replicates for
the control. Thus, the numerator degrees of freedom is 8 and the denominator
degrees of freedom is 7. For a two-tailed test at the 0.01 level of
significance, the critical F value is obtained from a table of the F
308
-------
TABLE H.I. CERIODAPHNIA DUBIA REPRODUCTION DATA
FROM AN EFFLUENT SCREENING TEST
Replicate
8
10
Control 36 38 35 35 28 41 37 33
100% Effluent 23 14 21 7 12 17 23 8 18
35.4 14.5
15.9 36.6
distribution (Snedecor and Cochran, 1980). The critical F value for this test
is 8.68. Since 2.52 is not greater than 8.68, the conclusion is that the
variances of the control and 100% effluent are homogeneous.
9. EQUAL VARIANCE T-TEST
9.1 To perform the t-test, calculate the following test statistic:
t = ?1~I*
Where:
s I A+_L
"N *i *2
Y, = Mean for the control
Y2 = Mean for the effluent concentration
ni+n2-2
S, = Estimate of the variance for the control
2 ' '
S2 = Estimate of the Variance for the effluent
concentration
n, = Number of replicates for the control
n2 = Number of replicates for the effluent
concentration
9.2 Since we are usually concerned with a decreased response from the
309
-------
control, such as a decrease in survival or a decrease in reproduction, a
one-tailed test is appropriate. Thus, compare the calculated t with a
critical t, where the critical t is at the 5% level of significancewithn, +
n - 2 degrees of freedom. If the calculated t exceeds the critical t, the
mean responses are declared different.
9.3 Using the data from Table H.I to illustrate the t-test, the calculation
of t is as follows:
35.4-15.9 = 7 go
Cป ~~ *"* "
Where:
(8-1)14.5+(9-l)36.6 = 5 13
(8+9-2)
9 4 For an 0.05 level of significance test with 15 degrees of freedom the
critical t is 1.754 (Note: Table D.5 for K = 1 includes the critical t values
for comparing two groups). Since 7.82 is greater than 1.754, the conclusion
is that the reproduction in the 100% effluent concentration is significantly
lower than the control reproduction.
10. UNEQUAL VARIANCE T-TEST
10 1 If the F test for equality of variance fails, the t-test is still a
valid test. However, the denominator of the t statistic is adjusted as
f ol1ows:
t =
n2
Where: Y., = Mean for the control
Mean for the effluent concentration
Estimate of the variance for the control
Estimate of the variance for the effluent
concentration
n1 = Number of replicates for the control
n2 = Number of replicates for the effluent
concentration
310
-------
10.2 Additionally, the degrees of freedom for the test are adjusted using the
following formula: i
df =
ca+(l-O2(n1-l)
Where:
C =
10.3 The modified degrees of freedom is usually not an integer.
practice is to round down to the nearest integer.
Common
10.4 The t-test is then conducted as the equal variance t-test. The
calculated t is compared to the critical t at the 0.05 significance level with
the modified degrees of freedom. If the calculated t exceeds the critical t,
the mean responses are found to be statistically different.
311
-------
APPENDIX I
PROBIT ANALYSIS
1. This program calculates the EC1 and EC50 (or LCI and LC50), and the
associated 95% confidence intervals.
2. The program is written in IBM PC Basic for the IBM compatible PC by
Computer Sciences Corporation, 26 W. Martin Luther King Drive, Cincinnati, OH
45268. A compiled, executable version of the program can be obtained from
EMSL-Cincinnati by sending a written request to EMSL at 3411 Church Street,
Cincinnati, OH 45244.
2.1 Data input is illustrated by a set of total mortality data (Figure I.I)
from a fathead minnow embryo-larval survival and teratogenicity test. The
program requests the following input:
1. Desired output of abbreviated (A) or full (F) output? (Note: only
abbreviated output is shown below.)
2. Output designation (P = printer, D = disk file).
3. Title for the output.
4*. The number of exposure concentrations.
5. Toxicant concentration data.
2.2 The program output for the abbreviated output includes the following:
1. A table of the observed proportion responding and the proportion
responding adjusted for the controls (see Figure 1.2).
2. The calculated chi-square statistic for heterogeneity and the tabular
value. This test is one indicator of how well the data fit the model.
The program will issue a warning when the test indicates that the data
do not fit the model.
3. Estimated LCI and LC50 values and associated 95% confidence intervals
(see Figure 1.2).
312
-------
USEPA PROSIT ANALYSIS PROGRAM
USED FOR CALCULATING LC/EC VALUES
Version 1.5
Do you wish abbreviated (A) or full ? P
Title ? Example of Probit Analysis
Number responding in the control group = ? 2
Number of animals exposed in the concurrent control group = ? 20
Number of exposure concentrations, exclusive of controls ? 5
Input data starting with the lowest exposure concentration
Concentration = ? 0.5
Number responding = ? 2
Number exposed = ? 20
Concentration = ? 1.0
Number responding = ? 1
Number exposed = ? 20
Concentration = ? 2.0
Number responding = ? 4
Number exposed = ? 20
Concentration = ? 4.0
Number responding = ? 16
Number exposed = ? 20
Concentration = ? 8.0
Number responding = ? 20
Number exposed = ? 20
Number
1
2
3
4
5
Cone.
0.5000
1.0000
2.0000
4.0000
8.0000
Number
Resp.
