Short-term Methods for Estimating

the Chronic Toxicity of Effluents and

Receiving Waters to Marine and

Estuarine Organisms

Third Edition

October 2002
U.S. Environmental Protection Agency

Office of Water (4303T)

1200 Pennsylvania Avenue, NW

Washington, DC 20460

EPA-821-R-02-014
ii

DISCLAIMER

The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and

approved this report for publication. Neither the United States Government nor any of its employees, contractors,

or their employees make any warranty, expressed or implied, or assumes any legal liability or responsibility for any

third party’s use of or the results of such use of any information, apparatus, product, or process discussed in this

report, or represents that its use by such party would not infringe on privately owned rights.
iii

CONTENTS

Page

Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3. Health and Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

General Precautions ........................................................... 7

Safety Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

General Laboratory and Field Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Disease Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Safety Manuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Waste Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. Quality Assurance .................................................................... 9

Introduction ................................................................. 9

Facilities, Equipment, and Test Chambers .......................................... 9

Test Oranisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Laboratory Water Used for Culturing and and Test Dilution Water . . . . . . . . . . . . . . . . . . . . . . 9

Effluent and Receiving Water Sampling and Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Test Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Quality of Test Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Food Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Acceptability of Chronic Toxicity Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Calibration and Standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Replication and Test Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Variability in Toxicity Test Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Test Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Demonstrating Acceptable Laboratory Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Documenting Ongoing Laboratory Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Reference Toxicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Record Keeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5. Facilities, Equipment, and Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

General Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Test Chambers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Cleaning Test Chambers and Laboratory Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Apparatus and Equipment for Culturing and Toxicity Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Reagents and Consumable Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Test Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

6. Test Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Test Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Sources of Test Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
CONTENTS (CONTINUED)

Section Number Page

iv

Life Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Laboratory Culturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Holding and Handling Test Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Transportation to the Test Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Test Organism Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

7. Dilution Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Types of Dilution Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Standard, Synthetic Dilution Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Use of Receiving Water as Dilution Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Use of Tap Water as Dilution Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Dilution Water Holding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

8. Effluent and Receiving Water Sampling, Sample Handling,and Sample Preparation for

Toxicity Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Effluent Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Effluent Sample Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Effluent Sampling Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Receiving Water Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Effluent and Receiving Water Sample Handling, Preservation, and Shipping . . . . . . . . . . . . . 33

Sample Receiving . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

 Persistence of Effluent Toxicity During Sample Shipment and Holding . . . . . . . . . . . . . . . . . . 34

Preparation of Effluent and Receiving Water Samples for Toxicity Tests . . . . . . . . . . . . . . . . . 34

Preliminary Toxicity Range-finding Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Multiconcentration (Definitive) Effluent Toxicity Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Receiving Water Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

9. Chronic Toxicity Test Endpoints and Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Relationship between Endpoints Determined by Hypothesis Testing and Point

Estimation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Choice of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Hypothesis Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Point Estimation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

10. Report Preparation and Test Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Report Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Test Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

11. Test Method: Sheepshead Minnow, Cyprinodon variegatus, Larval 

Survival and GrowthTest Method 1004.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Scope and Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Summary of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Interferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Apparatus and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Reagents and Consumable Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Effluent and Receiving Water Collection, Preservation, and Storage . . . . . . . . . . . . . . . . . . . . . 67

Calibration and Standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
CONTENTS (CONTINUED)

Section Number Page

v

Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Summary of Test Conditions and Test Acceptability Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Acceptability of Test Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Precision and Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

12. Test Method: Sheepshead Minnow, Cyprinodon variegatus, Embryo-larval Survival and

Teratogenicity Test Method 1005.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Scope and Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Summary of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Interferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Apparatus and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Reagents and Consumable Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Effluent and Receiving Water Collection, Preservation, and Storage . . . . . . . . . . . . . . . . . . . . 130

Calibration and Standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Acceptability of Test Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Summary of Test Conditions and Test Acceptability Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Precision and Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

13. Test Method: Inland Silverside, Menidia beryllina, Larval

Survival and Growth Method 1006.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Scope and Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Summary of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Interferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Apparatus and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Reagents and Consumable Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Effluent and Receiving Water Collection, Preservation, and Storage . . . . . . . . . . . . . . . . . . . . 168

Calibration and Standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

Summary of Test Conditions and Test Acceptability Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Acceptability of Test Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Precision and Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

14. Test Method: Mysid, Mysidopsis bahia, Survival, Growth, and Fecundity

Test Method 1007.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

Scope and Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

Summary of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

Interferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

Apparatus and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

Reagents and Consumable Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

Effluent and Receiving Water Collection, Preservation, and Storage . . . . . . . . . . . . . . . . . . . . 226

Calibration and Standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
CONTENTS (CONTINUED)

Section Number Page

vi

Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Summary of Test Conditions and Test Acceptability Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Acceptability of Test Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

Precision and Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

15. Test Method: Sea Urchin, Arbacia punctulata, Fertilization Test Method 1008.0 . . . . . . . . . . . . . . . . . 293

Scope and Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Summary of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Interferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Apparatus and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Reagents and Consumable Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Effluent and Receiving Water Collection, Preservation, and Storage . . . . . . . . . . . . . . . . . . . . 303

Calibration and Standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Summary of Test Conditions and Test Acceptability Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . 307

Acceptability of Test Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307

Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

Precision and Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

16. Test Method: Red Macroalga, Champia parvula, Reproduction Test Method 1009.0 . . . . . . . . . . . . . 332

Scope and Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

Summary of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

Interferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

Apparatus and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

Reagents and Consumable Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

Effluent and Receiving Water Collection, Preservation, and Storage . . . . . . . . . . . . . . . . . . . . 341

Calibration and Standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

Summary of Test Conditions and Test Acceptability Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . 346

Acceptability of Test Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

Precision and Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

Cited References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376

Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383

A. Independence, Randomization, and Outliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

B. Validating Normality and Homogeneity of Variance Assumptions . . . . . . . . . . . . . . . . . . . . . 390

C. Dunnett's Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

D. T test with Bonferroni's Adjustment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414

E. Steel's Many-one Rank Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420

F. Wilcoxon Rank Sum Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425

G. Single Concentration Toxicity Test - Comparison of Control with 100% Effluent or 

Receiving Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
CONTENTS (CONTINUED)

Section Number Page

vii

H. Probit Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436

I. Spearman-Karber Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439

J. Trimmed Spearman-Karber Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444

K. Graphical Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448

L. Linear Interpolation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

 Cited References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
viii

FIGURES

SECTION 1-10 

Number Page

1. Control charts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2. Flowchart for statistical analysis of test data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

SECTION 11

Number Page

1. Embryonic development of sheepshead minnow, Cyprinodon variegatus . . . . . . . . . . . . . . . . . . . . . . . . 65

2. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Daily

record of larval survival and test conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

3. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Dry

weights of larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

4. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Summary

of test results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

5. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus, larval

survival data by hypothesis testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

6. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus, larval

survival data by point estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

7. Plot of mean survival proportion data in Table 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

8. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus,

larval growth data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

9. Plot of weight data from sheepshead minnow, Cyprinodon variegatus, larval survival 

and growth test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

10. Plot of raw data, observed means, and smoothed means for the sheepshead minnow,

Cyprinodon variegatus, growth data from Tables 4 and 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

11. ICPIN program output for the IC25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

12. ICPIN program output for the IC50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
FIGURES (CONTINUED)

SECTION 12

Number Page

ix

1. Data form for sheepshead minnow, Cyprinodon variegatus, embryo-larval survival/teratogenicity

test. Daily record of embryo-larval survival/terata and test conditions. . . . . . . . . . . . . . . . . . . . . . . . . 122

2. Embryonic development of sheepshead minnow, Cyprinodon variegatus . . . . . . . . . . . . . . . . . . . . . . . 134

3. Flowchart for statistical analysis of sheepshead minnow, Cyprinodon variegatus, 

embryo-larval survival and teratogenicity test. Survival and terata data . . . . . . . . . . . . . . . . . . . . . . . . 140

4. Plot of sheepshead minnow, Cyprinodon variegatus, total mortality data from the 

embryo-larval test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

5. Output for USEPA Probit Analysis Program, Version 1.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

SECTION 13

Number Page

1. Glass chamber with sump area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

2. Inland silverside, Menidia beryllina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

3. Data form for the inland silverside, Menidia beryllina, larval survival and growth test. Daily

record of larval survival and test conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

4. Data form for the inland silverside, Menidia beryllina, larval survival and growth test. Dry

weights of larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

5. Data form for the inland silverside, Menidia beryllina, larval survival and growth test.

Summary of test results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

6. Flowchart for statistical analysis of the inland silverside, Menida beryllina, survival data by hypothesis

 testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

7. Flowchart for statistical analysis of the inland silverside, Menida beryllina, survival data by

point estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

8. Plot of mean survival proportion of the inland silverside, Menidia beryllina, larvae . . . . . . . . . . . . . . . 183

9. Output for USEPA Probit Analysis Program, Version 1.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

10. Flowchart for statistical analysis of the inland silverside, Menidia beryllina, growth data . . . . . . . . . . . 193

11. Plot of mean weights of inland silverside, Menidia beryllina, larval survival and growth test . . . . . . . . 195

12. Plot of the raw data, observed means, and smoothed means from Tables 12 and 19 . . . . . . . . . . . . . . . 204
FIGURES (CONTINUED)

SECTION 13

Number Page

x

13. ICPIN program output for the IC25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

14. ICPIN program output for the IC50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

SECTION 14

Number Page

1. Apparatus (brood chamber) for collection of juvenile mysids, Mysidopsis bahia . . . . . . . . . . . . . . . . 225

2. Data form for the mysid, Mysidopsis bahia, water quality measurements . . . . . . . . . . . . . . . . . . . . . . . 231

3. Mature female mysid, Mysidopsis bahia, with eggs in oviducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

4. Mature female mysid, Mysidopsis bahia, with eggs in oviducts and developing 

embryos in the brood sac . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

5. Mature male mysid, Mysidopsis bahia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

6. Immature mysid, Mysidopsis bahia, (A) lateral view, (B) dorsal view . . . . . . . . . . . . . . . . . . . . . . . . . 236

7. Data form for the mysid, Mysidopsis bahia, survival and fecundity data . . . . . . . . . . . . . . . . . . . . . . . . 237

8. Data form for the mysid, Mysidopsis bahia, dry weight measurements . . . . . . . . . . . . . . . . . . . . . . . . . 239

9. Flowchart for statistical analysis of mysid, Mysidopsis bahia, survival data by 

hypothesis testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

10. Flowchart for statistical analysis of mysid, Mysidopsis bahia, survival data by 

point estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

11. Plot of survival proportions of mysids, Mysidopsis bahia, at each treatment level . . . . . . . . . . . . . . . . 247

12. Output for USEPA Probit Analyis Program, Version 1.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

13. Flowchart for statistical analysis of mysid, Mysidopsis bahia, growth data . . . . . . . . . . . . . . . . . . . . . . 256

14. Plot of mean growth data for mysid, Mysidopsis bahia, test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

15. Plot of raw data, observed means, and smoothed means for the mysid, Mysidopsis bahia,

growth data from Tables 13 and 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

16. ICPIN program output for the IC25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

17. ICPIN program output for the IC50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
FIGURES (CONTINUED)

SECTION 14

Number Page

xi

18. Flowchart for statistical analysis of mysid, Mysidopsis bahia, fecundity data . . . . . . . . . . . . . . . . . . . . 270

19. Proportion of female mysids, Mysidopsis bahia, with eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

20. Plot of the mean proportion of females mysids, Mysidopsis bahia, with eggs . . . . . . . . . . . . . . . . . . . . 282

21. ICPIN program output for the IC25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

22. ICPIN program output for the IC50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

SECTION 15

Number Page

1. Data form (1) for fertilization test using sea urchin, Arbacia punctulata . . . . . . . . . . . . . . . . . . . . . . . . 297

2. Data form (2) for fertilization test using sea urchin, Arbacia punctulata . . . . . . . . . . . . . . . . . . . . . . . . 298

3. Data form (3) for fertilization test using sea urchin, Arbacia punctulata . . . . . . . . . . . . . . . . . . . . . . . . 299

4. Flowchart for statistical analysis of sea urchin, Arbacia punctulata, by point estimation . . . . . . . . . . . 312

5. Plot of mean percent of fertilized sea urchin, Arbacia punctulata, eggs . . . . . . . . . . . . . . . . . . . . . . . . 313

6. ICPIN program output for the IC25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

7. ICPIN program output for the IC50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

SECTION 16

Number Page

1. Life history of the red macroalga, Champia parvula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338

2. Apex of branch of female plant, showing sterile hairs and reproductive 

hairs (trichogynes) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

3. A portion of the male thallus showing spermatial sori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

4. A magnified portion of a spermatial sorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

5. Apex of a branch on a mature female plant that was exposed to spermatia from a male plant . . . . . . . . 340

6. Data form for the red macroalga, Champia parvula, sexual reproduction test. Receiving 

water summary sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
FIGURES (CONTINUED)

SECTION 16

Number Page

xii

7. A mature cystocarp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

8. Comparison of a very young branch and an immature cystocarp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

9. An aborted cystocarp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

10. Data form for the red macroalga, Champia parvula, sexual reproduction test. Cystocarp

data sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347

11. Flowchart for statistical analysis of the red macroalga, Champia parvula, data . . . . . . . . . . . . . . . . . . 351

12. Plot of the number of cystocarps per plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

13. ICPIN program output for the IC25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

14. ICPIN program output for the IC50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
xiii

TABLES

SECTION 1- 10

Number Page

1. National interlaboratory study of chronic toxicity test precision, 1991: Summary of responses using two

reference toxicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2. National interlaboratory study of chronic toxicity test precision, 2000: Precision of responses using

effluent, receiving water, and reference toxicant sample types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

3. Preparation of GP2 artificial seawater using reagent grade chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

4. Oxygen solubility (mg/L) in water at equilibrium with air at 760 mm Hg . . . . . . . . . . . . . . . . . . . . . . . . 36

5. Percent unionized NH3 in aqueous ammonia solutions: temperature 15-26EC and pH 6.0-8.9 . . . . . . . . 37

6. Variability criteria (upper and lower PMSD bounds) for sublethal hypothesis testing endpoints submitted

under NPDES permits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

SECTION 11

Number Page

1. Reagent grade chemicals used in the preparation of GP2 artificial seawater for the sheepshead minnow,

Cyprinodon variegatus, toxicity test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

2. Preparation of test solutions at a salinity of 20‰, using 20‰ salinity dilution water prepared from natural

seawater, hypersaline brine, or artificial sea salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

3. Summary of test conditions and test acceptability criteria for sheepshead minnow, Cyprinodon variegatus,

larval survival and growth test with effluents and receiving waters (Test Method 1004.0) . . . . . . . . . . . 78

4. Summary of survival and growth data for sheepshead minnow, Cyprinodon variegatus, larvae exposed to

an effluent for seven days . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

5. Sheepshead minnow, Cyprinodon variegatus, survival data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

6. Centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

7. Ordered centered observations for the Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

8. Coefficients and differences for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

9. Assigning ranks to the control and 6.25% effluent concentration for Steel's Many-one Rank Test . . . . . 88

10. Table of ranks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

11. Rank sums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
TABLES (CONTINUED)

SECTION 11 

Number Page

xiv

12. Data for example of Spearman-Karber Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

13. Sheepshead minnow, Cyprinodon variegatus, growth data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

14. Centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

15. Ordered centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

16. Coefficients and differences for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

17. ANOVA table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

18. ANOVA table for Dunnett's Procedure example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

19. Calculated t values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

20. Sheepshead minnow, Cyprinodon variegatus, mean growth response after smoothing . . . . . . . . . . . . . 102

21. Single-laboratory precision of the sheepshead minnow, Cyprinodon variegatus, larval survival and growth

test performed in FORTY FATHOMS® artificial seawater, using larvae from fish maintained and spawned

in FORTY FATHOMS® artificial seawater, using copper (Cu) sulfate as a reference 

toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

22. Single-laboratory precision of the sheepshead minnow, Cyprinodon variegatus, larval survival and growth

test performed in FORTY FATHOMS® artificial seawater, using larvae from fish maintained and spawned

in FORTY FATHOMS® artificial seawater, using sodium dodecyl sulfate (SDS) as a reference 

toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

23. Single-laboratory precision of the sheepshead minnow, Cyprinodon variegatus, larval survival and growth

test performed in natural seawater, using larvae from fish maintained and spawned in natural seawater,

using copper (Cu) sulfate as a reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

24. Single-laboratory precision of the sheepshead minnow, Cyprinodon variegatus, larval survival and growth

test performed in natural seawater, using larvae from fish maintained and spawned in natural seawater,

using sodium dodecyl sulfate (SDS) as a reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

25. Single-laboratory precision of the sheepshead minnow, Cyprinodon variegatus, larval survival and growth

test performed in FORTY FATHOMS® artificial seawater, using larvae from fish maintained and spawned

in FORTY FATHOMS® artificial seawater, and hexavalent chromium as the reference toxicant . . . . . 113