2~
1
4
16
20
Number
Exposed
20
20
20
20
20
Do you wish to modify your data ? N
The number of control animals which responded = 2
The number of control animals exposed = 20
Do you wish to modify these values ? N
Figure1.I. Sample Data Input for USEPA Probit Analysis Program, Version
J. , 0- i
313
-------
Example of Probit Analysis
Cone.
Control
0.5000
1.0000
2.0000
4.0000
8.0000
Number
Exposed
20
20
20
20
20
20
Number
Resp.
2
2
1
4
16
20
Observed
Proportion
Responding
0.1000
0.1000
0.0500
0.2000
0.8000
1.0000
Proportion
Responding
Adjusted for
Controls
0.0000
0.0174
-.0372
0.1265
0.7816
1.0000
Chi - Square for Heterogeneity (calculated)
Chi - Square for Heterogeneity
(tabular value at 0.05 level)
0.441
7.815
Example of Probit Analysis
Estimated LC/EC Values and Confidence Limits
Point
LC/EC 1.00
LC/EC 50.00
Exposure
Cone.
1.346
3.018
Lower Upper
95% Confidence Limits
0.453
2.268
1.922
3.672
Figure 1.2.
USEPA Probit Analysis Program Used for Calculating LC/EC
Values, Version 1.5.
314
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APPENDIX J
SPEARMAN-KARBER METHOD
i
1. The Spearman-Karber Method is a nonparametric statist!ca'l procedure for
estimating the LC50 and the associated 95% confidence interval (Finney, 1978).
The Spearman-Karber Method estimates the mean of the distribution of the Iog10
of the tolerance. If the log tolerance distribution is symmetric, this
estimate of the mean is equivalent to an estimate of the median of the log
tolerance distribution.
2. If the response proportions are not monotonically non-decreasing with
increasing concentration (constant or steadily increasing with concentration),
the data must be smoothed. Abbott's procedure is used to "adjust" the
concentration response proportions for mortality occurring in the control
replicates. i
3. Use of the Spearman-Karber Method is recommended when partial mortalities
occur in the test solutions, but the data do not fit the Probit model.
4. To calculate the LC50 using the Spearman-Karber Method, the following must
be true: 1) the smoothed adjusted proportion mortality for the lowest
effluent concentration (not including the control) must be zero, and 2) the
smoothed adjusted proportion mortality for the highest effluent concentration
must be one.
5. To calculate the 95% confidence interval for the LC50 estimate, one or
more of the smoothed adjusted proportion mortalities must be between zero and
one. !
i
6. The Spearman-Karber Method is illustrated below using a set of mortality
data from a Fathead Minnow Larval Survival and Growth test. These data are
listed in Table J.I.
TABLE J.I. EXAMPLE OF SPEARMAN-KARBER METHOD: MORTALITY DATA FROM
A FATHEAD MINNOW LARVAL SURVIVAL AND GROWTH TEST
(40 ORGANISMS PER CONCENTRATION)
Eff 1 uent
Concentration
Control
6.25%
12.5%
25.0%
50.0%
100.0%
Number of
Mortalities
2
2
0
0
26
40
Mortality
Proportion
0.05
0.05
0.00
0.00
0.65
1.00
315
-------
7. Let p0, pr ..., pk denote the observed response proportion mortalities
for the control and k effluent concentrations. The first step is to smooth
the p,. if they do not satisfy p0 < p1 < ... < pk. The smoothing process
replaces any adjacent p('s that do not conform to p0 < p., < ... < pk with their
average. For example, if p,- is less than p,-.., then:
P/-I -Pi = (Pi+Pi-i)/2
Where: p? -= the smoothed observed proportion mortality for effluent
concentration i.
7.1 For the data in this example, because the observed mortality proportions
for the control and the 6.25% effluent concentration are greater than the
observed response proportions for the 12.5% and 25.0% effluent concentrations,
the responses for these four groups must be averaged:
Pcf = P/ = P/ = P/ = 0-05+0-05*ฐ-00+0-00 - -^ =0.025
7.2 Since p4 - 0.65 is larger than pj, set p^ = 0.65. Similarly, p5 = 1.00 is
larger than p^, so set p^ = 1.00. Additional smoothing is not necessary. The
smoothed observed proportion mortalities are shown in Table J.2.
8. Adjust the smoothed observed proportion mortality in each effluent
concentration for mortality in the control group using Abbott's formula
(Finney, 1971). The adjustment takes the form:
Where: p? = (p* - p*} / (1 - PQ)
Po = the smoothed observed proportion mortality for the control
p? = the smoothed observed proportion mortality for effluent
concentration i .
8.1 For the data in this example, the data for each effluent concentration
must be adjusted for control mortality using Abbott's formula, as follows:
ซa a _ _a _ & _ Pi~PoS _ 0.025-0.025 _ 0.0 _
Po =Pi -P2 -Pa -
i-o.025 975
a_P4-Po_ 0.650-0.025, 0 . 0625 ,
P4 - 1-0.025 " 0.975
316
-------
~~ t j* n nr\n_n nr>c n a *~7 si
1.000
a_^s-Po 1.000-0.025 _ 0.975
l-p<
1-0.025 0.975
The smoothed, adjusted response proportions for the effluent concentrations
are shown in Table J.2. A plot of the smoothed, adjusted data is shown in
Figure J.I.