26. Comparison of larval survival (LC50) and growth (IC50) values for the sheepshead minnow, Cyprinodon

variegatus, exposed to sodium dodecyl sulfate (SDS) and copper (Cu) sulfate in GP2 artificial seawater

medium or natural seawater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
TABLES (CONTINUED)

SECTION 11 

Number Page

xv

27. Data from interlaboratory study of the sheepshead minnow, Cyprinodon variegatus, larval

survival and growth test using an industrial effluent as a reference toxicant . . . . . . . . . . . . . . . . . . . . . 115

28. Precision of point estimates for various sample types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

29. Frequency distribution of hypothesis testing results for various sample types . . . . . . . . . . . . . . . . . . . . 116

SECTION 12

Number Page

1. Preparation of test solutions at a salinity of 20‰, using 20‰ natural or artificial seawater,

hypersaline brine, or artificial sea salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

2. Summary of test conditions and test acceptability criteria for the sheepshead minnow, 

Cyprinodon variegatus, embryo-larval survival and teratogenicity test with effluents and 

receiving waters (Test Method 1005.0) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

3. Sheepshead minnow, Cyprinodon variegatus, embryo-larval total mortality data . . . . . . . . . . . . . . . . . 141

4 Centered observation for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

5. Ordered centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

6. Coefficients and differences for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

7. ANOVA table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

8. ANOVA table for Dunnett's Procedure example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

9. Calculated t values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

10. Data for Probit Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

11. Single-laboratory precision of the sheepshead minnow, Cyprinodon variegatus, embryo-larval survival and

teratogenicity test performed in HW MARINEMIX® artificial seawater, using embryos from fish

maintained and spawned in HW MARINEMIX® artificial seawater using copper (Cu) sulfate as reference

toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

12. Single-laboratory precision of the sheepshead minnow, Cyprinodon variegatus, embryo-larval survival and

teratogenicity test performed in HW MARINEMIX® artificial seawater, using embryos from fish

maintained and spawned in HW MARINEMIX® artificial seawater using sodium dodecyl sulfate (SDS) as

reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
TABLES (CONTINUED)

SECTION 13 

Number Page

xvi

1. Preparation of 3 L saline water from deionized water and a hypersaline brine of 100‰ needed for test

solutions at 20‰ salinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

2. Reagent grade chemicals used in the preparation of GP2 artificial seawater for the inland silverside,

Menidia beryllina, toxicity test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

3. Summary of test conditions and test acceptability criteria for the inland silverside, Menidia beryllina, larval

survival and growth test with effluents and receiving waters (Test Method 1006.0) . . . . . . . . . . . . . . . 178

4. Inland silverside, Menidia beryllina, larval survival data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

5. Centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

6. Ordered centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

7. Coefficients and differences for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

8. ANOVA table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

9. ANOVA table for Dunnett's Procedure example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

10. Calculated t values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

11. Data for Probit Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

12. Inland silverside, Menidia beryllina, growth data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

13. Centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

14. Ordered centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

15. Coefficients and differences for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

16. ANOVA table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

17. ANOVA table for Dunnett's Procedure example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

18. Calculated t values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

19. Inland silverside mean growth response after smoothing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

20. Single-laboratory precision of the inland silverside, Menidia beryllina, survival and growth test performed

in natural seawater, using larvae from fish maintained and spawned in natural seawater, and copper (Cu) as

a reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
TABLES (CONTINUED)

SECTION 13 

Number Page

xvii

21. Single-laboratory precision of the inland silverside, Menidia beryllina, survival and growth test performed

in natural seawater, using larvae from fish maintained and spawned in natural seawater, and sodium

dodecyl sulfate (SDS) as a reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

22. Comparison of the single-laboratory precision of the inland silverside, Menidia beryllina, larval survival

(LC50) and growth (IC50) values exposed to sodium dodecyl sulfate (SDS) or copper (Cu) sulfate, in GP2

artificial seawater medium or natural seawater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

23. Precision of point estimates for various sample types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

24. Frequency distribution of hypothesis testing results for various sample types . . . . . . . . . . . . . . . . . . . . 213

SECTION 14

Number Page

1. Reagent grade chemicals used in the preparation of GP2 artificial seawater for the mysid,

Mysidopsis bahia, toxicity test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

2. Quantities of effluent, deionized water, and hypersaline brine (100‰) needed to prepare

1800 mL volumes of test solution with a salinity of 20‰ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

3. Summary of test conditions and test acceptability criteria for the mysid, Mysidopsis bahia, seven day

survival, growth, and fecundity test with effluents and receiving waters (Test Method 1007.0) . . . . . . 241

4. Data for Mysidopsis bahia 7-day survival, growth, and fecundity test . . . . . . . . . . . . . . . . . . . . . . . . . . 243

5. Mysid, Mysidopsis bahia, survival data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

6. Centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

7. Ordered centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

8. Coefficients and differences for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

9. Assigning ranks to the control and 50 ppb concentration level for 

Steel's Many-one Rank Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

10. Table of ranks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

11. Rank sums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

12. Data for Probit Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
TABLES (CONTINUED)

SECTION 14 

Number Page

xviii

13. Mysid, Mysidopsis bahia, growth data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

14. Centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

15. Ordered centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

16. Coefficients and differences for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

17. ANOVA table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

18. ANOVA table for Dunnett's Procedure example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

19. Calculated t values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

20. Mysid, Mysidopsis bahia, mean growth response after smoothing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

21. Mysid, Mysidopsis bahia, fecundity data: Percent females with eggs . . . . . . . . . . . . . . . . . . . . . . . . . . 273

22. Centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

23. Ordered centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

24. Coefficients and differences for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

25. ANOVA table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

26. ANOVA table for the t test with Bonferroni's Adjustment example . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

27. Calculated t values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

28. Mysid, Mysidopsis bahia, mean proportion of females with eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280

29. Single-laboratory precision of the mysid, Mysidopsis bahia, survival, growth,and fecundity test performed

in natural seawater, using juveniles from mysids cultured and spawned in natural seawater, and copper

(Cu) sulfate as a reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

30. Single-laboratory precision of the mysid, Mysidopsis bahia, survival, growth, and fecundity test performed

in natural seawater, using juveniles from mysids cultured and spawned in natural seawater, and sodium

dodecyl sulfate (SDS) as a reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

31. Comparison of survival (LC50), growth and fecundity (IC50) results from 7-day tests with the mysid,

Mysidopsis bahia, using natural seawater (NSW) and artificial seawater (GP2) as dilution water and

sodium dodecyl sulfate (SDS) as a reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
TABLES (CONTINUED)

SECTION 14 

Number Page

xix

32. Comparison of survival (LC50), growth and fecundity (IC50) results from 7-day tests with the mysid,

Mysidopsis bahia, using natural seawater (NSW) and artificial seawater (GP2) as dilution water and copper

(Cu) sulfate as a reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

33. Control results from 7-day survival, growth, and fecundity tests with the mysid, Mysidopsis bahia, using

natural seawater and artificial seawater (GP2) as a dilution water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

34. Precision of point estimates for various sample types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

35. Frequency distribution of hypothesis testing results for various sample types . . . . . . . . . . . . . . . . . . . . 292

SECTION 15

Number Page

1. Preparation of test solutions at a salinity of 30‰ using natural seawater, hypersaline brine, or artificial sea

salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301

2. Reagent grade chemicals used in the preparation of GP2 artificial seawater for the sea urchin, Arbacia

punctulata toxicity test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

3. Summary of test conditions and test acceptability criteria for sea urchin, Arbacia punctulata, fertilization

test with effluent and receiving waters (Test Method 1008.0) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

4. Data from sea urchin, Arbacia punctulata, fertilization test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

5. Sea urchin, Arbacia punctulata, fertilization data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

6. Centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

7. Ordered centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

8. Coefficients and differences for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

9. ANOVA table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317

10. ANOVA table for Dunnett's Procedure example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

11. Calculated t values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

12. Sea urchin, Arbacia punctulata, mean proportion of fertilized eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
TABLES (CONTINUED)

SECTION 15

Number Page

xx

13. Single-laboratory precision of the sea urchin, Arbacia punctulata, fertilization test performed in FORTY

FATHOMS® artificial seawater, using gametes from adults maintained in FORTYFATHOMS® artificial

seawater, or obtained directly from natural sources, and copper (Cu) sulfate as a reference toxicant . . 326

14. Single-laboratory precision of the sea urchin, Arbacia punctulata, fertilization test performed in

FORTY FATHOMS® artificial seawater, using gametes from adults maintained in FORTY

FATHOMS® artificial seawater, or obtained directly from natural sources, and sodium dodecyl

sulfate (SDS) as a reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

15. Single-laboratory precision of the sea urchin, Arbacia punctulata, fertilization test performed in natural

seawater, using gametes from adults maintained in natural seawater and copper (Cu) sulfate as a reference

toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

16. Single-laboratory precision of the sea urchin, Arbacia punctulata, fertilization test performed in natural

seawater, using gametes from adults maintained in natural seawater and sodium dodecyl sulfate (SDS) as a

reference toxicant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

17. Single-laboratory precision of the sea urchin, Arbacia punctulata, fertilization test performed in GP2, using

gametes from adults maintained in GP2 artificial seawater and copper (Cu) sulfate and sodium dodecyl

sulfate (SDS) as reference toxicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

18. Single-laboratory precision of the sea urchin, Arbacia punctulata, fertilization test performed in natural

seawater, using gametes from adults maintained in natural seawater and copper (Cu) sulfate and sodium

dodecyl sulfate (SDS) as reference toxicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

SECTION 16

Number Page

1. Nutrients to be added to natural seawater and to artificial seawater (GP2) described in 

Table 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

2. Reagent grade chemicals used in the preparation of GP2 artificial seawater for use in conjunction with

natural seawater for the red macroalga, Champia parvula, culturing and toxicity testing . . . . . . . . . . . 337

3. Summary of test conditions and test acceptability criteria for the red macroalga, Champia parvula, sexual

reproduction test with effluents and receiving waters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

4. Data from the red macroalga, Champia parvula, effluent toxicity test. Cystocarp counts for individual

plants and mean count per test chamber for each effluent concentration . . . . . . . . . . . . . . . . . . . . . . . . 350

5. Red macroalga, Champia parvula, sexual reproduction data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

6. Centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
TABLES (CONTINUED)

SECTION 16

Number Page

xxi

7. Ordered centered observations for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354

8. Coefficients and differences for Shapiro-Wilk's example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354

9. ANOVA table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

10. ANOVA table for Dunnett's Procedure example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

11. Calculated t values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

12. Red macroalga, Champia parvula, mean number of cystocarps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

13. Single-laboratory precision of the red macroalga, Champia parvula, reproduction test performed in a 50/50

mixture of natural seawater and GP2 artificial seawater, using gametes from adults cultured in natural

seawater. The reference toxicant used was copper (Cu) sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

14. Single-laboratory precision of the red macroalga, Champia parvula, reproduction test performed in a 50/50

mixture of natural seawater and GP2 artificial seawater, using gametes from adults cultured in natural

seawater. The reference toxicant used was sodium dodecyl sulfate (SDS) . . . . . . . . . . . . . . . . . . . . . . 365

15. Single-laboratory precision of the red macroalga, Champia parvula, reproduction test in natural seawater

(30‰ salinity). The reference toxicant used was copper (Cu) sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . 366

16. Single-laboratory precision of the red macroalga, Champia parvula, reproduction test in

natural seawater (30‰ salinity). The reference toxicant used was sodium dodecyl sulfate (SDS) . . . . 366
1

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. With the exception of the Red Macroalga, Champia parvula, Reproduction Test Method 1009.0, 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. The Red Macroalga, Champia parvula, Reproduction Test Method 1009.0 is not listed at

40 CFR Part 136 for nationwide use.

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, IC25, or IC50 (see Section 9, Chronic Toxicity Test 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, 2002a), the short-term chronic toxicity test methods for freshwater organisms (USEPA,

2002b), and the manual for evaluation of laboratories performing aquatic toxicity tests (USEPA, 1991c). In 2002,

EPA revised previous editions of each of the three methods manuals (USEPA, 1993a; USEPA, 1994a; USEPA,

1994b).

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 marine and estuarine short-term toxicity tests are similar to those developed for the freshwater organisms

to evaluate the toxicity of effluents discharged to estuarine and coastal marine waters under the NPDES permit

program. Methods are presented in this manual for five species from four phylogenetic groups. Five of the six

methods were developed and extensively field tested by Environmental Research Laboratory-Narragansett

(ERL-N). The methods vary in duration from one hour and 20 minutes to nine days.

1.7 The five species for which toxicity test methods are provided are: the sheepshead minnow, Cyprinodon

variegatus; the inland silverside, Menidia beryllina; the mysid, Mysidopsis bahia; the sea urchin, Arbacia

punctulata; and the red macroalga, Champia parvula. 

1.7.1 Four of the methods incorporate the chronic endpoints of growth or reproduction (or both) in addition to

lethality. The sheepshead minnow 9-day embryo-larval survival and teratogenicity test incorporates teratogenic

effects in addition to lethality. The sea urchin sperm cell test uses fertilization as an endpoint and has the advantage

of an extremely short exposure period (1 h and 20 min). 

1.8 The validity of the marine/estuarine methods in predicting adverse ecological impacts of toxic discharges was
2

demonstrated in field studies (USEPA, 1986d).

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 testing 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 The manual was prepared in the established EMSL-Cincinnati format (USEPA, 1983). 
3

SECTION 2 

SHORT-TERM METHODS FOR ESTIMATING CHRONIC TOXICITY

2.1 INTRODUCTION

2.1.1 The objective of aquatic toxicity tests with effluents or pure compounds is to estimate the "safe" or "noeffect" concentration of these substances, which is defined as the concentration which will permit normal

propagation of fish and other aquatic life in the receiving waters. The endpoints that have been considered in tests

to determine the adverse effects of toxicants include death and survival, decreased reproduction and growth,

locomotor activity, gill ventilation rate, heart rate, blood chemistry, histopathology, enzyme activity, olfactory

function, and terata. Since it is not feasible to detect and/or measure all of these (and other possible) effects of toxic

substances on a routine basis, observations in toxicity tests generally have been limited to only a few effects, such as

mortality, growth, and reproduction. 

2.1.2 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 promelas, 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. 

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 fish 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- to 90-day ELS toxicity tests for estimating chronically safe concentrations in the late 1970s. In
4

1980, USEPA adopted the policy that ELS test data could be used in establishing water quality criteria if data from

full life-cycle tests were not available (USEPA, 1980a). 

2.1.8 Published reports of the results of ELS tests indicate that the relative sensitivity of growth and survival as

endpoints may be species dependent, toxicant dependent, or both. Ward and Parrish (1980) examined the literature

on ELS tests that used embryos and juveniles of the sheepshead minnow, Cyprinodon variegatus, and found that

growth was not a statistically sensitive indicator of toxicity in 16 of 18 tests. They suggested that the ELS tests be

shortened to 14 days posthatch and that growth be eliminated as an indicator of toxic effects. 

2.1.9 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, Woltering (1984) found that at the lowest effect concentration, significant reductions were

observed in fry survival in 57%, fry growth in 36%, and egg hatchability in 19% of the tests. He also found that fry

survival and growth were very often equally sensitive, and concluded that the growth response could be deleted

from routine application of the ELS tests. The net result would be a significant reduction in the duration and cost of

screening tests with no appreciable impact on estimating MATCs for chemical hazard assessments. Benoit et al.

(1982), however, 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.

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 al., 1985), and a seven-day larval survival and growth

test (Norberg and Mount, 1985).

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 and

Chemistry (Norberg and Mount, 1983). This test was subsequently used by Mount and associates in field

demonstrations at Lima, Ohio (USEPA, 1984), and at many other locations (USEPA, 1985c, USEPA, 1985d;

USEPA, 1985e; USEPA, 1986a; USEPA, 1986b; USEPA, 1986c; USEPA, 1986d). 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 DURSBAN®, 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 USEPA (1987b) and USEPA (1987c) adapted the fathead minnow larval growth and survival test for use

with the sheepshead minnow and the inland silverside, respectively. When daily renewal 7-day sheepshead minnow

larval growth and survival tests and 28-day ELS tests were performed with industrial and municipal effluents,

growth was more sensitive than survival in seven out of 12 larval growth and survival tests, equally sensitive in four

tests, and less sensitive in only one test. In four cases, the ELS test may have been three to 10 times more sensitive

to effluents than the larval growth and survival test. In tests using copper, the No Observable Effect Concentrations

(NOECs) were the same for both types of test, and growth was the most sensitive endpoint for both. In a four

laboratory comparison, six of seven tests produced identical NOECs for survival and growth (USEPA, 1987a). 

Data indicate that the inland silverside is at least equally sensitive or more sensitive to effluents and single

compounds than the sheepshead minnow, and can be tested over a wider salinity range, 5-30 ‰ (USEPA, 1987a). 

2.1.15 Lussier et al. (1985) and USEPA (1987e) determined that survival and growth are often as sensitive as

reproduction in 28-day life-cycle tests with the mysid, Mysidopsis bahia.
5

2.1.16 Nacci and Jackim (1985) and USEPA (1987g) compared the results from the sea urchin fertilization test,

using organic compounds, with results from acute toxicity tests using the freshwater organisms, fathead minnows,

Pimphales promelas, and Daphnia magna. The test was also compared to acute toxicity tests using Atlantic

silverside, Menidia menidia, and the mysid, Mysidopsis bahia, and five metals. For six of the eight organic

compounds, the results of the fertilization test and the acute toxicity test correlated well (r2

 = 0.85). However, the

results of the fertilization test with the five metals did not correlate well with the results from the acute tests. 

2.1.17 USEPA (1987f) evaluated two industrial effluents containing heavy metals, five industrial effluents

containing organic chemicals (including dyes and pesticides), and 15 domestic wastewaters using the two-day red

macroalga, Champia parvula, sexual reproduction test. Nine single compounds were used to compare the effects on

sexual reproduction using a two-week exposure and a two-day exposure. For six of the nine compounds tested, the

chronic values were the same for both tests. 

2.1.18 The use of short-term toxicity 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 28-day mysid 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.

2.2.2 Effluent chronic toxicity is generally measured using a multi-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 that affects the hatchability, gross morphological

abnormalities, survival, growth, and/or reproduction within the prescribed period of time (one hour and 20 minutes

to nine days). The results of the tests are expressed in terms of either 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 ensure 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/4. More specifically, if the RWC = 50%, appropriate effluent concentrations may 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 other 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 static non-renewal or static renewal. Individual

methods specify which static type of test is to be conducted.
6

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. 

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.

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.

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. 
7

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 and includes: (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 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 a lack of

oxygen or the presence of noxious gases. 

3.1.3 Prior to sample collection and laboratory work, personnel should determine that all necessary safety

equipment and materials have been obtained and are in good condition. 

3.1.4 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 must 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 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 listed in Section 3, Health and Safety, Subsection 3.5). It is recommended

that personnel collecting samples and performing toxicity tests should not work alone.

3.3.2 Because the chemical composition of effluents is usually only poorly known, they should be considered as

potential health hazards, and exposure to them should be minimized. 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 should be adequately labeled to indicate their contents.
8

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 toxicity testing activities. Local fire officials

should be notified of any potentially hazardous conditions.
9

SECTION 4

QUALITY ASSURANCE

4.1 INTRODUCTION

4.1.1 Development and maintenance of a toxicity test laboratory quality assurance (QA) program (USEPA, 1991b)

requires an ongoing commitment by laboratory management. Each toxicity test laboratory should (1) appoint a quality

assurance officer with the responsibility and authority to develop and maintain a QA program, (2) prepare a quality

assurance plan with stated data quality objectives (DQOs), (3) prepare written descriptions 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 toxicity 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 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); USEPA (1991c); DeWoskin (1984); and Taylor (1987). 

4.1.4 Guidelines for the evaluation of laboratory performing toxicity tests and laboratory evaluation criteria are found

in USEPA (1991c).

4.2 FACILITIES, EQUIPMENT, AND TEST CHAMBERS

4.2.1 Separate test organism culturing and toxicity testing areas should 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).

4.3 TEST ORGANISMS 

4.3.1 The test organisms used in the procedures described in this manual are the sheepshead minnow, Cyprinodon

variegatus; the inland silverside, Menidia beryllina; the mysid, Mysidopsis bahia; the sea urchin, Arbacia punctulata;

and the red macroalga, Champia parvula. The organisms used should be disease-free and appear healthy, behave

normally, feed well, and have low mortality in cultures, during holding, and in test control. Test organisms should be

positively identified to species (see Section 6, Test Organisms).

4.4 LABORATORY WATER USED FOR CULTURING AND TEST DILUTION WATER 

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
10

tests will depend on the objectives of the study and logistical constraints, as discussed in Section 7, Dilution Water. 

The dilution water used in the toxicity tests may be natural seawater, hypersaline brine (100‰) prepared from natural

seawater, or artificial seawater prepared from commercial sea salts, such as FORTY FATHOMS® or HW

MARINEMIX®, if recommended in the method. GP2 synthetic seawater, made from reagent grade chemical salts

(30‰) in conjunction with natural seawater, may also be used if recommended. Hypersaline brine and artificial

seawater can be used with Champia parvula only if they are accompanied by at least 50% natural seawater. Types of

water are discussed in Section 5, Facilities, Equipment, and Supplies. Water used for culturing and test dilution water

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, Zn, expressed as total metal, should not exceed 1 μg/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 National Ambient Water Quality chronic

criteria values where available.

4.5 EFFLUENT AND RECEIVING WATER 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

4.6.1 Water temperature and salinity should 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 vessel during the duration of each test. Test

solution temperatures should be maintained within the limits specified for each test. DO concentrations and pH should

be checked at the beginning of the test and daily throughout the test period. 

4.7 QUALITY OF TEST ORGANISMS

4.7.1 The health of test organisms is primarily assessed by the performance (survival, growth, and/or reproduction) of

organisms in control treatments of individual tests. The health and sensitivity of test organisms is also assessed by

reference toxicant testing. In addition to documenting the sensitivity and health of test organisms, reference toxicant

testing is used to initially demonstrate acceptable laboratory performance (Subsection 4.15) and to document ongoing

laboratory performance (Subsection 4.16). 

4.7.2 Regardless of the source of test organisms (in-house cultures or purchased from external suppliers), the testing

laboratory must perform at least one acceptable reference toxicant test per month for each toxicity test method

conducted in that month (Subsection 4.16). If a test method is conducted only monthly, or less frequently, a reference

toxicant test must be performed concurrently with each effluent toxicity test.

4.7.3 When 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 toxicant and test conditions (see Section 6, Test Organisms). 

4.7.4 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. 

4.7.5 If a routine reference toxicant test fails to meet test acceptability criteria, then the reference toxicant test must be

immediately repeated. 
11

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 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.

4.8.3 New batches of food used in culturing and testing should 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 μg/g wet weight, or the concentration of total

organochlorine pesticides plus PCBs exceeds 0.30 μg/g wet weight, or toxic metals (Al, As, Cr, Cd, Cu, Pb, Ni, Zn,

expressed as total metal) exceed 20 μg/g wet weight, the food should not be used (for analytical methods, see AOAC,

1990; and USDA, 1989).

4.84 For foods (e.g., 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 CHRONIC TOXICITY TESTS

4.9.1 The results of the sheepshead minnow, Cyprinodon variegatus, inland silverside, Menidia beryllina, or mysid,

Mysidopsis bahia, tests are acceptable if survival in the controls is 80% or greater. The sea urchin, Arbacia punctulata,

test requires control egg fertilization equal to or exceeding 70%. However, greater than 90% fertilization may result in

masking toxic responses. The red macroalga, Champia parvula, test is acceptable if survival is 100%, and the mean

number of cystocarps per plant should equal or exceed 10. If the sheepshead minnow, Cyprindon variegatus, larval

survival and growth test is begun with less-than-24-h old larvae, the mean dry weight of the surviving larvae in the

control chambers at the end of the test must equal or exceed 0.60 mg, if the weights are determined immediately, or

0.50 mg if the larvae are preserved in a 4% formalin or 70% ethanol solution. If the inland silverside, Menidia

beryllina, larval survival and growth test is begun with larvae seven days old, the mean dry weight of the surviving

larvae in the control chambers at the end of the test must equal or exceed 0.50 mg, if the weights are determined

immediately, or 0.43 mg if the larvae are preserved in a 4% formalin or 70% ethanol solution. The mean mysid dry

weight of survivors must be at least 0.20 mg. Automatic or hourly feeding will generally provide control mysids with a

dry weight of 0.30 mg. At least 50% of the females should bear eggs at the end of the test, but mysid fecundity is not a

factor in test acceptability. However, fecundity must equal or exceed 50% to be used as an endpoint in the test. 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 conditions and

test acceptability criteria summaries). The acceptability of the test will 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 a test.
12

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.

4.11 CALIBRATION AND STANDARDIZATION

4.11.1 Instruments used for routine measurements of chemical and physical parameters, such as pH, DO, temperature,

conductivity, and salinity, must be calibrated and standardized according to instrument manufacturers procedures as

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. The minimum 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; (5) and 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 or more 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 Interlaboratory precision data from a 1991 study of chronic toxicity tests using two reference toxicants with the

mysid, Mysidopsis bahia, and the inland silverside, Menidia beryllina, is listed in Table 1. Table 2 shows

interlaboratory precision data from a study of three chronic toxicity test methods using effluent, receiving water, and

reference toxicant sample types (USEPA, 2001a; USEPA, 2001b). For the Mysidopsis bahia and the Cyprinodon

variegatus test methods, the effluent sample was a municipal wastewater spiked with KCl, the receiving water sample

was a river water spiked with KCl, and the reference toxicant sample was bioassay-grade FORTY FATHOMS®
13

synthetic seawater spiked with KCl. For the Menidia beryllina test method, the effluent sample was an industrial

wastewater spiked with CuSO4, the receiving water sample was a natural seawater spiked with CuSO4, and the

reference toxicant sample was bioassay-grade FORTY FATHOMS® synthetic seawater spiked with CuSO4. Additional

precision data for each of the tests described in this manual are presented in the sections describing the individual test

methods. 

4.14.4 Additional information on toxicity test precision is provided in the Technical Support Document for Water

Quality-based Toxic 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 precision has been attained. The "true" no effect concentration could fall anywhere

within the interval, NOEC ± (LOEC minus NOEC). 

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 uncertainty as high as ± 300%. With a

dilution factor of 0.5, the NOEC could be considered to have a relative variability of ± 100%. As a result of the

variability of different dilution factors, USEPA recommends the use of a $ 0.5 dilution factor. Other factors which

can affect test precision include: test organism age, condition, and sensitivity; temperature control; and feeding.
14

TABLE 1. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST PRECISION,

1991: SUMMARY OF RESPONSES USING TWO REFERENCE TOXICANTS1,2

Organism Endpoint No. Labs KCl(mg/L)4 SD CV(%)3

Mysidopsis

 bahia

Survival, NOEC

Growth, IC25

Growth, IC50

Growth, NOEC

Fecundity, NOEC

34

26

22

32

25

NA

480

656

NA

NA

NA

3.47

3.17

NA

NA

NA

28.9

19.3

NA

NA

Organism Endpoint No. Labs Cu(mg/L)4 SD CV(%)3

Menidia

 beryllina

Survival, NOEC

Growth, IC25

Growth, IC50

Growth, NOEC

19

13

12

17

NA

0.144

0.180

NA

NA

1.56

1.87

NA

NA

43.5

41.6

NA

1 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. 2 Static renewal test, using 25 ‰ modified GP2 artificial seawater. 3 Percent coefficient of variation = (standard deviation X 100)/mean. 4 Expressed as mean.

TABLE 2. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST PRECISION,

2000: PRECISION OF RESPONSES USING EFFLUENT, RECEIVING WATER, AND

REFERENCE TOXICANT SAMPLE TYPES1

Organism Endpoint Number of Tests2 CV (%)3

Cyprinodon variegatus Growth, IC25 21 10.5

Menidia beryllina Growth, IC25 30 43.8

Mysidopsis bahia Growth, IC25 36 41.3

1 From EPA’s WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b). 2 Represents the number of valid tests (i.e., those that met test acceptability criteria) that were used in the analysis

of precision. Invalid tests were not used. 3 CVs based on total interlaboratory variability (including both within-laboratory and between-laboratory

components of variability) and averaged across sample types. IC25s or IC50s were pooled for all laboratories

to calculate the CV for each sample type. The resulting CVs were then averaged across sample types.
15

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 a

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 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 conducted in the laboratory during that month. 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. Each laboratory’s reference toxicity data will reflect conditions unique to that facility, including dilution

water, culturing, and other variables; however, each laboratory’s reference toxicity results should reflect good

repeatability.

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 (X1) are

within prescribed limits (Figure 1). The chart should plot logarithm of concentration on the vertical axis against the

date of the test or test number on the horizontal axis. The types of control charts illustrated (see USEPA, 1979a) are

used to evaluate the cumulative trend of results from a series of samples, thus reference toxicant test results should not

be used as a de facto criterion for rejection of individual effluent or receiving water tests. For endpoints that are point

estimates (LC50s and IC25s), the cumulative mean and uppe (X ̄) r and lower control limits (± 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

representing the central tendency. After two years of data collection, or a minimum of 20 data points, the control chart

should be maintained using only the 20 most recent data points.

4.16.3 Laboratories should compare the calculated CV (i.e., standard deviation / mean) of the IC25 for the 20 most

recent data points to the distribution of laboratory CVs reported nationally for reference toxicant testing (Table 3-2 in

USEPA, 2000b). If the calculated CV exceeds the 75th percentile of CVs reported nationally, the laboratory should use

the 75th and 90th percentiles to calculate warning and control limits, respectively, and the laboratory should investigate

options for reducing variability. Note: Because NOECs can only be a fixed number of discrete values, the mean,

standard deviation, and CV cannot be interpreted and applied in the same way that these descriptive statistics are

interpreted and applied for continuous variables such as the IC25 or LC50.

4.16.4 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 (LC50s and IC25s), at the

P0.05 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 laboratory should investigate sources of

variability, take corrective actions to reduce identified sources of variability, and perform an additional reference

toxicant test during the same month. Control limits for the NOECs will also be exceeded occasionally, regardless of

how well a laboratory performs. In those instances when the laboratory can document the cause for the outlier (e.g.,

operator error, culture health or test system failure), the outlier should be excluded from the future calculations of the
16

control limits. If two or more consecutive tests do not fall within the control limits, the results must be explained and

the reference toxicant test must be immediately repeated. Actions taken to correct the problem must be reported.

4.16.5 If the toxicity value from a given test with a reference toxicant fall well outside the expected range for the test

organisms when using the standard dilution water and other test conditions, the laboratory should investigate sources of

variability, take corrective actions to reduce identified sources of variability, and perform an additional reference

toxicant test during the same month. Performance should improve with experience, and the control limits for endpoints

that are point estimates should gradually narrow. However, control limits of ± 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 in determining whether or not a reference toxicant

test result falls “well” outside the expected range. The width of the control limits may be evaluated by comparing the

calculated CV (i.e., standard deviation / mean) of the IC25 for the 20 most recent data points to the distribution of

laboratory CVs reported nationally for reference toxicant testing (Table 3-2 in USEPA, 2000b). In determining

whether or not a reference toxicant test result falls “well” outside the expected range, the result also may be compared

with upper and lower bounds for ± 3S, as any result outside these control limits would be expected to occur by chance

only 1 out of 100 tests (Environment Canada, 1990). When a result from a reference toxicant test is outside the 99%

confidence intervals, the laboratory must conduct an immediate investigation to assess the possible causes for the

outlier. 

4.16.6 Reference toxicant test results should not be used as a de facto criterion for rejection of individual effluent or

receiving water tests. Reference toxicant testing is used for evaluating the health and sensitivity of organisms

over time and for documenting initial and ongoing laboratory performance. While reference toxicant test results should

not be used as a de facto criterion for test rejection, effluent and receiving water test results should be reviewed and

interpreted in the light of reference toxicant test results. The reviewer should consider the degree to which the

reference toxicant test result fell outside of control chart limits, the width of the limits, the direction of the deviation

(toward increased test organism sensitivity or toward decreased test organism sensitivity), the test conditions of both

the effluent test and the reference toxicant test, and the objective of the test.

4.17 REFERENCE TOXICANTS

4.17.1 Reference toxicants such as sodium chloride (NaCl), potassium chloride (KCl), cadmium chloride (CdCl2),

copper sulfate (CuSO4), sodium dodecyl sulfate (SDS), and potassium dichromate (K2Cr2O7), are suitable for use in the

NPDES Program and other Agency programs requiring aquatic toxicity tests. EMSL-Cincinnati plans 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. Standard reference materials 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

X '

j

n

i%1

Xi

n

S '

j

n

i'1

X 2

i &

(j

n

i'1

Xi)

2

n

n&1

Where: = Successive toxicity values from toxicity tests. Xi

n = Number of tests.

X ̄ = Mean toxicity value.

S = Standard deviation.