TABLE J.2. EXAMPLE OF SPEARMAN-KARBER METHOD: SMOOTHED, ADJUSTED
MORTALITY DATA FROM A FATHEAD MINNOW LARVAL SURVIVAL
AND GROWTH TEST
Effluent
Concentration
Control
6.25%
12.5%
25.0%
50.0%
100.0%
Mortality
Proportion
0.05
0.05
0.00
0.00
0.65
1.00
Smoothed
Mortality
Proportion
0.025
0.025
0.025
0.025
0.650
1.000
Smoothed ,
| Adjusted
Mortal i ty
Proportion
0.000
; 0.000
0.000
0.000
0.641
1.000
9. Calculate the Iog10 of the estimated LC50, m, as follows:
Where: p? = the smoothed adjusted proportion mortality at concentration i
X,- = the Iog10 of concentration i
k = the number of effluent concentrations tested, not including the
control.
9.1 For this example, the Iog10 of the estimated LC50, m, is calculated as
follows:
m = [(0.000 - 0.000) (0.7959 + 1.0969)]/2 +
[(0.000 - 0.000) (1.0969 + 1.3979)]/2 +
[(0.641 - 0.000) (1.3979 + 1.6990)]/2 +
[(1.000 - 0.641) (1.6990 + 2.0000)3/2
= 1.656527
317
-------
CO
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*
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8
ง
.
to
CM
111
o
o
o
T- 111
ti-
ll.
Ill
in
UCM
re
C4-
cu
s-
o
ns
+->
nj
O)
CO
=3
TJ
OJ
O
o
oo
cu
O
51
s-
3
en
-------
10. Calculate the estimated variance of m as follows:
i=2
4
Where: X,- = the Iog10 of concentration i
n,- = the number of organisms tested at effluent concentration i
p* = the smoothed adjusted observed proportion mortality at effluent
concentration i
k = the number of effluent concentrations tested, not including the
control. !
10.1 For this example, the estimated variance of m, V(m), is calculated as
fol1ows:
V(m) = (0.000)(1.000)(1.3979 - 0.7959)?/4(39) +
(0.000)(1.000)(1.6990 - 1.0969)V4(39) +
(0.641)(0.359)(2.0000 - 1.3979)74(39)
= 0.00053477
QjV(m)
is calculated as
11. Calculate the 95% confidence interval for m: mฑ2.
11.1 For this example, the 95% confidence interval for m
follows:
1.656527ฑ2^0-. 00053477 = (1.610277, 1.702777)
12. The estimated LC50 and a 95% confidence interval for the estimated LC50
can be found by taking base10 antilogs of the above values,,
12.1 For this example, the estimated LC50 is calculated as follows:
LC50 = antilog(m) = antilog(1.656527) = 45.3%.
12.2 The limits of the 95% confidence interval for the estimated LC50 are
calculated by taking the antilogs of the upper and lower limits of the 95%
confidence interval for m as follows:
lower limit: antilog(l.610277) = 40.8%
upper limit: antilog(l.702777) = 50.4%
319
-------
APPENDIX K
TRIMMED SPEARMAN-KARBER METHOD
1. The Trimmed Spearman-Karber Method is a modification of the Spearman-
Karber Method, a nonparametric statistical procedure for estimating the LC50
and the associated 95% confidence interval (Hamilton et al ; 1977). Appendix
The Trimmed Spearman-Karber Method estimates the trimmed mean of the
distribution of the Iog10 of the tolerance. If the log tolerance distribution
is symmetric, this estimate of the trimmed mean is equivalent to an estimate
of the median of the log tolerance distribution.
2. If the response proportions are not monotonically non-decreasing with
increasing concentration (constant or steadily increasing with concentration),
the data must be smoothed. Abbott's procedure is used to "adjust" the
concentration response proportions for mortality occurring in the control
replicates.
3. Use of the Trimmed Spearman-Karber Analysis is recommended only when the
requirements for the Probit Method and the Spearman-Karber Method are not met.
4. To calculate the LC50 using the Trimmed Spearman-Karber Method, the
smoothed, adjusted, observed proportion mortalities must bracket 0.5.
5. To calculate the 95% confidence interval for the LC50 estimate, one or
more of the smoothed, adjusted, observed proportion mortalities must be
between zero and one.
6. Let p0, pซ, ..., p. denote the observed proportion mortalities for the
control and the k effluent concentrations. The first step is to smooth the p,-
if they do not satisfy p0 < p, < ... < pk. The smoothing process replaces any'
adjacent p,-'s that do not conform to p0 < p1 < . . . < pk, with their average.
For example, if p,. is less than p,..,, then:
Where: p'., ซ p? = (p, + p^J/Z
p* - the smoothed observed proportion mortality for effluent
concentration i .
7. Adjust the smoothed observed proportion mortality in each effluent
concentration for mortality in the control group using Abbott's formula
(Finney, 1971). The adjustment takes the form:
Where: p* = (p? - p*) / (1 - p*)
Po = the smoothed observed proportion mortality for the control
pฎ = the smoothed observed proportion mortality for effluent
concentration i.
320
-------
8. Calculate the amount of trim to use in the estimation of the LC50 as
follows:
Where: Trim = max(p*, l-pฃ)
p^ = the smoothed, adjusted proportion mortality for the lowest
effluent concentration, exclusive of the control
pฃ = the smoothed, adjusted proportion mortality for the highest
effluent concentration
k = the number of effluent concentrations, exclusive of the
control.
The minimum trim should be calculated for each data set rather than using a
fixed amount of trim for each data set.