Figure 1. Control charts. (A) hypothesis testing results; (B) point estimates (LC, EC, or IC).
18

4.18 RECORD KEEPING 

4.18.1 Proper record keeping is important. A complete file must be maintained for each individual toxicity test or

group of tests on closely related samples. This file must 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)].
19

SECTION 5

FACILITIES, EQUIPMENT, AND SUPPLIES

5.1 GENERAL REQUIREMENTS

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 natural seawater

or water made up from hypersaline brine derived from natural seawater, or water made up from reagent grade

chemicals (GP2) or commercial (FORTY FATHOMS® or HW MARINEMIX®) artificial sea salts when specifically

recommended in the method. 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 BALSTON® Grade BX or equivalent filters, and

oil and other organic vapors can be removed using activated carbon filters (BALSTON®, C-1 filter, or equivalent). 

5.1.2 The facilities must be well ventilated and free of fumes. Laboratory ventilation systems should be checked to

ensure that return air from chemistry laboratories and/or sample handling 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 testing areas should be separated to avoid cross-contamination 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., which come in contact with the effluent and dilution water,

should be carefully chosen. Tempered glass and perfluorocarbon plastics (TEFLON®) 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, TYGON®,

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 might 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 as

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 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, 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 with effluent samples and test solutions. Some materials, such as several types of

neoprene rubber (commonly used for stoppers) may be toxic and should be tested before use.

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. 
20

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 generally does not require thorough

cleaning before use. It is sufficient to rinse new sample containers once with dilution water before use. New,

disposable, plastic test chambers may have to be rinsed with dilution water before use. New glassware must be soaked

overnight in 10% acid (see below) and also should be rinsed well in deionized water and seawater.

5.3.2 All non-disposable sample containers, test vessels, pumps, tanks, and other equipment that has come in contact

with effluent must be washed after use to remove surface contaminants, as described below. 

1. Soak 15 minutes 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 acid or 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 deionized 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 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 CULTURING AND TOXICITY TESTS 

5.4.1 Apparatus and equipment requirements for culturing and toxicity tests are specified in each toxicity test method. 

Also, see USEPA, 2002a.

5.4.2 WATER PURIFICATION SYSTEM

5.4.2.1 A good quality, laboratory grade deionized water, providing a resistance of 18 megaohm-cm, must be

available in the laboratory and in sufficient quantity for laboratory needs. Deionized water may be obtained from

MILLIPORE®, MILLI-Q®, MILLIPORE® QPAKTM

2 or equivalent system. If large quantities of high quality deionized

water are needed, it may be advisable to supply the laboratory grade water deionizer with preconditioned water from a

Culligan®, Continental®, or equivalent mixed-bed water treatment system.

5.5 REAGENTS AND CONSUMABLE MATERIALS

5.5.1 SOURCES OF FOOD FOR CULTURE AND TOXICITY TESTS

1. Brine Shrimp, Artemia sp. cysts -- Many commercial sources of brine shrimp cysts are available.

2. Frozen Adult Brine Shrimp, Artemia -- Available from most pet supply shops or other commercial

sources.

3. Flake Food -- TETRAMIN® and BIORIL® or equivalent are available at most pet supply shops.

4. Feeding requirements and other specific foods are indicated in the specific toxicity test method.

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).
21

5.5.2 Reagents and consumable materials are specified in each toxicity test method. Also, see Section 4, Quality

Assurance.

5.6 TEST ORGANISMS

5.6.1 Test organisms are obtained from inhouse cultures or commercial suppliers (see specific toxicity test method;

Sections 4, Quality Assurance and 6, Test Organisms).

5.7 SUPPLIES

5.7.1 See toxicity test methods (see Sections 11-16) for specific supplies. 
22

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 toxicity tests must be identified to species. If there is any doubt as to the identity of the test

organisms, 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, 2002a).

6.1.3 The organisms used in the short-term tests described in this manual are the sheepshead minnow, Cyprinodon

variegatus; the inland silverside, Menidia beryllina; the mysid, Mysidopsis bahia; the sea urchin, Arbacia punctulata;

and the red macroalga, Champia parvula. 

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 species in Subsection 6.1.3. Where

state regulations prohibit importation of non-native fishes or use of the 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 one or more of the

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 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 < 1‰, freshwater organisms are used regardless of the

salinity of the effluent.

2. Where the salinity of the receiving water is $1‰, 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. Also, see USEPA (2002a). 

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 should be purchased from experienced commercial suppliers (see USEPA,

1993b). 
23

6.2.3 Sheepshead minnows, inland silversides, mysids, and sea urchins may be purchased from commercial suppliers. 

However, some of these organisms (e.g., adult sheepshead minnows or adult inland silversides) may not always be

available from commercial suppliers and may have to be collected in the field and brought back to the laboratory for

spawning to obtain eggs and larvae.

6.2.4 If, because of their source, there is any uncertainty concerning the identity of the organisms, it is advisable to

have them examined by a taxonomic specialist to confirm their identification. For detailed guidance on identification,

see the individual toxicity test methods. 

6.2.5 FERAL (NATURAL OCCURRING, WILD CAUGHT) ORGANISMS

6.2.5.1 The use of test organisms taken from the receiving water has strong appeal, and would seem to be the 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 collection 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. Fish such as sheepshead minnows and silversides, and invertebrates such as mysids, are easily

reared in the laboratory or purchased. 

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 test organisms is known to the species level, it would be

necessary to examine each organism caught in the wild to confirm its identity, which 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 ensure that they are free of signs of parasitic or bacterial infections and other adverse

effects. Fish captured by electroshocking must not be used in toxicity testing.

6.2.5.2 Guidelines for collection of natural occurring organisms are provided in USEPA (1973); USEPA (1990a); and

USEPA (1993b).

6.2.6 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, such as trout, can be obtained from

stocks 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 juvenile mysids and larval fish, is required for all tests. 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 were used throughout

a monitoring program at a given facility. 

6.4 LABORATORY CULTURING 

6.4.1 Instructions for culturing and/or holding the recommended test organisms are included in specified test methods

(also, see USEPA, 2002a). 
24

6.5 HOLDING AND HANDLING TEST ORGANISMS 

6.5.1 Test organisms should not be subjected to changes of more than 3°C in water temperature or 3‰ in salinity in

any 12 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 dry surfaces or are injured

during handling must be discarded. Dipnets are best for handling larger organisms. These nets are commercially

available or can be made from small-mesh nylon netting, silk bolting cloth, plankton netting, or similar material.

Wide-bore, smooth glass tubes (4 to 8 mm ID) with rubber bulbs or pipettors (such as a PROPIPETTE® or other

pipettor) should be used for transferring smaller organisms such as mysids, and larval fish. 

6.5.3 Holding tanks for fish are supplied with a good quality water (see Section 5, Facilities, Equipment, and

Supplies) with a flow-through rate of at least two tank-volumes per day. Otherwise, use a recirculation system where

the water flows through an activated carbon or undergravel filter to remove dissolved metabolites. Culture water can

also be piped through high intensity ultraviolet light sources for disinfection, and to photo-degrade dissolved organics. 

6.5.4 Crowding should be avoided because it will stress the organisms and lower the DO concentrations to

unacceptable levels. The DO must be maintained at a minimum of 4.0 mg/L. The solubility of oxygen depends on

temperature, salinity, and altitude. 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 TETRAMIN® or BIORIL® 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 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

the bags. Another method commonly used to maintain sufficient DO during shipment is to aerate with an airstone

which is supplied from a portable pump. The DO concentration must not fall below 4.0 mg/L. 

6.6.2 Upon arrival at the test site, 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 siphoning, and

replaced slowly over a 10 to 15 minute period with dilution water. If receiving water is used as dilution water, caution

must be exercised in exposing the test organisms to it, because of the possibility that it might be toxic. For this reason,

it is recommended that only 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

are 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 receiving
25

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.

6.6.4 The marine organisms can be used at all concentrations of effluent by adjusting the salinity of the effluent to

salinities specified for the appropriate species test condition or to the salinity approximating that of the receiving water,

by adding sufficient dry ocean salts, such as FORTY FATHOMS®, or equivalent, GP2, or hypersaline brine.

6.6.5 Saline dilution water can be prepared with deionized water or a freshwater such as well water or a suitable

surface water. If dry ocean salts are used, care must be taken to ensure that the added salts are completely dissolved

and the solution is aerated 24 h before the test organisms are placed in the solutions. The test organisms should be

acclimated in synthetic saline water prepared with the dry salts. Caution: addition of dry ocean salts to dilution water

may result in an increase in pH. (The pH of estuarine and coastal saline waters is normally 7.5-8.3).

6.6.6 All effluent concentrations and the control(s) used in a test should have the same salinity. The change in salinity

upon acclimation at the desired test dilution should not exceed 6‰. The required salinities for culturing and toxicity

tests with estuarine and marine species are listed in the test method sections.

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.
26

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 absolute chronic toxicity of the effluent, a synthetic (standard)

dilution 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 outside the influence of the outfall, or with other uncontaminated natural water (surface water) or standard

dilution water having approximately the same salinity as the receiving water. Seasonal variations in the quality of

receiving waters may affect effluent toxicity. Therefore, the salinity of saline 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 the additive or mitigating effects of the discharge on already

contaminated receiving water, the test is performed using dilution water consisting of receiving water collected outside

the influence of the outfall. A second set of controls, using culture water, should be included in the test. 

7.1.2 An acceptable dilution water is one which is appropriate for the objectives of the test; supports adequate

performance of the test organisms with respect to survival, growth, reproduction, or other responses that may be

measured in the test (i.e., consistently meets test acceptability criteria for control responses); is consistent in quality;

and does not contain contaminants that could produce toxicity. Receiving waters, synthetic waters, or synthetic waters

adjusted to approximate receiving water characteristics may be used for dilution provided that the water meets the

above listed qualifications for an acceptable dilution water. USEPA (2000a) provides additional guidance on selecting

appropriate dilution waters.

7.1.3 When dual controls (one control using culture water and one control using dilution water) are used (see

Subsections 7.1.1.1 - 7.1.1.3 above), the dilution water control should be used to determine test acceptability. It is also

the dilution water control that should be compared to effluent treatments in the calculation and reporting of test results. 

The culture water control should be used to evaluate the appropriateness of the dilution water source. Significant

differences between organism responses in culture water and dilution water controls could indicate toxicity in the

dilution water and may suggest an alternative dilution water source. USEPA (2000a) provides additional guidance on

dual controls.

7.2 STANDARD, SYNTHETIC DILUTION WATER 

7.2.1 Standard, synthetic, dilution water is prepared with deionized water and reagent grade chemicals (GP2) or

commercial sea salts (FORTY FATHOMS®, HW MARINEMIX®) (Table 3). 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 MILLIPORE MILLI-Q®, MILLIPORE® QPAKTM

2 or equivalent system. It

is advisable to provide a preconditioned (deionized) feed water by using a Culligan®, Continental®, or equivalent

system in front of the MILLI-Q® System to extend the life of the MILLI-Q® cartridges (see Section 5, Facilities,

Equipment, and Supplies).
27

7.2.2.2 The recommended order of the cartridges in a four-cartridge deionizer (i.e., MILLI-Q® System or equivalent)

is: (1) ion exchange, (2) ion exchange, (3) carbon, and (4) organic cleanup (such as ORGANEX-Q®, or equivalent),

followed by a final bacteria filter. The QPAKTM

2 water system is a sealed system which does not allow for the

rearranging of the cartridges. However, the final cartridge is an ORGANEX-Q® filter, followed by a final bacteria

filter. Commercial laboratories using this system have not experienced any difficulty in using the water for culturing or

testing. Reference to the MILLI-Q® systems throughout the remainder of the manual includes all MILLIPORE® or

equivalent systems.

7.2.3 STANDARD, SYNTHETIC SEAWATER

7.2.3.1 To prepare 20 L of a standard, synthetic, reconstituted seawater (modified GP2), using reagent grade

chemicals (Table 3), with a salinity of 31‰, follow the instructions below. Other salinities can be prepared by making

the appropriate dilutions. Larger or smaller volumes of modified GP2 can be prepared by using proportionately larger

or smaller amounts of salts and dilution water.

1. Place 20 L of MILLI-Q® or equivalent deionized water in a properly cleaned plastic carboy.

2. Weigh reagent grade salts listed in Table 3 and add, one at a time, to the deionized water. Stir well

after adding each salt.

3. Aerate the final solution at a rate of 1 L/h for 24 h. 

4. Check the pH and salinity. 

7.2.3.2 Synthetic seawater can also be prepared by adding commercial sea salts, such as FORTY FATHOMS®, HW

MARINEMIX®, or equivalent, to deionized water. For example, thirty-one parts per thousand (31‰) FORTY

FATHOMS® can be prepared by dissolving 31 g of sea salts per liter of deionized water. The salinity of the resulting

solutions should be checked with a refractometer.

7.2.4 Artificial seawater is to be used only if specified in the method. EMSL-Cincinnati has found FORTY

FATHOMS® artificial sea salts suitable for maintaining and spawning the sheepshead minnow, Cyprinodon variegatus,

and for its use in the sheepshead minnow larval survival and growth test, suitable for maintaining and spawning the

inland silverside, Menidia beryllina, and for its use in the inland silverside larval survival and growth test, suitable for

culturing and maintaining mysid shrimp, Mysidopsis bahia, and its use in the mysid shrimp survival, growth, and

fecundity test, and suitable for maintaining sea urchins, Arbacia punctulata, and for its use in the sea urchin fertilization

test. The USEPA Region 6 Houston Laboratory has successfully used HW MARINEMIX® sea salts to maintain and

spawn sheepshead minnows, and perform the larval survival and growth test and the embryo-larval survival and

teratogenicity test. Also, HW MARINEMIX® sea salts has been used successfully to culture and maintain the mysid

brood stock and perform the mysid survival, growth, fecundity test. An artificial seawater formulation, GP2 (Spotte et

al., 1984), Table 3, has been used by the Environmental Research Laboratory-Narragansett, RI for all but the embryolarval survival and teratogenicity test. The suitability of GP2 as a medium for culturing organisms has not been

determined. 
28

TABLE 3. PREPARATION OF GP2 ARTIFICIAL SEAWATER USING REAGENT GRADE CHEMICALS1,2,3

Compound 

Concentration

 (g/L)

Amount (g)

Required for

 20 L

 NaCl 21.03 420.6

 Na2SO4 3.52 70.4

 KCl 0.61 12.2

 KBr 0.088 1.76

 Na2B4O7·10 H2O 0.034 0.68

 MgCl2 ·6 H2O 9.50 190.0

 CaCl2 ·2 H2O 1.32 26.4

 SrCl2 · 6 H2O 0.02 0.400

 NaHCO3 0.17 3.40

1 Modified GP2 from Spotte et al. (1984). 2 The constituent salts and concentrations were taken from USEPA (2002a). The salinity is 30.89 g/L. 3 GP2 can be diluted with deionized (DI) water to the desired test salinity.

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 close to the outfall,

but should be away from or beyond the influence of the effluent. However, if the receiving water is contaminated, it

may be necessary to collect the sample in 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 0-6°C during or immediately following collection, and maintained at that temperature

prior to use in the test. 

7.3.3 The investigator should collect uncontaminated water having a salinity as near as possible to the salinity of

the receiving water at the discharge site. Water should be collected at slack high tide, or within one hour after high

tide. If there is reason to suspect contamination of the water in the estuary, it is advisable to collect uncontaminated

water from an adjacent estuary. At times it may be necessary to collect water at a location closer to the open sea,

where the salinity is relatively high. In such cases, deionized water or uncontaminated freshwater is added to the

saline water to dilute it to the required test salinity. Where necessary, the salinity of a surface water can be increased

by the addition of artificial sea salts, such as FORTY FATHOMS®, HW MARINEMIX®, or equivalent, GP2, a
29

natural seawater of higher salinity, or hypersaline brine. Instructions for the preparation of hypersaline brine by

concentrating natural seawater are provided below. 

7.3.4 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 μm mesh openings prior to use.

7.3.5 HYPERSALINE BRINE

7.3.5.1 Hypersaline brine (HSB) has several advantages that make it desirable for use in toxicity testing. It can be

made from any high quality, filtered seawater by evaporation, and can be added to deionized water to prepare

dilution water, or to effluents or surface waters to increase their salinity. 

7.3.5.2 The ideal container for making HSB from natural seawater is one that (l) has a high surface to volume ratio,

(2) is made of a noncorrosive material, and (3) is easily cleaned (fiberglass containers are ideal). Special care should

be used to prevent any toxic materials from coming in contact with the seawater being used to generate the brine. If

a heater is immersed directly into the seawater, ensure that the heater materials do not corrode or leach any

substances that would contaminate the brine. One successful method used is a thermostatically controlled heat

exchanger made from fiberglass. If aeration is used, use only oil-free air compressors to prevent contamination. 

7.3.5.3 Before adding seawater to the brine generator, thoroughly clean the generator, aeration supply tube, heater,

and any other materials that will be in direct contact with the brine. A good quality biodegradable detergent should

be used, followed by several thorough deionized water rinses. High quality (and preferably high salinity) seawater

should be filtered to at least 10 mm before placing into the brine generator. Water should be collected on an

incoming tide to minimize the possibility of contamination.

7.3.5.4 The temperature of the seawater is increased slowly to 40°C. The water should be aerated to prevent

temperature stratification and to increase water evaporation. The brine should be checked daily (depending on the

volume being generated) to ensure that the salinity does not exceed 100‰ and that the temperature does not exceed

40°C. Additional seawater may be added to the brine to obtain the volume of brine required.

7.3.5.5 After the required salinity is attained, the HSB should be filtered a second time through a l-μm filter and

poured directly into portable containers (20-L CUBITAINERS® or polycarbonate water cooler jugs are suitable). 

The containers should be capped and labelled with the date the brine was generated and its salinity. Containers of

HSB should be stored in the dark and maintained under room temperature until used.

7.3.5.6 If a source of HSB is available, test solutions can be made by following the directions below. Thoroughly

mix together the deionized water and brine before mixing in the effluent. 

7.3.5.7 Divide the salinity of the HSB by the expected test salinity to determine the proportion of deionized water

to brine. For example, if the salinity of the brine is 100‰ and the test is to be conducted at 25‰, 100‰ divided by 

25‰ = 4.0. The proportion of brine is 1 part in 4 (one part brine to three parts deionized water). 

7.3.5.8 To make 1 L of seawater at 25‰ salinity from a hypersaline brine of 100‰, 250 mL of brine and 750 mL

of deionized water are required. 

7.4 USE OF TAP WATER AS DILUTION WATER

7.4.1 The use of tap water in the reconstituting of synthetic (artificial) seawater as dilution water is discouraged

unless it is dechlorinated and fully treated. 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 dechlorination, total residual chlorine should not exceed 0.01 mg/L. Because of the possible
30

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 dechlorination, 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.
31

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,

l988b). Conditions for exception would be: (l) better access to a sampling point between the final treatment and the

discharge outfall; (2) if the 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, 2002a).

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. 

Disadvantages: 

1. Samples are collected over a very short period of time and on a relatively infrequent basis. The

chances of detecting a spike in toxicity would depend on the frequency of sampling, and the

probability of missing spikes is high. 
32

8.2.1.2 COMPOSITE SAMPLES: 

Advantages: 

1. A single effluent sample is collected over a 24-h period. 

2. The sample is collected over a much longer period of time than grab samples 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.

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 must be collected to perform the required toxicity and chemical tests. A 4-L (1-gal)

CUBITAINER® will provide sufficient sample volume for most tests.

8.3.4 THE FOLLOWING EFFLUENT SAMPLING METHODS ARE RECOMMENDED: 

8.3.4.1 Continuous Discharges 

8.3.4.1.1 If the facility discharge is continuous, a single 24-h composite sample is to be taken.

8.3.4.2 Intermittent Discharges 

8.3.4.2.1 If the facility discharge is intermittent, a composite sample is to be collected for the duration of the

discharge but not more than 24 hours.

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 samples of receiving water is collected for use in the test.

8.4.2 The sampling point is determined by the objectives of the test. At estuarine and marine sites, samples should

be collected at mid-depth.

8.4.3 To determine the extent of the zone of toxicity in the receiving water at estuarine and marine effluent sites,

receiving water samples are collected at several distances away from the discharge. The time required for the

effluent-receiving-water mixture to travel to sampling points away from the effluent, and the rate and degree of

mixing, may be difficult to ascertain. Therefore, it may not be possible to correlate receiving water toxicity with

effluent toxicity at the discharge point unless a dye study is performed. The toxicity of receiving water samples

from five stations in the discharge plume can be evaluated using the same number of test vessels and test organisms

as used in one effluent toxicity test with five effluent dilutions.
33

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 (or hand delivered to the testing

laboratory for use on the day of collection), it is recommended that they be held at 0-6°C 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. Grab samples should be chilled immediately

following collection.

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 each grab or composite sample must not exceed 36 h. EPA believes that 36 h is adequate time to

deliver the sample 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), should 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, each grab or composite

sample may also be used to prepare test solutions for renewal at 24 h and/or 48 h after first use, if stored at 0-6°C,

with minimum head space, as described in Subsection 8.5. If shipping problems (e.g., unsuccessful Saturday

delivery) are encountered with renewal samples after a test has been initiated, the permitting authority may allow the

continued use of the most recently used sample for test renewal. 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 0-6°C 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 should not be placed between the ice and the sample in the shipping container unless required to

prevent breakage of glass sample containers.

8.5.7.2 Samples may be shipped in one or more 4-L (l-gal) CUBITAINERS® 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, CUBITAINERS® and plastic jugs are punctured to prevent reuse. 

8.5.7.3 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. 
34

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 0-6°C 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),

information on the effects of the extension in holding time on the toxicity of the 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 must 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 Adjust the sample salinity to the level appropriate for objectives of the study using hypersaline brine or

artificial sea salts. 

8.8.2 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 CUBITAINER® used), or by using an appropriate discharge

valve (spigot). 

8.8.3 It may be necessary to first coarse-filter samples through a NYLON® sieve having 2 to 4 mm mesh openings

to remove debris and/or break up large floating or suspended solids. If samples contain indigenous organisms that

may attack or be confused with the test organisms, the samples should be filtered through a sieve with 60-μm mesh

openings. Since filtering may increase the dissolved oxygen (DO) in an effluent, the DO should be checked both

before and after filtering. Low dissolved oxygen concentrations will indicate a potential problem in performing the

test. Caution: filtration may remove some toxicity.

8.8.4 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, samples may be warmed slowly in

open test containers. If DO is still above 100% saturation, based on temperature and salinity (Table 4), after

warming to test temperature, samples should be aerated moderately (approximately 500 mL/min) for a few minutes

using an airstone. If DO is below 4.0 mg/L, the solutions must be 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.

8.8.4.1 Aeration during the test may alter the results and should be used 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 the pH. However, the DO in the test solution should not be

permitted to fall below 4.0 mg/L.

8.8.4.2 In static tests (non-renewal or renewal) low DOs may commonly occur in the higher concentrations of

wastewater. Aeration is accomplished by bubbling air through a pipet at the rate of 100 bubbles/min. If aeration is

necessary, all test solutions must be aerated. It is advisable to monitor the DO closely during the first few hours of

the test. Samples with a potential DO problem generally show a downward trend in DO within 4 to 8 h after the test
35

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 or salinity, 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 and the dilution water.

8.8.6 Total ammonia is measured in effluent and receiving water samples where toxicity may be contributed by

unionized ammonia (i.e., where total ammonia $ 5 mg/L). The concentration (mg/L) of unionized (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 than

the amount needed to dechlorinate tap water, (see Section 7, Dilution Water). 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 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. 

8.8.9 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, and marine samples are adjusted to pH 8.0, by adding 1N NaOH or 1N

HCl dropwise, as required, being careful to avoid overadjustment.
36

TABLE 4. OXYGEN SOLUBILITY (MG/L) IN WATER AT EQUILIBRIUM WITH AIR AT 760 MM HG

(AFTER Richards and Corwin, 1956) 

TEMP SALINITY (‰)

(C°) 0 5 10 15 20 25 30 35 40 43

 0 14.2 13.8 13.4 12.9 12.5 12.1 11.7 11.2 10.8 10.6

 1 13.8 13.4 13.0 12.6 12.2 11.8 11.4 11.0 10.6 10.3

 2 13.4 13.0 12.6 12.2 11.9 11.5 11.1 10.7 10.3 10.0

 3 13.1 12.7 12.3 11.9 11.6 11.2 10.8 10.4 10.0 9.8

 4 12.7 12.3 12.0 11.6 11.3 10.9 10.5 10.1 9.8 9.5

 5 12.4 12.0 11.7 11.3 11.0 10.6 0.2 9.8 9.5 9.3

 6 12.1 11.7 11.4 11.0 10.7 10.3 0.0 9.6 9.3 9.1

 8 11.5 11.2 10.8 10.5 10.2 9.8 9.5 9.2 8.9 8.7

10 10.9 10.7 10.3 10.0 9.7 9.4 9.1 8.8 8.5 8.3

12 10.5 10.2 9.9 9.6 9.3 9.0 8.7 8.4 8.1 7.9

14 10.0 9.7 9.5 9.2 8.9 8.6 8.3 8.1 7.8 7.6

16 9.6 9.3 9.1 8.8 8.5 8.3 8.0 7.7 7.5 7.3

18 9.2 9.0 8.7 8.5 8.2 8.0 7.7 7.5 7.2 7.1

20 8.9 8.6 8.4 8.1 7.9 7.7 7.4 7.2 6.9 6.8

22 8.6 8.4 8.1 7.9 7.6 7.4 7.2 6.9 6.7 6.6

24 8.3 8.1 7.8 7.6 7.4 7.2 6.9 6.7 6.5 6.4

26 8.1 7.8 7.6 7.4 7.2 7.0 6.7 6.5 6.3 6.1

28 7.8 7.6 7.4 7.2 7.0 6.8 6.5 6.3 6.1 6.0

30 7.6 7.4 7.1 6.9 6.7 6.5 6.3 6.1 5.9 5.8

32 7.3 7.1 6.9 6.7 6.5 6.3 6.1 5.9 5.7 5.6
37

TABLE 5. PERCENT UNIONIZED NH3 IN AQUEOUS AMMONIA SOLUTIONS: TEMPERATURE 

15-26°C AND pH 6.0-8.91

pH TEMPERATURE (°C)

15 16 17 18 19 20 21 22 23 24 25 26

6.0 0.0274 0.0295 0.0318 0.0343 0.0369 0.0397 0.0427 0.0459 0.0493 0.0530 0.0568 0.0610

6.1 0.0345 0.0372 0.0400 0.0431 0.0464 0.0500 0.0537 0.0578 0.0621 0.0667 0.0716 0.0768

6.2 0.0434 0.0468 0.0504 0.0543 0.0584 0.0629 0.0676 0.0727 0.0781 0.0901 0.0901 0.0966

6.3 0.0546 0.0589 0.0634 0.0683 0.0736 0.0792 0.0851 0.0915 0.0983 0.1134 0.1134 0.1216

6.4 0.0687 0.0741 0.0799 0.0860 0.0926 0.0996 0.107 0.115 0.124 0.133 0.143 0.153

6.5 0.0865 0.0933 0.1005 0.1083 0.1166 0.1254 0.135 0.145 0.156 0.167 0.180 0.19

6.6 0.109 0.117 0.127 0.136 0.147 0.158 0.170 0.182 0.196 0.210 0.226 0.242

6.7 0.137 0.148 0.159 0.171 0.185 0.199 0.214 0.230 0.247 0.265 0.284 0.305

6.8 0.172 0.186 0.200 0.216 0.232 0.250 0.269 0.289 0.310 0.333 0.358 0.384

6.9 0.217 0.234 0.252 0.271 0.292 0.314 0.338 0.363 0.390 0.419 0.450 0.482

7.0 0.273 0.294 0.317 0.342 0.368 0.396 0.425 0.457 0.491 0.527 0.566 0.607

7.1 0.343 0.370 0.399 0.430 0.462 0.497 0.535 0.575 0.617 0.663 0.711 0.762

7.2 0.432 0.466 0.502 0.540 0.581 0.625 0.672 0.722 0.776 0.833 0.893 0.958

7.3 0.543 0.586 0.631 0.679 0.731 0.786 0.845 0.908 0.975 1.05 1.12 1.20

7.4 0.683 0.736 0.793 0.854 0.918 0.988 1.061 1.140 1.224 1.31 1.41 1.51

7.5 0.858 0.925 0.996 1.07 1.15 1.24 1.33 1.43 1.54 1.65 1.77 1.89

7.6 1.08 1.16 1.25 1.35 1.45 1.56 1.67 1.80 1.93 2.07 2.21 2.37

7.7 1.35 1.46 1.57 1.69 1.82 1.95 2.10 2.25 2.41 2.59 2.77 2.97

7.8 1.70 1.83 1.97 2.12 2.28 2.44 2.62 2.82 3.02 3.24 3.46 3.71

7.9 2.13 2.29 2.46 2.65 2.85 3.06 3.28 3.52 3.77 4.04 4.32 4.62

8.0 2.66 2.87 3.08 3.31 3.56 3.82 4.10 4.39 4.70 5.03 5.38 5.75

8.1 3.33 3.58 3.85 4.14 4.44 4.76 5.10 5.46 5.85 6.25 6.68 7.14

8.2 4.16 4.47 4.80 5.15 5.52 5.92 6.34 6.78 7.25 7.75 8.27 8.82

8.3 5.18 5.56 5.97 6.40 6.86 7.34 7.85 8.39 8.96 9.56 10.2 10.9

8.4 6.43 6.90 7.40 7.93 8.48 9.07 9.69 10.3 11.0 11.7 12.5 13.3

8.5 7.97 8.54 9.14 9.78 10.45 11.16 11.90 12.7 13.5 14.4 15.2 16.2

8.6 9.83 10.5 11.2 12.0 12.8 13.6 14.5 15.5 16.4 17.4 18.5 19.5

8.7 12.07 12.9 13.8 14.7 15.6 16.6 17.6 18.7 19.8 21.0 22.2 23.4

8.8 14.7 15.7 16.7 17.8 18.9 20.0 21.2 22.5 23.7 25.1 26.4 27.8

8.9 17.9 19.0 20.2 21.4 22.7 24.0 25.3 26.7 28.2 29.6 31.1 32.6

1 Table provided by Teresa Norberg-King, Duluth, Minnesota. Also see Emerson et al. (1975), Thurston et al.

(1974), and USEPA (1985a). 
38

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 fullscale 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 MULTICONCENTRATION (DEFINITIVE) EFFLUENT TOXICITY TESTS 

8.10.1 The tests recommended for use in determining discharge permit compliance in the NPDES program are

multiconcentration, 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. USEPA recommends the use

of a $0.5 dilution factor for selecting effluent test concentrations. Effluent test concentrations of 6.25%, 12.5%,

25%, 50%, and 100% are commonly used, however, test concentrations should be selected independently for each

test based on the objective of the study, the expected range of toxicity, the receiving water concentration, and any

available historical testing information on the effluent. USEPA (2000a) provides additional guidance on choosing

appropriate test concentrations.

8.10.3 When these tests are used in determining compliance with permit limits, effluent test concentrations should

be selected to bracket the receiving water concentration. This may be achieved by selecting effluent test

concentrations in the following manner: (1) 100% effluent, (2) [RWC + 100]/2, (3) RWC, (4) RWC/2, and (5)

RWC/4. For example, where the RWC = 50%, appropriate effluent concentrations may 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.

8.11 RECEIVING WATER TESTS 

8.11.1 Receiving water toxicity tests generally consist of 100% receiving water and a control. The total salinity 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).
39

8.11.3 In cases where the objective of the test is to estimate the degree of toxicity of the receiving water, a

definitive, multiconcentration test is performed by preparing dilutions of the receiving water, using a $0.5 dilution

series, with a suitable control water. 
40

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 test 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 the observable 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 (IC) - The toxicant concentration that would cause a given percent reduction in a

nonquantal 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 in the mean population responses.

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
41

Sum Test with Bonferroni adjustment), whereas LCs, ICs, and ECs are determined by point estimation techniques

(Probit Analysis, the Spearman-Karber Method, the Trimmed Spearman-Karber Method, the 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, IC, EC, or other point estimates derived

from curve fitting, interpolation, etc.

9.2.2 Most point estimates, such as the LC, IC, or EC are derived from a mathematical model that assumes a

continuous dose-response relationship. By definition, any LC, IC, 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 effect. 

The purpose of the statistical analysis in this case is to estimate as closely as possible where that threshold lies.

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.

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 noncontinuous 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.
42

9.2.5 A better understanding of the relationship between endpoints derived by hypothesis testing (NOECs) and

point estimation techniques (LCs, ICs, 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 sea urchin, Arbacia punctulata,

the sheepshead minnow, Cyprinodon variegatus, and the red macroalga, Champia parvula. Birge et al. (1985)

reported that LC1s derived from Probit Analyses of data from short-term embryo-larval tests with reference

toxicants were comparable to NOECs for several organisms. Similarly, USEPA (1988d) reported that the IC25s

were comparable to the NOECs for a set of daphnia, 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 interval

above 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.

9.3.2.2 It should be noted that software used to calculate point estimates occasionally may not provide associated

95% confidence intervals. This situation may arise when test data do not meet specific assumptions required by the

statistical methods, when point estimates are outside of the test concentration range, and when specific limitations

imposed by the software are encountered. USEPA (2000a) provides guidance on confidence intervals under these

circumstances.

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
43

assumptions, a statistician should be consulted. Analysts who are not proficient in statistics are strongly advised to

seek the assistance of a statistician before selecting the method of analysis and using any of the results.

9.4.1.2 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 computerintensive computations. Alternative point estimations 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 ensure 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 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 data.

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.
44

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 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 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 tests

of assumptions, as illustrated in the flowchart. A specific flow chart is included in the analysis section for each test.