9. Due to the intensive nature of the calculation for the estimated LC50 and
the calculation of the associated 95% confidence interval using the Trimmed
Spearman-Karber Method, it is recommended that the data be analyzed by
computer.
10. A computer program which estimates the LC50 and associated 95% confidence
interval using the Trimmed Spearman-Karber Method, can be obtained from EMSL-
Cincinnati by sending a written request to EMSL, 3411 Church Street,
Cincinnati, OH 45244.
11. The Trimmed Spearman-Karber program automatically performs the following
functions:
a. Smoothing.
b. Adjustment for mortality in the control.
c. Calculation of the necessary trim.
d. Calculation of the LC50.
e. Calculation of the associated 95% confidence interval.
12. To illustrate the Trimmed Spearman-Karber method using the Trimmed
Spearman-Karber computer program, a set of data from a Fathead Minnow Larval
Survival and Growth test will be used. The data are listed in Table K.I.
i
12.1 The program requests the following input (Figure K.I):
au Output destination (D = disk file, P = printer).
b. Control data.
c. Data for each toxicant concentration.
' i
12.2 The program output includes the following (Figure K.2):
a. A table of the concentrations tested, number of organisms
exposed, and mortalities.
b. The amount of trim used in the calculation.
c. The estimated LC50 and the associated 95% confidence interval.
321
-------
TABLE K.I. EXAMPLE OF TRIMMED SPEARMAN-KARBER METHOD: MORTALITY
DATA FROM A FATHEAD MINNOW LARVAL SURVIVAL AND GROWTH
TEST (40 ORGANISMS PER CONCENTRATION)
Effluent Number of Mortality
Concentration Mortalities Proportion
Control
6.25
12.5
25.0
50.0
100.0
2
0
2
0
0
32
0.05
0.00
0.05
0.00
0.00
0.80
322
-------
A:>spearman
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5
ENTER DATE OF TEST:
1
ENTER TEST NUMBER:
2
WHAT IS TO BE ESTIMATED?
(ENTER "L" FOR LC50 AND "E" FOR EC50)
L '
ENTER TEST SPECIES NAME:
Fathead minnow
ENTER TOXICANT NAME:
Eff1uent
ENTER UNITS FOR EXPOSURE CONCENTRATION OF TOXICANT:
f
ENTER THE NUMBER OF INDIVIDUALS IN THE CONTROL:
40
ENTER THE NUMBER OF MORTALITIES IN THE CONTROL:
2
ENTER THE NUMBER OF CONCENTRATIONS
(NOT INCLUDING THE CONTROL; MAX = 10):
5
ENTER THE 5 EXPOSURE CONCENTRATIONS (IN INCREASING ORDER):
6.25 12.5 25 50 100
ARE THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION EQUAL(Y/N)?
ENTER THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION:
40
ENTER UNITS FOR DURATION OF EXPERIMENT
(ENTER "H" FOR HOURS, "D" FOR DAYS, ETC.):
Days
ENTER DURATION OF TEST:
7
ENTER THE NUMBER OF MORTALITIES AT EACH EXPOSURE CONCENTRATION:
0 2 0 0 32
WOULD YOU LIKE THE AUTOMATIC TRIM CALCULATION(Y/N)?
y !
Figure K.I. Example input for Trimmed Spearman-Karber Method.
323
-------
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5
DATE: 1
TOXICANT: effluent
SPECIES: fathead minnow
RAW DATA: Concentration
TEST NUMBER: 2
DURATION: 7 Days
.00
6.25
12.50
25.00
50.00
100.00
SPEARMAN-KARBER TRIM:
SPEARMAN-KARBER ESTIMATES:
Number
Exposed
40
40
40
40
40
40
20.41%
LC50:
Mortal i
2
0
2
0
0
32
77.;
95% CONFIDENCE LIMITS
ARE NOT RELIABLE.
NOTE: MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING.
ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION.
Figure K.2. Example output for Trimmed Spearman-Karber Method.
324
-------
APPENDIX L
GRAPHICAL METHOD
1. The Graphical Method is used to calculate the LC50. It is a mathematical
procedure which estimates the LC50 by linearly interpolating between points of
a plot of observed percent mortality versus the base 10 logarithm (Iog10) of
percent effluent concentration. This method does not provide a confidence
interval for the LC50 estimate and its use is only recommended when there are
no partial mortalities. The only requirement for the Graphical Method is that
the observed percent mortalities bracket 50%.
2. For an analysis using the Graphical Method the data must first be smoothed
and adjusted for mortality in the control replicates. The procedure for
smoothing and adjusting the data is detailed in the following steps.
I
3. The Graphical Method is illustrated below using a set of mortality data
from an Fathead Minnow Larval Survival and Growth test. These data are listed
in Table L.I.
TABLE L.I. EXAMPLE OF GRAPHICAL METHOD: MORTALITY DATA FROM A
FATHEAD MINNOW LARVAL SURVIVAL AND GROWTH TEST (40
ORGANISMS PER CONCENTRATION)
Effluent
Concentration
%
Control
6.25
12.5
25.0
50.0
100.0
Number of
Mortalities
2
0
0
0
40
40
Mortality
Proportion
0.05
0.00
0.00
0.00
1.00
1.00
4. Let p0, p., ..., p,< denote the observed proportion mortalities for the
control and the k effluent concentrations. The first step is to smooth the p,
if they do not satisfy p0 < p, < ... < pk. The smoothing process replaces any
adjacent pf's that do not conform to p0 < p., < ... < pk with their average.