9.5.2 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 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 sheepshead minnow, Cyprinodon variegatus, and inland silverside, Menidia

beryllina, larval survival and growth tests, and the mysid, Mysidopsis bahia, survival, growth, and fecundity test, are

analyzed using hypothesis testing 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

concentrations that have tested significantly different from the control for survival are excluded from further

hypothesis tests concerning growth effects. Growth is defined as the change in dry weight of the orginal number of

test organisms when group weights are obtained. When analyzing the data using point estimating techniques, data

from all concentrations are included in the analysis.

9.5.3.2 Fecundity data from the mysid, Mysidopsis bahia, test may be analyzed using hypothesis testing after an

arc sine transformation according to the flowchart in Figure 2. The fecundity data from the mysid test may also be

analyzed by generating a point estimate with the Linear Interpolation Method.

9.5.3.3 Reproduction data from the red macroalga, Champia parvula, test are analyzed using hypothesis testing as

illustrated in Figure 2. The reproduction data from the red macroalga test may also be analyzed by generating a

point estimate with the Linear Interpolation Method.
45

Figure 2. Flowchart for statistical analysis of test data
46

9.5.4 ANALYSIS OF THE SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION DATA

9.5.4.1 Data from the sea urchin, Arbacia punctulata, fertilization test may be analyzed by hypothesis testing after

an arc sine transformation according to the flowchart in Figure 2. The fertilization data from the sea urchin test may

also be analyzed by generating a point estimate with the Linear Interpolation Method.

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 HK) (see discussion below). The mortality data can also be analyzed by hypothesis testing, after an arc sine square

root transformation (see Appendices B-F), according to the flowchart in Figure 2.

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 The t test with the Bonferroni 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 the Bonferroni 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 test is reported in terms of the NOEC. A

step-by-step example of the use of a t-test with the Bonferroni adjustment is provided in Appendix D.
47

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 is a nonparametric test for comparing a treatment 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 an LC1, 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. 

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 Probit Analysis to determine if use of the

analysis is appropriate. The chi-square test for heterogeneity provides a good test of appropriateness of the analysis. 

The computer program (see discussion, Appendix H) 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 H. 
48

9.7.1.4 In cases where Probit Analysis is not appropriate, the LC50 and confidence interval may be estimated by

the Spearman-Karber Method (Appendix I) or the Trimmed Spearman-Karber Method (Appendix J). If a 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 K).

9.7.2 LINEAR INTERPOLATION METHOD

9.7.2.1 The Linear Interpolation Method (see Appendix L) is a procedure to calculate a point estimate of the

effluent or other toxicant concentration [Inhibition Concentration, (IC)] that causes a given percent reduction (e.g.,

25%, 50%, etc.) in the reproduction, growth, fertilization, or fecundity 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 piece-wise linear response function, and (3) are from a

random, independent, and representative sample of test data. The assumption for piece-wise 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, the smoothing process

may result in a large upward adjustment in the control mean.

9.7.2.3 The inability to test the monotonicity and piece wise 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 L for a more detailed discussion of the use of this method and a computer program available for

performing calculations.
49

SECTION 10

REPORT PREPARATION AND TEST REVIEW

10.1 REPORT PREPARATION

The toxicity data are reported, together with other appropriate data. The following general format and content are

recommended for the report: 

10.1.1 INTRODUCTION

1. Permit number

2. Toxicity testing requirements of permit

3. Plant location

4. Name of receiving water body

5. Contract Laboratory (if the test was performed under contract)

a. Name of firm

b. Phone number

c. Address

6. Objective of test

10.1.2 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

10.1.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION WATER

1. Effluent Samples

a. Sampling point (including latitude and longitude)

b. Collection dates and times

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 (including latitude and longitude)

b. Collection dates and times

c. Sample collection method

d. Physical and chemical data

 e. Tide stages

f. Sample temperature when received at the laboratory

 g. Lapsed time from sample collection to delivery
50

3. Dilution Water Samples

a. Source

b. Collection date and time

c. Pretreatment

d. Physical and chemical characteristics 

10.1.4 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 of volume and test chambers

 7. Volume of solution used per chamber

 8. Number of organisms used per test chamber

 9. Number of replicate test chambers per treatment

10. Acclimation of test organisms (temperature and salinity mean and range)

11. Test temperature (mean and range)

12. Specify if aeration was needed

13. Feeding frequency, and amount and type of food

14. Test salinity (mean and range)

15. Specify if (and how) pH control measures were implemented

10.1.5 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

10.1.6 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); report percent

minimum significant difference (PMSD) calculated for sublethal endpoints determined by hypothesis

testing in reference toxicant test

5. Physical and chemical methods used

10.1.7 RESULTS

1. Provide raw toxicity data in tabular form, including daily records of affected organisms in each 

concentration (including controls) and replicate, and in graphical form (plots of toxicity data)

2. Provide table of LC50s, NOECs, IC25, IC50, etc. (as required in the applicable NPDES permit)

3. Indicate statistical methods to calculate endpoints

4. Provide summary table of physical and chemical data

5. Tabulate QA data

6. Provide percent minimum significant difference (PMSD) calculated for sublethal endpoints
51

10.1.8 CONCLUSIONS AND RECOMMENDATIONS

1. Relationship between test endpoints and permit limits.

2. Action to be taken.

10.2 TEST REVIEW

10.2.1 Test review is an important part of an overall quality assurance program (Section 4) and is necessary for

ensuring that all test results are reported accurately. Test review should be conducted on each test by both the

testing laboratory and the regulatory authority. 

10.2.2 SAMPLING AND HANDLING 

10.2.2.1 The collection and handling of samples are reviewed to verify that the sampling and handling procedures

given in Section 8 were followed. Chain-of-custody forms are reviewed to verify that samples were tested within

allowable sample holding times (Subsection 8.5.4). Any deviations from the procedures given in Section 8 should

be documented and described in the data report (Subsection 10.1). 

10.2.3 TEST ACCEPTABILITY CRITERIA

10.2.3.1 Test data are reviewed to verify that test acceptability criteria (TAC) requirements for a valid test have

been met. Any test not meeting the minimum test acceptability criteria is considered invalid. All invalid tests must

be repeated with a newly collected sample. 

10.2.4 TEST CONDITIONS

10.2.4.1 Test conditions are reviewed and compared to the specifications listed in the summary of test condition

tables provided for each method. Physical and chemical measurements taken during the test (e.g., temperature, pH,

and DO) also are reviewed and compared to specified ranges. Any deviations from specifications should be

documented and described in the data report (Subsection 10.1).

10.2.4.2 The summary of test condition tables presented for each method identify test conditions as required or

recommended. For WET test data submitted under NPDES permits, all required test conditions must be met or the

test is considered invalid and must be repeated with a newly collected sample. Deviations from recommended test

conditions must be evaluated on a case-by-case basis to determine the validity of test results. Deviations from

recommended test conditions may or may not invalidate a test result depending on the degree of the departure and

the objective of the test. The reviewer should consider the degree of the deviation and the potential or observed

impact of the deviation on the test result before rejecting or accepting a test result as valid. For example, if

dissolved oxygen is measured below 4.0 mg/L in one test chamber, the reviewer should consider whether any

observed mortality in that test chamber corresponded with the drop in dissolved oxygen.

10.2.4.3 Whereas slight deviations in test conditions may not invalidate an individual test result, test condition

deviations that continue to occur frequently in a given laboratory may indicate the need for improved quality control

in that laboratory. 

10.2.5 STATISTICAL METHODS

10.2.5.1 The statistical methods used for analyzing test data are reviewed to verify that the recommended

flowcharts for statistical analysis were followed. Any deviation from the recommended flowcharts for selection of

statistical methods should be noted in the data report. Statistical methods other than those recommended in the

statistical flowcharts may be appropriate (see Subsection 9.4.1.2), however, the laboratory must document the use of

and provide the rationale for the use of any alternate statistical method. In all cases (flowchart recommended
52

methods or alternate methods), reviewers should verify that the necessary assumptions are met for the statistical

method used. 

10.2.6 CONCENTRATION-RESPONSE RELATIONSHIPS

10.2.6.1 The concept of a concentration-response, or more classically, a dose-response relationship is “the most

fundamental and pervasive one in toxicology” (Casarett and Doull, 1975). This concept assumes that there is a

causal relationship between the dose of a toxicant (or concentration for toxicants in solution) and a measured

response. A response may be any measurable biochemical or biological parameter that is correlated with exposure

to the toxicant. The classical concentration-response relationship is depicted as a sigmoidal shaped curve, however,

the particular shape of the concentration-response curve may differ for each coupled toxicant and response pair. In

general, more severe responses (such as acute effects) occur at higher concentrations of the toxicant, and less severe

responses (such as chronic effects) occur at lower concentrations. A single toxicant also may produce multiple

responses, each characterized by a concentration-response relationship. A corollary of the concentration-response

concept is that every toxicant should exhibit a concentration-response relationship, given that the appropriate

response is measured and given that the concentration range evaluated is appropriate. Use of this concept can be

helpful in determining whether an effluent possesses toxicity and in identifying anomalous test results.

10.2.6.2 The concentration-response relationship generated for each multi-concentration test must be reviewed to

ensure that calculated test results are interpreted appropriately. USEPA (2000a) provides guidance on evaluating

concentration-response relationships to assist in determining the validity of WET test results. All WET test results

(from multi-concentration tests) reported under the NPDES program should be reviewed and reported according to

USEPA guidance on the evaluation of concentration-response relationships (USEPA, 2000a). This guidance

provides review steps for 10 different concentration-response patterns that may be encountered in WET test data. 

Based on the review, the guidance provides one of three determinations: that calculated effect concentrations are

reliable and should be reported, that calculated effect concentrations are anomalous and should be explained, or that

the test was inconclusive and the test should be repeated with a newly collected sample. It should be noted that the

determination of a valid concentration-response relationship is not always clear cut. Data from some tests may

suggest consultation with professional toxicologists and/or regulatory officials. Tests that exhibit unexpected

concentration-response relationships also may indicate a need for further investigation and possible retesting. 

10.2.7 REFERENCE TOXICANT TESTING

10.2.7.1 Test review of a given effluent or receiving water test should include review of the associated reference

toxicant test and current control chart. Reference toxicant testing and control charting is required for documenting

the quality of test organisms (Subsection 4.7) and ongoing laboratory performance (Subsection 4.16). The reviewer

should verify that a quality control reference toxicant test was conducted according to the specified frequency

required by the permitting authority or recommended by the method (e.g., monthly). The test acceptability criteria,

test conditions, concentration-response relationship, and test sensitivity of the reference toxicant test are reviewed to

verify that the reference toxicant test conducted was a valid test. The results of the reference toxicant test are then

plotted on a control chart (see Subsection 4.16) and compared to the current control chart limits (± 2 standard

deviations).

10.2.7.2 Reference toxicant tests that fall outside of recommended control chart limits are evaluated to determine

the validity of associated effluent and receiving water tests (see Subsection 4.16). An out of control reference

toxicant test result does not necessarily invalidate associated test results. The reviewer should consider the degree to

which the reference toxicant test result fell outside of control chart limits, the width of the limits, the direction of the

deviation (toward increasing test organism sensitivity or toward decreasing test organism sensitivity), the test

conditions of both the effluent test and the reference toxicant test, and the objective of the test. More frequent

and/or concurrent reference toxicant testing may be advantageous if recent problems (e.g., invalid tests, reference

toxicant test results outside of control chart limits, reduced health of organism cultures, or increased within-test

variability) have been identified in testing. 
53

10.2.8 TEST VARIABILITY

10.2.8.1 The within-test variability of individual tests should be reviewed. Excessive within-test variability may

invalidate a test result and warrant retesting. For evaluating within-test variability, reviewers should consult EPA

guidance on upper and lower percent minimum significant difference (PMSD) bounds (USEPA, 2000b). 

10.2.8.2 When NPDES permits require sublethal hypothesis testing endpoints from Methods 1006.0 or 1007.0

(e.g., growth NOECs and LOECs), within-test variability must be reviewed and variability criteria must be applied

as described in this section (10.2.8.2). When the methods are used for non-regulatory purposes, the variability

criteria herein are recommended but are not required, and their use (or the use of alternative variability criteria) may

depend upon the intended uses of the test results and the requirements of any applicable data quality objectives and

quality assurance plan.

10.2.8.2.1 To measure test variability, calculate the percent minimum significant difference (PMSD) achieved in

the test. The PMSD is the smallest percentage decrease in growth or reproduction from the control that could be

determined as statistically significant in the test. The PMSD is calculated as 100 times the minimum significant

difference (MSD) divided by the control mean. The equation and examples of MSD calculations are shown in

Appendix C. PMSD may be calculated legitimately as a descriptive statistic for within-test variability, even when

the hypothesis test is conducted using a non-parametric method. The PMSD bounds were based on a representative

set of tests, including tests for which a non-parametric method was required for determining the NOEC or LOEC. 

The conduct of hypothesis testing to determine test results should follow the statistical flow charts provided for each

method. That is, when test data fail to meet assumptions of normality or heterogeneity of variance, a non-parametric

method (determined following the statistical flowchart for the method) should be used to calculate test results, but

the PMSD may be calculated as described above (using parametric methods) to provide a measure of test variability. 

10.2.8.2.2 Compare the PMSD measured in the test with the upper PMSD bound variability criterion listed in

Table 6. When the test PMSD exceeds the upper bound, the variability among replicates is unusually large for the

test method. Such a test should be considered insufficiently sensitive to detect toxic effects on growth or

reproduction of substantial magnitude. A finding of toxicity at a particular concentration may be regarded as

trustworthy, but a finding of "no toxicity" or "no statistically significant toxicity" at a particular concentration should

not be regarded as a reliable indication that there is no substantial toxic effect on growth or reproduction at that

concentration.

10.2.8.2.3 If the PMSD measured for the test is less than or equal to the upper PMSD bound variability criterion in

Table 6, then the test's variability measure lies within normal bounds and the effect concentration estimate (e.g.,

NOEC or LOEC) would normally be accepted unless other test review steps raise serious doubts about its validity.

10.2.8.2.4 If the PMSD measured for the test exceeds the upper PMSD bound variability criterion in Table 6, then

one of the following two cases applies (10.2.8.2.4.1, 10.2.8.2.4.2).

10.2.8.2.4.1 If toxicity is found at the permitted receiving water concentration (RWC) based upon the value of the

effect concentration estimate (NOEC or LOEC), then the test shall be accepted and the effect concentration estimate

may be reported, unless other test review steps raise serious doubts about its validity.

10.2.8.2.4.2 If toxicity is not found at the permitted RWC based upon the value of the effect concentration estimate

(NOEC or LOEC) and the PMSD measured for the test exceeds the upper PMSD bound, then the test shall not be

accepted, and a new test must be conducted promptly on a newly collected sample.

10.2.8.2.5 To avoid penalizing laboratories that achieve unusually high precision, lower PMSD bounds shall also

be applied when a hypothesis test result (e.g., NOEC or LOEC) is reported. Lower PMSD bounds, which are based

on the 10th percentiles of national PMSD data, are presented in Table 6. The 10th percentile PMSD represents a

practical limit to the sensitivity of the test method because few laboratories are able to achieve such precision on a
54

regular basis and most do not achieve it even occasionally. In determining hypothesis test results (e.g., NOEC or

LOEC), a test concentration shall not be considered toxic (i.e., significantly different from the control) if the relative

difference from the control is less than the lower PMSD bounds in Table 6. See USEPA, 2000b for specific

examples of implementing lower PMSD bounds. 

10.2.8.3 To assist in reviewing within-test variability, EPA recommends maintaining control charts of PMSDs

calculated for successive effluent tests (USEPA, 2000b). A control chart of PMSD values characterizes the range of

variability observed within a given laboratory, and allows comparison of individual test PMSDs with the

laboratory’s typical range of variability. Control charts of other variability and test performance measures, such as

the MSD, standard deviation or CV of control responses, or average control response, also may be useful for

reviewing tests and minimizing variability. The log of PMSD will provide an approximately normal variate useful

for control charting.

TABLE 6. VARIABILITY CRITERIA (UPPER AND LOWER PMSD BOUNDS) FOR SUBLETHAL

HYPOTHESIS TESTING ENDPOINTS SUBMITTED UNDER NPDES PERMITS.1

Test Method Endpoint Lower PMSD

Bound

Upper PMSD

Bound

Method 1006.0, Inland Silverside

Larval Survival and Growth Test growth 11 28

Method 1007.0, Mysidopsis bahia

Survival, Growth, and Fecundity Test growth 11 37

1

 Lower and upper PMSD bounds were determined from the 10th and 90th percentile, respectively, of PMSD

data from EPA’s WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2000b).
55

SECTION 11

TEST METHOD

SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS

LARVAL SURVIVAL AND GROWTH TEST

METHOD 1004.0

11.1 SCOPE AND APPLICATION

11.1.1 This method, adapted in part from USEPA (1987b), estimates the chronic toxicity of effluents and receiving

waters to the sheepshead minnow, Cyprinodon variegatus, 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

species.