For example, if p,- is less than pf., then: i
Where:
P/-I = Pi
p? = the smoothed observed proportion mortality for effluent
concentration i.
4.1 For the data in this example, because the observed mortality proportions
for the 6.25%, 12.5%, and 25.0% effluent concentrations are less than the
325 !
-------
observed response proportion for the control, the values for these four groups
must be averaged:
Po
P!S =
- 0.05+0.00+0.00+0.00 0.05 _ ...-_
- - = 0 . 0125
4 4
4.2 Since p4 = p5 - 1.00 are larger then 0.0125, set p^ = pg = 1.00.
Additional smoothing is not necessary. The smoothed observed proportion
mortalities are shown in Table L.2.
TABLE L.2. EXAMPLE OF GRAPHICAL METHOD: SMOOTHED, ADJUSTED
MORTALITY DATA FROM A FATHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
Effluent
Concentration
V
/o
Control
6.25
12.5
25.0
50.0
100.0
Mortality
Proportion
0.05
0.00
0.00
0.00
1.00
1.00
Smoothed
Mortality
Proportion
0.0125
0.0125
0.0125
0.0125
1.0000
1.0000
Smoothed,
Adjusted
Mortality
Proportion
0.00
0.00
0.00
0.00
1.00
1.00
5. Adjust the smoothed observed proportion mortality in each effluent
concentration for mortality in the control group using Abbott's formula
(Finney, 1971). The adjustment takes the form:
Where:
Pi
Pi = (P/-P03) /(l-PoS)
= the smoothed observed proportion mortality for the control
= the smoothed observed proportion mortality for effluent
concentration i.
5.1 Because the smoothed observed proportion mortality for the control group
is greater than zero, the responses must be adjusted using Abbott's formula,
as follows:
326
-------
a_ a_ a _ a _ Pi ~Pv _ 0 . 0125 - 0 . 0125 = 0.0
Po -Pi -Pa -Ps - s 1 T 0.0125 "0.9875
a = a _ P/~Pog _ 1.00 -0.0125 _ 0.9875 _ ฑ 00
P4 5 i _ns 1-0.0125 0.9875
A table of the smoothed, adjusted response proportions for the effluent
concentrations are shown in Table L.2.
^
5.2 Plot the smoothed, adjusted data on 2-cycle semi-log graph paper with the
logarithmic axis (the y axis) used for percent effluent concentration and the
linear axis (the x axis) used for observed percent mortality. A plot of the
smoothed, adjusted data is shown in Figure L.I.
6. Locate the two points on the graph which bracket 50% mortality and connect
them with a straight line.
7. On the scale for percent effluent concentration, read the value for the
point where the plotted line and the 50% mortality line intersect. This value
is the estimated LC50 expressed as a percent effluent concentration.
7.1 For this example, the two points on the graph which bracket the 50%
mortality line (0% mortality at 25% effluent, and 100% mortality at 50%
effluent) are connected with a straight line. The point at which the plotted
line intersects the 50% mortality line is the estimated LC50. The estimated
LC50 = 35% effluent.
327
-------
LLJ
H
LU
ID
LL
LJL
LU
H
LLI
O
DC
LU
Q.
100
50
10
1
0 10 20 30 40 50 60 70 80 90 100
PERCENT MORTALITY
Figure L.I Plot of the smoothed adjusted response proportions for fathead
minnow, PimephaTes promeTas, survival data.
328
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APPENDIX M
LINEAR INTERPOLATION METHOD j
1. GENERAL PROCEDURE
1.1 The Linear Interpolation Method is used to calculate a point estimate of
the effluent or other toxicant concentration that causes a given percent
reduction (e.g., 25%, 50%, etc.) in the reproduction or growth of the test
organisms (Inhibition Concentration, or 1C). The procedure was designed for
general applicability in the analysis of data from short-term chronic toxicity
tests, and the generation of an endpoint from a continuous model that allows a
traditional quantitative assessment of the precision of the endpoint, such as
confidence limits for the endpoint of a single test, and a mean and
coefficient of variation for the endpoints of multiple tests.
1.2 The Linear Interpolation Method assumes that the responses (1)
monotonically non-increasing, where the mean response for each higher
concentration is less than or equal to the mean response for the previous
concentration, (2) follow a piecewise linear response function, and (3) are
from a random, independent, and representative sample of test data. If the
data are not monotonically nonincreasing, they are adjusted by smoothing
(averaging). In cases where the responses at the low toxicant concentrations
are much higher than in the controls, the smoothing process may result in a
large upward adjustment in the control mean. Also, no assumption is made
about the distribution of the data except that the data within a group being
resampled are independent and identically distributed. ,
i
2. DATA SUMMARY AND PLOTS
2.1 Calculate the mean responses for the control and each toxicant
concentration, construct a summary table, and plot the data.
3. MONOTONICITY
3.1 If the assumption of monotonicity of test results is met, the observed
response means (Y,-) should stay the same or decrease as the toxicant
concentration increases. If the means do not decrease monotonically, the
responses are "smoothed" by averaging (pooling) adjacent means.