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 chemical 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

volatile and highly degradable toxicants present in the source may not be detected in the test.

11.1.5 This 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 Sheepshead minnow, Cyprinodon variegatus, larvae (preferably less than 24-h old) are exposed in a static

renewal system for seven days to different concentrations of effluent or to receiving water. Test results are based on

the survival and 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 concentrations (DO), high concentrations of suspended and/or

dissolved solids, and extremes of pH, may mask the effects of toxic substances. 

11.3.3 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). 

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 reduce the apparent toxicity

of the test substance. However, in a growth test the nutritional needs of the organisms must be satisfied, even if

feeding has the potential to confound test results. 
56

11.3.6 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-dependent toxicants

(such as metals) are present. As pH increases, the toxicity of ammonia also increases (see Subsection 8.8.6), so

upward pH drift may increase sample toxicity. For metals, toxicity may increase or decrease with increasing pH. 

Lead and copper were found to be more acutely toxic at pH 6.5 than at pH 8.0 or 8.5, while nickel and zinc were

more toxic at pH 8.5 than at pH 6.5 (USEPA, 1992). In situations where sample toxicity is confirmed to be

artifactual and due to pH drift (as determined by parallel testing as described in Subsection 11.3.6.1), the regulatory

authority may allow for control of sample pH during testing using procedures outlined in Subsection 11.3.6.2. It

should be noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large (more than 1 pH

unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold for toxicity. 

11.3.6.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one with

controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to drift during the

test. In the controlled-pH treatment, the pH is maintained using the procedures described in Subsection 11.3.6.2. 

The pH to be maintained in the controlled-pH treatment (or target pH) will depend on the objective of the test. If the

objective of the WET test is to determine the toxicity of the effluent in the receiving water, the pH should be

maintained at the pH of the receiving water (measured at the edge of the regulatory mixing zone). If the objective of

the WET test is to determine the absolute toxicity of the effluent, the pH should be maintained at the pH of the

sample after adjusting the sample salinity for use in marine testing. 

11.3.6.1.1 During parallel testing, the pH must be measured in each treatment at the beginning (i.e., initial pH) and

end (i.e., final pH) of each 24-h exposure period. For each treatment, the mean initial pH (e.g., averaging the initial

pH measured each day for a given treatment) and the mean final pH (e.g., averaging the final pH measured each day

for a given treatment) must be reported. pH measurements taken during the test must confirm that pH was

effectively maintained at the target pH in the controlled-pH treatment. For each treatment, the mean initial pH and

the mean final pH should be within ±0.3 pH units of the target pH. Test procedures for conducting toxicity

identification evaluations (TIEs) also recommend maintaining pH within ± 0.3 pH units in pH-controlled tests

(USEPA, 1996).

11.3.6.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing. Total

ammonia concentrations greater than 5 mg/L in the 100% effluent are an indicator that toxicity observed in the test

may be due to ammonia (USEPA, 1992). 

11.3.6.1.3 Results from both of the parallel tests (pH-controlled and uncontrolled treatments) must be reported to

the regulatory authority. If the uncontrolled test meets test acceptability criteria and shows no toxicity at the

permitted instream waste concentration, then the results from this test should be used for determining compliance. If

the uncontrolled test shows toxicity at the permitted instream waste concentration, then the results from the pHcontrolled test should be used for determining compliance, provided that this test meets test acceptability criteria and

pH was properly controlled (see Subsection 11.3.6.1.1). 

11.3.6.1.4 To confirm that toxicity observed in the uncontrolled test was artifactual and due to pH drift, the results

of the controlled and uncontrolled-pH tests are compared. If toxicity is removed or reduced in the pH-controlled

treatment, artifactual toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of

artifactual toxicity is representative of a given effluent, the regulatory authority may require additional information

or additional parallel testing before pH control (as described in Subsection 11.3.6.2) is applied routinely to

subsequent testing of the effluent. 

11.3.6.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-controlled

atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding 1N NaOH or 1N HCl

(see Subsection 8.8.9). The addition of acids and bases should be minimized to reduce the amount of additional ions

(Na or Cl) added to the sample. pH is then controlled using the CO2-controlled atmosphere technique. This may be

accomplished by placing test solutions and test organisms in closed headspace test chambers, and then injecting a

predetermined volume of CO2 into the headspace of each test chamber (USEPA, 1991b; USEPA, 1992); or by

placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and air (USEPA, 1996). Prior
57

experimentation will be needed to determine the appropriate CO2/air ratio or the appropriate volume of CO2 to inject. 

This volume will depend upon the sample pH, sample volume, container volume, and sample constituents. If more

than 5% CO2 is needed, adjust the solutions with acids (1N HCl) and then flush the headspace with no more than 5%

CO2 (USEPA, 1992). If the objective of the WET test is to determine the toxicity of the effluent in the receiving

water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH of the receiving water

(measured at the edge of the regulatory mixing zone). If the objective of the WET test is to determine the absolute

toxicity of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH of the

sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and Mount (1992)

provide techniques and guidance for controlling test pH using a CO2-controlled atmosphere. In pH-controlled

testing, control treatments must be subjected to all manipulations that sample treatments are subjected to. These

manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-controlled testing,

the pH also must be measured in each treatment at the beginning and end of each 24-h exposure period to confirm

that pH was effectively controlled at the target pH level. 

11.4 SAFETY

11.4.1 See Section 3, Health and Safety.

11.5 APPARATUS AND EQUIPMENT

11.5.1 Facilities for holding and acclimating test organisms.

11.5.2 Brine shrimp, Artemia, culture unit -- see Subsection 11.6.14 below and Section 4, Quality Assurance.

11.5.3 Sheepshead minnow culture unit -- see Subsection 11.6.15 below. The maximum number of larvae required

per test will range from a maximum of 360, if 15 larvae are used in each of four replicates, to a minimum of 240 per

test, if 10 larvae are used in each of four replicates. It is preferable to obtain the test organisms from an in-house

culture unit. If it is not feasible to culture fish in-house, embryos or newly hatched larvae can be obtained from

other sources if shipped in well oxygenated saline water in insulated containers.

11.5.4 Samplers -- automatic sampler, preferably with sample cooling capability, that can collect a 24-h composite

sample of 5 L.

11.5.5 Environmental chamber or equivalent facility with temperature control (25 ± 1°C).

11.5.6 Water purification system -- Millipore Milli-Q®, deionized water (DI) or equivalent.

11.5.7 Balance -- Analytical, capable of accurately weighing to 0.00001 g.

11.5.8 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.

11.5.9 Drying oven -- 50-105°C range, for drying larvae.

11.5.10 Air pump -- for oil-free air supply.

11.5.11 Air lines, and air stones -- for aerating water containing embryos or larvae, or for supplying air to test

solutions with low DO.

11.5.12 Meters, pH and DO -- for routine physical and chemical measurements. 

11.5.13 Standard or micro-Winkler apparatus -- for determining DO (optional).
58

11.5.14 Dissecting microscope -- for checking embryo viability.

11.5.15 Desiccator -- for holding dried larvae.

11.5.16 Light box -- for counting and observing larvae. 

11.5.17 Refractometer -- for determining salinity. 

11.5.18 Thermometers, glass or electronic, laboratory grade -- for measuring water temperatures. 

11.5.19 Thermometers, bulb-thermograph or electronic-chart type -- for continuously recording temperature. 

11.5.20 Thermometer, National Bureau of Standards Certified (see USEPA Method 170.1, USEPA, 1979b) -- to

calibrate laboratory thermometers. 

11.5.21 Test chambers -- four for each concentration and control. Borosilicate glass 1000 mL beakers or modified

Norberg and Mount (1985) glass chambers used in the short-term inland silverside test may be used. It is

recommended that each chamber contain a minimum of 50 mL/larvae and allow adequate depth of test solution (5.0

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).

11.5.22 Beakers -- six Class A, borosilicate glass or non-toxic plasticware, 1000 mL for making test solutions.

11.5.23 Wash bottles -- for deionized water, for washing embryos from substrates and containers, and for rinsing

small glassware and instrument electrodes and probes.

11.5.24 Crystallization dishes, beakers, culture dishes (1 L), or equivalent -- for incubating embryos.

11.5.25 Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-toxic plastic labware,

10-1000 mL for making test solutions.

11.5.26 Separatory funnels, 2-L -- two to four for culturing Artemia nauplii.

11.5.27 Pipets, volumetric -- Class A, 1-100 mL.

11.5.28 Pipets, automatic -- adjustable, 1-100 mL.

11.5.29 Pipets, serological -- 1-10 mL, graduated.

11.5.30 Pipet bulbs and fillers -- PROPIPET®, or equivalent.

11.5.31 Droppers, and glass tubing with fire polished edges, 4 mm ID -- for transferring larvae.

11.5.32 Siphon with bulb and clamp -- for cleaning test chambers.

11.5.33 Forceps -- for transferring dead larvae to weighing boats.

11.5.34 NITEX® or stainless steel mesh sieves (# 150 μm, 500 μm, 3 to 5 mm) -- for collecting Artemia nauplii

and fish embryos, and for spawning baskets, respectively. 
59

11.6 REAGENTS AND CONSUMABLE MATERIALS

11.6.1 Sample containers -- for sample shipment and storage (see Section 8, Effluent and Receiving Water

Sampling, Sample Handling, and Sample Preparation for Toxicity Tests).

11.6.2 Data sheets (one set per test) -- for data recording.

11.6.3 Vials, marked-- 24 per test, containing 4% formalin or 70% ethanol, to preserve larvae (optional). 

11.6.4 Weighing pans, aluminum -- 24 per test. 

11.6.5 Tape, colored -- for labeling test chambers. 

11.6.6 Markers, waterproof -- for marking containers, etc. 

11.6.7 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument manufacturer) -- for standards and

calibration check (see USEPA Method 150.1, USEPA, 1979b). 

11.6.8 Membranes and filling solutions for dissolved oxygen probe (see USEPA Method 360.l, USEPA, 1979b),

or reagents -- for modified Winkler analysis.

11.6.9 Laboratory quality control samples and standards -- for calibration of the above methods. 

11.6.10 Reference toxicant solutions -- see Section 4, Quality Assurance. 

11.6.11 Ethanol (70%) or formalin (4%) -- for use as a preservative for the fish larvae. 

11.6.12 Reagent water -- defined as distilled or deionized water that does not contain substances which are toxic to

the test organisms. 

11.6.13 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. 

11.6.13.1 Saline test and dilution water -- The salinity of the test water must be in the range of 20 to 32‰. The

salinity should vary by no more than ± 2‰ among the chambers on a given day. If effluent and receiving water

tests are conducted concurrently, the salinities of these tests should be similar. This test is not recommended for

salinities less than 20‰.

11.6.13.2 The overwhelming majority of industrial and sewage treatment effluents entering marine and estuarine

systems contain little or no measurable salts. Exposure of sheepshead minnow larvae to these effluents will require

adjustments in the salinity of the test solutions. It is important to maintain a constant salinity across all treatments. 

In addition, it may be desirable to match the test salinity with that of the receiving water. Two methods are

available to adjust salinities -- a hypersaline brine derived from natural seawater or artificial sea salts. 

11.6.13.3 Hypersaline brine (HSB): HSB has several advantages that make it desirable for use in toxicity testing.

It can be made from any high quality, filtered seawater by evaporation, and can be added to the effluent or to

deionized water to increase the salinity. HSB derived from natural seawater contains the necessary trace metals,

biogenic colloids, and some of the microbial components necessary for adequate growth, survival, and/or

reproduction of marine and estuarine organisms, and may be stored for prolonged periods without any apparent

degradation. However, if 100‰ HSB is used as a diluent, the maximum concentration of effluent that can be tested

will be 80% at 20‰ salinity and 70% at 30‰ salinity. 
60

11.6.13.3.1 The ideal container for making brine from natural seawater is one that (1) has a high surface to volume

ratio, (2) is made of a non-corrosive material, and (3) is easily cleaned (fiberglass containers are ideal). Special care

should be used to prevent any toxic materials from coming in contact with the seawater being used to generate the

brine. If a heater is immersed directly into the seawater, ensure that the heater materials do not corrode or leach any

substances that would contaminate the brine. One successful method used is a thermostatically controlled heat

exchanger made from fiberglass. If aeration is used, use only oil-free air compressors to prevent contamination.

11.6.13.3.2 Before adding seawater to the brine generator, thoroughly clean the generator, aeration supply tube,

heater, and any other materials that will be in direct contact with the brine. A good quality biodegradable detergent

should be used, followed by several (at least three) thorough deionized water rinses. 

11.6.13.3.3 High quality (and preferably high salinity) seawater should be filtered to at least 10 μm before placing

into the brine generator. Water should be collected on an incoming tide to minimize the possibility of

contamination.

11.6.13.3.4 The temperature of the seawater is increased slowly to 40°C. The water should be aerated to prevent

temperature stratification and to increase water evaporation. The brine should be checked daily (depending on

volume being generated) to ensure that the salinity does not exceed 100‰ and that the temperature does not exceed

40°C. Additional seawater may be added to the brine to obtain the volume of brine required. 

11.6.13.3.5 After the required salinity is attained, the HSB should be filtered a second time through a l μm filter

and poured directly into portable containers (20-L cubitainers or polycarbonate water cooler jugs are suitable). The

containers should be capped and labelled with the date the HSB was generated and its salinity. Containers of HSB

should be stored in the dark and maintained at room temperature until used. 

11.6.13.3.6 If a source of HSB is available, test solutions can be made by following the directions below. 

Thoroughly mix together the deionized water and HSB before adding the effluent. 

11.6.13.3.7 Divide the salinity of the HSB by the expected test salinity to determine the proportion of deionized

water to brine. For example, if the salinity of the brine is 100‰ and the test is to be conducted at 20‰, 100‰

divided by 20‰ = 5.0. The proportion of brine is 1 part in 5 (one part brine to four parts deionized water). To

make 1 L of seawater at 20‰ salinity from a HSB of 100‰, divide 1 L (1000 mL) by 5.0. The result, 200 mL, is

the quantity of brine needed to make 1 L of seawater. The difference, 800 mL, is the quantity of deionized water

required. 

11.6.13.4 Artificial sea salts: FORTY FATHOMS® brand sea salts have been used successfully at the

EMSL-Cincinnati to maintain and spawn sheephead minnows and perform the larval survival and growth test (see

Section 7, Dilution Water). HW MARINEMIX® sea salts have been used successfully at the USEPA Region 6

Houston Laboratory to maintain and spawn sheephead minnows and perform the larval growth and survival test and

the embryo-larval survival and teratogenicity test. In addition, a slightly modified version of the GP2 medium

(Spotte et al., 1984) has been successfully used to perform the sheepshead minnow survival and growth test (Table

1). Artifical sea salts may be used for culturing sheepshead minnows and for the larval survival and growth test if

the criteria for acceptability of test data are satisfied (see Subsection 11.12).

11.6.13.4.1 Synthetic sea salts are packaged in plastic bags and mixed with deionized water or equivalent. The

instructions on the package of sea salts should be followed carefully, and the salts should be mixed in a separate

container -- not in the culture tank. The deionized water used in hydration should be in the temperature range of

21-26°C. Seawater made from artificial sea salts is conditioned (Spotte, 1973; Spotte et al., 1984; Bower, 1983)

before it is used for culturing or testing. After adding the water, place an air stone in the container, cover, and

aerate the solution mildly for 24 h before use.

11.6.13.4.2 The GP2 reagent grade chemicals (Table 1) should be mixed with deionized (DI) water or its

equivalent in a container other than the culture or testing tanks. The deionized water used for hydration should be
61

between 21-26°C. The artificial seawater must be conditioned (aerated) for 24 h before use as the testing medium. 

If the solution is to be autoclaved, sodium bicarbonate is added after the solution has cooled. A stock solution of

sodium bicarbonate is made up by dissolving 33.6 g NaHCO3 in 500 mL of deionized water. Add 2.5 mL of this

stock solution for each liter of the GP2 artificial seawater.

TABLE 1. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2 ARTIFICIAL

SEAWATER FOR THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, TOXICITY

TEST1,2,3

Compound Concentration

 (g/L)

 Amount (g)

 Required for

 20 L

NaCl 21.03 420.6

Na2SO4 3.52 70.4

KCl 0.61 12.2

KBr 0.088 1.76

Na2B4O7 ·10 H2O 0.034 0.68

MgCl2·6 H2O 9.50 190.0

CaCl2·2 H 2O 1.32 26.4

SrCl2·6 H2O 0.02 0.400

NaHCO3 0.17 3.40

1 Modified GP2 from Spotte et al. (1984). 2 The constituent salts and concentrations were taken from USEPA (l990b). The salinity is 30.89 g/L. 3 GP2 can be diluted with deionized (DI) water to the desired test salinity.