3.2 Observed means at each concentration are considered in order of
increasing concentration, starting with the control mean (Y,). If the mean
observed response at the lowest toxicant concentration (Y2) is equal to or
smaller than the control mean (Y.,), it is used as the response. If it is
larger than the control mean, it is averaged with the control, and this
average is used for both the control response (M.,) and the lowest toxicant .
concentration response (M2) . This mean is then compared to the mean observed
response for the next higher toxicant concentration (Y3) . Again, if the mean
observed response for the next higher toxicant concentration is smaller than
the mean of the control and the lowest toxicant concentration, it is used as
the response. If it is higher than the mean of the first two, it is averaged
with the first two, and the mean is used as the. response for the control and
329
-------
two lowest concentrations of toxicant. This process is continued for data
from the remaining toxicant concentrations. A numerical example of smoothing
the data is provided below. (Note: Unusual patterns in the deviations from
monotonicity may require an additional step of smoothing). Where Yf decrease
monotonically, the Y,- become M,- without smoothing.
4. LINEAR INTERPOLATION METHOD
4.1 The method assumes a linear response from one concentration to the next.
Thus, the ICp is estimated by linear interpolation between two concentrations
whose responses bracket the response of interest, the (p) percent reduction
from the control.
4.2 To obtain the estimate, determine the concentrations Cj and CJ4.- which
bracket the response M, (1 - p/100), where M., is the smoothed control mean
response and p is the percent reduction in response relative to the control
response. These calculations can easily be done by hand or with a computer
program as described below. The linear interpolation estimate is calculated
as follows:
ICp = Cj + [ Mฑ (l - p/100) -
(g.7 + i -
UfcH-l -
Where: Cd = tested concentration whose observed mean response is
greater than M.,(l - p/100).
C, + . = tested concentration whose observed mean response is less
than M,(l - p/100).
M., = smoothed mean response for the control.
Mj = smoothed mean response for concentration J.
Mj+ 1 = smoothed mean response for concentration J + 1.
p = percent reduction in response relative to the control
response.
ICp = estimated concentration at which there is a percent
reduction from the smoothed mean control response.
The ICp is reported for the test, together with the 95%
confidence interval calculated by the ICPIN.EXE program
described below.
4.3 If the Cj is the highest concentration tested, the ICp would be specified
as greater than C,. If the response at the lowest concentration tested is
used to extrapolate the ICp value, the ICp should be expressed as a less than
the lowest test concentration.
330
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5. CONFIDENCE INTERVALS
5.1 Due. to the use of a linear interpolation technique to calculate an
estimate of the ICp, standard statistical methods for calculating confidence
intervals are not applicable for the ICp. This limitation is avoided by use a
technique known as the bootstrap method as proposed by Efron (1982) for
deriving point estimates and confidence intervals.
5.2 In the Linear Interpolation Method, the smoothed response means are used
to obtain the ICp estimate reported for the test. The bootstrap method is
used to obtain the 95% confidence interval for the true mean. In the
bootstrap method, the test data Yj,- is randomly resampled with replacement to
produce a new set of data Yj,-*, that is statistically equivalent to the
original data, but a new and slightly different estimate of the ICp (ICp*) is
obtained. This process is repeated at least 80 times (Marcus and Holtzman,
1988) resulting in multiple "data" sets, each with an associate ICp* estimate.
The distribution of the ICp* estimates derived from the sets of resampled data
approximates the sampling distribution of the ICp estimate. The standard
error of the ICp is estimated by the standard deviation of the individual ICp*
estimates. Empirical confidence intervals are derived from the quantiles of
the ICp* empirical distribution. For example, if the test data are resampled
a minimum of 80 time, the empirical 2.5% and the 97.5% confidence limits are
approximately the second smallest and second largest ICp* estimates (Marcus
and Holtzman, 1988).
5.3 The width of the confidence intervals calculated by the bootstrap method
is related to the variability of the data. When confidence intervals are
wide, the reliability of the 1C estimate is in question. !However, narrow
intervals do not necessarily indicate that the estimate is highly reliable,
because of undetected violations of assumptions and the fact that the
confidence limits based on the empirical quantiles of a bootstrap distribution
of 80 samples may be unstable.
5.4 The bootstrapping method of calculating confidence intervals is
computationally intensive. For this reason, all of the calculations
associated with determining the confidence intervals for the ICp estimate have
been incorporated into a computer program. Computations are most easily done
with a computer program such as the revision of the BOOTSTRP program (USEPA,
1988; USEPA, 1989) which is now called "ICPIN" which is described below in
subsection 7.
6. MANUAL CALCULATIONS
6.1 DATA SUMMARY AND PLOTS ;
6.1.1. The data used in this example are the Ceriodaphm'a dubia reproduction
data used in the example in Section 13. Table M.I includes the raw data and
the mean reproduction for each concentration. Data are included for all
animals tested regardless of death of the organism. If an animal died during
the test without producing young, a zero is entered. If death occurred after
producing young, the number of young produced prior to death is entered. A
plot of the data is provided in Figure M.I.
331
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TABLE M.I. CERIODAPHNIA DUBIA REPRODUCTION DATA
Replicate
1
2
3
4
5
6
7
8
9
10
Mean (Y,)
1
Control
27
30
29
31
16
15
18
17
14
27
22.4
1
1.56
32
35
32
26
18
29
27
16
35
13
26.3
2 .
Effluent
3.12
39
30
33
33
36
33
33
27
38
44
34.6
3
Concentration (%)
6.25
27
34
36
34
31
27
33
31
33
31
31.7
4
12.5
10
13
7
7
7
10 .
10
16
12
2
9.4
5
25.0
0
0
0
0
0
0
0
0
0
0
0
6
6.2 MONOTONICITY
6.2.1 As can be seen from the plot in Figure M.I, the observed means are not
monotonically non-increasing with respect to concentration. Therefore, the
means must be smoothed prior to calculating the 1C.
6.2.2 Starting with the control mean T, = 22.4 and T2 = 26.3, we see that
othed means:
L = M2 = ( TI + T) 2 /2 = 24.35
< T2 . Calculate the smoothed means:
6.2.3 Since T3 = 34.6 is larger than M2, average T3 with the previous
concentrations:
Mi = M2 = Af3 = (
M
) /3 = 27 .7
6.2.4 Additionally, T4 =31.7 is larger than M3, and is pooled with the first
three means. Thus,
M1 = M2 = M3 = M4 = ( ML + M2 + M3 + T^ ) /4 = 28 .7
6.2.5 Since M4 > T5 = 9.4, set M5 = 9.4. Likewise, M= > T6 = 0 and M6 becomes
0. Table M.2 contains the smoothed means and Figure M.I gives a plot of the
smoothed response curve.
332
-------
O)
S-
o
CD
E
-o
O
O
to a)
c: >
O) +->
E O
O) O
> S-
S- Q.
O) O)
00 S-
JO
o
10
"3
0-0
o
fj '~
O i.
i Ol
Q- O
O>
333
-------
TABLE M.2. CERIODAPHNIA DUBIA REPRODUCTION MEAN
RESPONSE AFTER SMOOTHING
Effluent
Concentration
Response
Mean (Y,-)
(Young/female)
Smoothed
Mean (Mf)
(Young/female)
Control
1.56
3.12
6.25
12.5
25.0
1
2
3
4
5
6
22.4
26.3
34.6
31.7
9.4
0.0
28.75
.28.75
28.75
28.75
9.40
0.00
6.3 LINEAR INTERPOLATION
6.3.1 Estimates of the IC25 and IC50 are calculated using the Linear
Interpolation Method. A 25% reduction in reproduction, compared to the
controls, would result in a mean reproduction of 21.56 young per adult, where
MJl-p/100) = 28.75(1-25/100). A 50% reduction in reproduction, compared to
the controls, would result in a mean reproduction of 14.38 young per adult,
where M^l-p/lOO) = 28.75(1-50/100). Examining the smoothed means and their
associated concentrations (Table M.2), the two effluent concentrations
bracketing the reproduction of 21.56 young per adult are C4 = 6.25% effluent
and C5 - 12.5% effluent. The two effluent concentrations bracketing a
response of 14.38 young per adult are also C4
effluent.
6.25% effluent and C5 = 12.5%
6.3.2 Using Equation 1 from 4.2, the estimate of the IC25 is calculated as
f ol 1 ows :
IC25 = 6.25 + [28.75 (1 - 25/100) - 28.75]
= 8.57% effluent
334
-------
6.3.3 Using the equation from section 4.2, the estimate of the IC50 is
calculated as follows:
ICp = Cj + [ ML (1 - p/100) -
(Mr
IC50 = 6.25 + [28.75 (1 - 50/100) - 28.75] (12.5 - 6.25
(9.40 - 28.7f
= 10.89% effluent
6.4 CONFIDENCE INTERVALS
i
6.4.1 Confidence intervals for the ICp are derived using the bootstrap
method. As described above, this method involves randomly resampling the
individual observations and recalculating the ICp at least 80 times, and
determining the mean ICp, standard deviation, and empirical 95% confidence
intervals. For this reason, the confidence intervals are calculated using a
computer program called ICPIN. This program is described below and is
available to carry out all the calculations of both the Interpolation estimate
(ICp) and the confidence intervals.
7. COMPUTER CALCULATIONS
I
7.1 The computer program, ICPIN, prepared for the Linear Interpolation Method
was written in TURBO PASCAL for IBM compatible PCs. The program (version 2.0)
has been modified by Computer Science Corporation, Duluth, MN with funding
provided by the Environmental Research Laboratory, Duluth, MN (Norberg-King,
1993). The program was originally developed by Battelle Laboratories,
Columbus, OH through a government contract supported by the Environmental
Research Laboratory, Duluth, MN (USEPA, 1988). To obtain the program and
supporting documentation, send a written request to EMSL-Cincinnati at 3411
Church Street, Cincinnati, OH 45244.
7.2 The ICPIN.EXE program performs the following functions:: 1) it calculates
the observed response means (Y,) (response means); 2) it calculates the
standard deviations; 3) checks the responses for monotoriicity; 4) calculates
smoothed means (M,) (pooled response means) if necessary; 5) uses the means,
M,-, to calculate the initial ICp of choice by linear interpolation; 6)
performs a user-specified number of bootstrap resamples between 80 and 1000
(as multiples of 40); 7) calculates the mean and standard deviation of the
bootstrapped ICp estimates; and 8) provides an original 95% confidence
intervals to be used with the initial ICp when the number of replicates per
concentration is over six and provides both original and expanded confidence
intervals when the number of replicates per concentration are less than seven
(Norberg-King, 1993).
7.3 For the ICp calculation, up to twelve treatments can be used (which
includes the control). There can be up to 40 replicates per concentration,
and the program does not require an equal number of replicates per
concentration. The value of p can range from 1% to 99%.
i
335
-------
7.4 DATA INPUT.
7.4.1 Data is entered directly into the program onscreen. A sample data
entry screen in shown in Figure M.2. The program documentation provides
guidance on the entering and analysis of data for the Linear Interpolation
Method (Norberg-King, 1993).
7.4.2 The user selects the ICp estimate desired (e.g., IC25 or IC50) and the
number of resamples to be taken for the bootstrap method of calculating the
confidence intervals. The program has the capability of performing any number
of resamples from 80 to 1000 as multiples of 40. However, Marcus and Holtzman
(1988) recommend a minimum of 80 resamples for the bootstrap method be used
and at least 250 resamples are better (Norberg-King, 1993).
7.5 DATA OUTPUT.
7.5.1 The program output includes the following (Figures M.3 and M.4):
1. A table of the concentration identification, the concentration tested
and raw data response for each replicate and concentration.
2. A table of test concentrations, number of replicates, concentration
(units), response means (?,), standard deviations for each response
mean, and the pooled response means (smoothed means; M,-).
3. The linear interpolation estimate of the ICp using the means (M,-). Use
this value for the ICp estimate.
4. The mean ICp and standard deviation from the bootstrap resampling.
5. The confidence intervals calculated by the bootstrap method for the ICp.
Provides an original 95% confidence intervals to be used with the
initial ICp when the number of replicates per concentration is over six
and provides both original and expanded confidence intervals when the
number of replicates per concentration are less than seven.
7.6 ICPIN program output for the analysis of the Ceriodaphnia dubia
reproduction data in Table M.I is provided in Figures M.3 and M.4.
7.6.1 When the ICPIN program was used to analyze this set of data, requesting
80 resamples, the estimate of the IC25 was 8.57% effluent. The empirical 95%
confidence intervals for the true mean were 8.30% to 8.85% effluent.
7.6.2 When the ICPIN program was used to analyze this set of data, requesting
80 resamples, the estimate of the IC50 was 10.89% effluent. The empirical 95%
confidence intervals for the true mean were 10.36% to 11.62% effluent.
336
-------
ICp Data Entry/Edit Screen
Current File:
Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
Response 9
Response 10
Response 11
Response 12
Response 13
Response 14
Response 15
Response 16
Response 17
Response 18
Response 19
Response 20
1
2
3
4
.
'trm w <**
5
F10 for Command Menu
Use Arrow Keys to Switch Fields
Figure M.2. ICp data entry/edit screen. Twelve concentration identifications
can be used. Data for concentrations are entered in columns 1
through 6. For concentrations 7 through 12 and responses 21-40
the data is entered in additional fields of the same screen.
337
-------
Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
Response 9
Response 10
1
0
27
30
29
31
16
15
18
17
14
27
2
1.56
32
35
32
26
18
29
27
16
35
13
3
3.12
39
30
33
33
36
33
33
27
38
44
4
6.25
27
34
36
34
31
27
33
31
33
31
5
12.5
10
13
7
7
7
10
10
16
12
2
6
25.0
0
0
0
0
0
0
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent:
Test Start Date: app M Test Ending Date:
Test Species: Ceriodaphnia dubia
Test Duration: 7-d
DATA FILE: cerioman.icp
OUTPUT FILE: cerioman.i25
Cone.
ID
1
2
3
4
5
6
Number
Replicates
10
10
10
10
10
10
Concentration
%
0.000
1.560
3.120
6.250
12.500
25.000
Response
Means
22.400
26.300
34.600
31.700
9.400
0.000
Std.
Dev. F
6.931
8.001
4.835
2.946
3.893
0.000
Pooled
Response Means
28.750
28.750
28.750
28.750
9.400
0.000
The Linear Interpolation Estimate: 8.5715 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean: 8.6014 Standard Deviation: 0.1467
Original Confidence Limits: Lower: 8.3040 Upper: 8.8496
Resampling time in Seconds: 2.53 Random Seed: -1652543090
Figure M.S. Example of ICPIN program output for the IC25.
338
-------
Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
Response 9
Response 10
1
0
27
30
29
31
16
15
18
17
14
27
2
1.56
32
35
32
26
18
29
27
16
35
13
3
3.12
39
30
33
33
36
33
33
27
38
44
4
6.25
27
34
36
34
31
27
33
31
33
31
5
12.5
10
13
7
7
i 7
10
10
16
12
2
6
25.0
0
0
0
0
0
0
0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent:
Test Start Date: app M Test Ending Date:
Test Species: Ceriodaphnia dubia
Test Duration: 7-d
DATA FILE: cerioman.icp
OUTPUT FILE: cerioman.i50
Cone.
ID
1
2
3
4
5
6
Number
Replicates
10
10
10
10
10
10
Concentration
%
0.000
1.560
3.120
6.250
12.500
25.000
Response
Means
22.400
26.300
34.600
31.700
9.400
0.000
Std.
Dev. 1
6.931
8.001
4.835
2.9415
3.893
0.000
Pooled
Response Means
28.750
28.750
28.750
28.750
9.400
0.000
The Linear Interpolation Estimate: 10.8931 Entered P Value: 50
Number of Resamplings: 80
The Bootstrap Estimates Mean: 10.9108 Standard Deviation: 0.3267
Original Confidence Limits: Lower: 10.3618 Upper: 11.6201
Resampling time in Seconds: 2.58 Random Seed: 340510286
Figure M.4. Example of ICPIN program output for
339
the IC50.
-------
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