11.6.14 BRINE SHRIMP, ARTEMIA, NAUPLII -- for feeding cultures and test organisms 

11.6.14.1 Newly-hatched Artemia nauplii (see USEPA, 2002a) are used as food for sheepshead minnow larvae in

toxicity tests and 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. 

11.6.14.2 Each new batch of Artemia cysts must be evaluated for size (Vanhaecke and Sorgeloos, 1980, and

Vanhaecke et al., 1980) and nutritional suitability (Leger et al., 1985, and 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. 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 pesticides exceeds 0.15 μg/g wet weight or the total

concentration of organochlorine pesticides plus PCBs exceeds 0.30 μg/g wet weight. (For analytical methods see

USEPA, 1982.)

11.6.14.3 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 of deionized water, to a 2-L separatory funnel, or equivalent. 

2. Add 10 mL Artemia cysts to the separatory funnel and aerate for 24 h at 27°C. (Hatching time varies
62

with incubation temperature and the geographic strain of Artemia used (USEPA, 1985a; USEPA,

2002a; 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 minutes. 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 μm NITEX® or stainless steel screen, and

rinse with seawater or equivalent before use.

11.6.14.4 Testing Artemia nauplii as food for toxicity test organisms. 

11.6.14.4.1 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 sheepshead 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. 

11.6.14.4.2 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.

11.6.14.4.3 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. 

11.6.15 TEST ORGANISMS, SHEEPSHEAD MINNOWS, CYPRINODON VARIEGATUS

11.6.15.1 Brood Stock 

11.6.15.1.1 Adult sheepshead minnows for use as brood stock may be obtained by seine in Gulf of Mexico and

Atlantic coast estuaries, from commercial sources, or from young fish raised to maturity in the laboratory. Feral

brood stocks and first generation laboratory fish are preferred, to minimize inbreeding. 

11.6.15.1.2 To detect disease and to allow time for acute mortality due to the stress of capture, field-caught adults

are observed in the laboratory a minimum of two weeks before using as a source of gametes. Injured or diseased

fish are discarded. 

11.6.15.1.3 Sheepshead minnows can be continuously cultured in the laboratory from eggs to adults. The larvae,

juvenile, and adult fish should be kept in appropriate size rearing tanks, maintained at ambient laboratory

temperature. The larvae should be fed sufficient newly-hatched Artemia nauplii daily to assure that live nauplii are

always present. Juveniles are fed frozen adult brine shrimp and a commercial flake food, such as TETRA SM-80®

or MARDEL AQUARIAN® Tropical Fish Flakes or equivalent. Adult fish (age one month) are fed flake food three

or four times daily, supplemented with frozen adult brine shrimp. 

11.6.15.1.3.1 Sheepshead minnows reach sexual maturity in three-to-five months after hatch, and have an average

standard length of approximately 27 mm for females and 34 mm for males. At this time, the males begin to exhibit

sexual dimorphism and initiate territorial behavior. When the fish reach sexual maturity and are to be used for

natural spawning, the temperature should be controlled at 18-20°C. 

11.6.15.1.4 Adults can be maintained in natural or artificial seawater in a flow-through or recirculating, aerated

system consisting of an all-glass aquarium , or equivalent.

11.6.15.1.5 The system is equipped with an undergravel or outside biological filter of shells (Spotte, 1973; Bower,

1983) for conditioning the biological filter), or a cartridge filter, such as a MAGNUM® Filter, or an EHEIM® Filter,
63

or equivalent, at a salinity of 20-30‰ and a photoperiod of 16 h light/8 h dark.

11.6.15.2 Obtaining Embryos for Toxicity Tests (See USEPA, 1978)

11.6.15.2.1 Embryos can be shipped to the laboratory from an outside source or obtained from adults held in the

laboratory. Ripe eggs can be obtained either by natural spawning or by intraperitoneal injection of the females with

human chorionic gonadotrophin (HCG) hormone. If the culturing system for adults is temperature controlled,

natural spawning can be induced. Natural spawning is preferred because repeated spawnings can be obtained from

the same brood stock, whereas with hormone injection, the brood stock is sacrificed in obtaining gametes.

11.6.15.2.2 It should be emphasized that the injection and hatching schedules given below are to be used only as

guidelines. Response to the hormone varies from stock to stock and with temperature. Time to hatch and percent

viable hatch also vary among stocks and among batches of embryos obtained from the same stock, and are

dependent on temperature, DO, and salinity. The coordination of spawning and hatching is further complicated by

the fact that, even under the most ideal conditions, embryos spawned over a 24-h period may hatch over a 72-h

period. Therefore, it is advisable (especially if natural spawning is used) to obtain fertilized eggs over several days

to ensure that a sufficient number of newly hatched larvae (less than 24 h old) will be available to initiate a test. 

11.6.15.2.3 Forced Spawning 

11.6.15.2.3.1 HCG is reconstituted with sterile saline or Ringer's solution immediately before use. The standard

HCG vial contains l,000 IU to be reconstituted in 10 mL of saline. Freeze-dried HCG which comes with

premeasured and sterilized saline is the easiest to use. Use of a 50 IU dose requires injection of 0.05 mL of

reconstituted hormone solution. Reconstituted HCG may be used for several weeks if kept in the refrigerator. 

11.6.15.2.3.2 Each female is injected intraperitoneally with 50 IU HCG on two consecutive days, starting at least

10 days prior to the beginning of a test. Two days following the second injection, eggs are stripped from the

females and mixed with sperm derived from excised macerated testes. At least ten females and five males are used

per test to ensure that there is a sufficient number (400) of viable embryos.

11.6.15.2.3.3 HCG is injected into the peritoneal cavity, just below the skin, using as small a needle as possible. A

50 IU dose is recommended for females approximately 27 mm in standard length. A larger or smaller dose may be

used for fish which are significantly larger or smaller than 27 mm. With injections made on days one and two,

females which are held at 25°C should be ready for stripping on days 4, 5, and 6. Ripe females should show

pronounced abdominal swelling, and release at least a few eggs in response to a gentle squeeze. Injected females

should be isolated from males. It may be helpful if fish that are to be injected are maintained at 20°C before

injection, and the temperature raised to 25°C on the day of the first injection. 

11.6.15.2.3.4 Prepare the testes immediately before stripping the eggs from the females. Remove the testes from

three-to-five males. The testes are paired, dark grey organs along the dorsal midline of the abdominal cavity. If the

head of the male is cut off and pulled away from the rest of the fish, most of the internal organs can be pulled out of

the body cavity, leaving the testes behind. The testes are placed in a few mL of seawater until the eggs are ready. 

11.6.15.2.3.5 Strip the eggs from the females, into a dish containing 50-100 mL of seawater, by firmly squeezing

the abdomen. Sacrifice the females and remove the ovaries if all the ripe eggs do not flow out freely. Break up any

clumps of ripe eggs and remove clumps of ovarian tissue and underripe eggs. Ripe eggs are spherical,

approximately 1 mm in diameter, and almost clear. 

11.6.15.2.3.6 While being held over the dish containing the eggs, the testes are macerated in a fold of NITEX®

screen (250-500 μm mesh) dampened with seawater. The testes are then rinsed with seawater to remove the sperm

from tissue, and the remaining sperm and testes are washed into the dish. Let the eggs and milt stand together for

10-15 min, swirling occasionally.
64

11.6.15.2.3.7 Pour the contents of the dish into a beaker, and insert an airstone. Aerate gently, such that the water

moves slowly over the eggs, and incubate at 25°C for 60-90 min. After incubation, wash the eggs on a NITEX®

screen and resuspend them in clean seawater. Examine the eggs periodically under a dissecting microscope until

they are in the 2-8 cell stage. (The stage at which it is easiest to tell the developing embryos from the abnormal

embryos and unfertilized eggs; see Figure 1). The eggs can then be gently rolled on a NITEX® screen and culled

(see Section 6, Test Organisms).

11.6.15.2.4 Natural Spawning

11.6.15.2.4.1 Cultures of adult fish to be used for spawning are maintained at 18-20°C until embryos are required. 

When embryos are required, raise the temperature to 25°C in the morning, seven or eight days before the beginning

of a test. That afternoon, transfer the adult fish (generally, at least five females and three males) to a spawning

chamber (approximately, 20 × 35 × 22 cm high; USEPA, 1978), which is a basket constructed of 3-5 mm NITEX®

mesh, made to fit a 57-L (15 gal) aquarium. Spawning generally will begin within 24 h or less. Embryos will fall

through the bottom of the basket and onto a collecting screen (250-500 μm mesh) or tray below the basket. Allow

the embryos to collect for 24 h. Embryos are washed from the screen, checked for viability, and placed in

incubation dishes. Replace the screens until a sufficient number of embryos have been collected. One-to-three

spawning aquaria can be used to collect the required number of embryos to run a toxicity test. To help keep the

embryos clean, the adults are fed while the screens are removed.

11.6.15.2.5 Incubation

11.6.15.2.5.1 Four hours post-fertilization, the embryos obtained by natural or forced spawning are rolled gently

with a finger on a 250-500 μm Nitex® screen to remove excess fibers and tissue. The embryos have adhesive

threads and tend to adhere to each other. Gentle rolling on the screen facilitates the culling process described

below. To reduce fungal contamination of the newly spawned embryos after they have been manipulated, they

should be placed in a 250 μm sieve and briskly sprayed with seawater from a squeeze bottle.

11.6.15.2.5.2 Under a dissecting microscope, separate and discard abnormal embryos and unfertilized eggs. While

they are checked, the embryos are maintained in seawater at 25°C. The embryos should be in Stages C-G, Figure 1. 

11.6.15.2.5.3 If the test is prepared with four replicates of 15 larvae at each of six treatments (five effluent

concentrations and a control), and the combined mortality of eggs and larvae prior to the start of the test is less than

20%, approximately 400 viable embryos are required at this stage. 
65

Figure 1. Embryonic development of sheepshead minnow, Cyprinodon variegatus: A. Mature unfertilized egg,

showing attachment filaments and micropyle, X33; B. Blastodisc fully developed; C,D. Blastodisc, 8 cells; E.

Blastoderm, 16 cells; F. Blastoderm, late cleavage stage; G. Blastoderm with germ ring formed, embryonic

shield developing; H. Blastoderm covers over 3/4 of yolk, yolk noticeably constricted; I. Early embryo. From

Kuntz (1916).
66

Figure 1. Embryonic development of sheepshead minnow, Cyprinodon variegatus: J.

Embryo 48 h after fertilization, now segmented throughout, pigment on yolk sac

and body, otoliths formed; K. Posterior portion of embryo free from yolk and

moves freely within egg membrane, 72 h after fertilization; L. Newly hatched

fish, actual length 4 mm; M. Larval fish 5 days after hatching, actual length 5 mm;

N. Young fish 9 mm in length; O. Young fish 12 mm in length (CONTINUED). 

From Kuntz (1916).
67

11.6.15.2.5.4 Embryos are demersal. They should be aerated and incubated at 25°C, at a salinity of 20-30‰ and a

16-h photoperiod. The embryos can be cultured in either a flow-through or static system, using aquaria or

crystallization dishes. However, if the embryos are cultured in dishes, it is essential that aeration and daily water

changes be provided, and the dishes be covered to reduce evaporation that may cause increased salinity. One-half to

three-quarters of the seawater from the culture vessels can be poured off and the incubating embryos retained. 

Embryos cultured in this manner should hatch in six or seven days. 

11.6.15.2.5.5 At 48 h post-fertilization, embryos are examined under a microscope to determine development and

survival. Embryos should be in Stages I and J, Figure 1. Discard dead embryos. Approximately 360 viable embryos

are required at this stage. 

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.

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. At estuarine and marine sites, samples are

usually collected at mid-depth. Receiving water toxicity is determined with samples used directly as collected or with

samples passed through a 60 μm NITEX® filter and compared without dilution, against a control. Using four replicate

chambers per test, each containing 500-750 mL, and 400 mL for chemical analysis, would require approximately 2.4-

3.4 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 ± 100%, and allows for testing of

concentrations between 6.25% and 100% effluent using only five effluent concentrations (6.25%, 12.5%, 25.0%,

50.0%, and 100%). Test precision shows little improvement as dilution factors are increased beyond 0.5 and declines

rapidly if smaller dilution factors are used. Therefore, USEPA recommends the use of the $ 0.5 dilution factor. If

100‰ HSB is used as a diluent, the maximum concentration of effluent that can be tested will be 80% at 20‰ salinity

and 70% at 30‰ salinity. 

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 at the lower range of effluent concentrations should be added. 

11.10.1.2.3 The volume of effluent required to initiate the test and for daily renewal of four replicates per

concentration for five concentrations of effluent and a control, each containing 750 mL of test solution, is

approximately 5 L. Prepare enough test solution (approximately 3400 mL) at each effluent concentration to provide
68

400 mL additional volume for chemical analyses (Table 2). 

11.10.1.2.4 The salinity of effluent and receiving water tests for sheepshead minnows should be between 20‰ and

30‰. If concurrent effluent and receiving water testing occurs, the effluent test salinity should closely approximate

that of the receiving water test. If an effluent is tested alone, select a salinity between 20‰ and 30‰, whichever comes

closest to the salinity of the receiving waters. Table 2 illustrates the quantities of effluent, artificial sea salts,

hypersaline brine, or seawater needed to prepare 3 L of test solution at each treatment level for tests performed at 20‰

salinity.

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 adjusted to the test temperature (25 ± 1°C) and maintained at that temperature during

the addition of dilution water.

11.10.1.2.6 Higher effluent concentrations (i.e., 25%, 50%, and 100%) may require aeration to maintain adequate

dissolved oxygen concentrations. However, if one solution is aerated, all concentrations must be aerated. Aerate

effluent as it warms and continue to gently aerate test solutions in the test chambers for the duration of the test. 

11.10.1.2.7 Effluent dilutions should be prepared for all replicates in each treatment in one beaker to minimize

variability among the replicates. The test chambers are labelled with the test concentration and replicate number. 

Dispense into the appropriate effluent dilution chamber.

11.10.1.3 Dilution Water

11.10.1.3.1 Dilution water may be uncontaminated natural seawater (receiving water), HSB prepared from natural

seawater, or artificial seawater prepared from FORTY FATHOMS® or GP2 sea salts (see Table 1 and Section 7,

Dilution Water). Other artificial sea salts may be used for culturing sheepshead minnows and for the larval survival

and growth test if the control criteria for acceptability of test data are satisfied. 

11.10.2 START OF THE TEST 

11.10.2.1 Tests should begin as soon as possible, preferably within 24 h after 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).
69

TABLE 2. PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 20‰ , USING 20‰ SALINITY 

DILUTION WATER PREPARED FROM NATURAL SEAWATER, HYPERSALINE BRINE,

OR ARTIFICIAL SEA SALTS

Effluent

Solution

Effluent

Conc. (%)

Solutions To Be Combined

Volume of

Effluent Solution

Volume of Diluent

Seawater (20‰)

1 1001,2 6800 mL ----

2 50 3400 mL Solution 1 + 3400 mL

3 25 3400 mL Solution 2 + 3400 mL

4 12.5 3400 mL Solution 3 + 3400 mL

5 6.25 3400 mL Solution 4 + 3400 mL

Control 0.0 3400 mL

Total 17000 mL

1 This illustration assumes: (1) the use of 750 mL of test solution in each of four replicates and 400 mL for chemical

analysis (total of 3,400 mL) for the control and each of five concentrations of effluent (2) an effluent dilution

factor of 0.5, and (3) the effluent lacks appreciable salinity. A sufficient initial volume (6,800 mL) of effluent is

prepared by adjusting the salinity to the desired level. In this example, the salinity is adjusted by adding artificial

sea salts to the 100% effluent, and preparing a serial dilution using 20‰ seawater (natural seawater, hypersaline

brine, or artificial seawater). Following addition of salts, the effluent is stirred for 1 h to ensure that the salts have

dissolved. The salinity of the initial 6,800 mL of 100% effluent is adjusted to 20‰ by adding 136 g of dry

artificial sea salts (FORTY FATHOMS®). Test concentrations are then made by mixing appropriate volumes of

salinity-adjusted effluent and 20‰ salinity dilution water to provide 6,800 mL of solution for each concentration. 

If hypersaline brine alone (100‰) is used to adjust the salinity of the effluent, the highest concentration of effluent

that could be achieved would be 80% at 20‰ salinity. When dry sea salts are used to adjust the salinity of the

effluent, it may be desirable to use a salinity control prepared under the same conditions and used to determine

survival and growth. 2 The same procedures would be followed in preparing test concentrations at other salinities between 20‰ and

30‰: (1) the salinity of the bulk (initial) effluent sample would be adjusted to the appropriate salinity using

artificial sea salts or hypersaline brine, and (2) the remaining effluent concentrations would be prepared by serial

dilution, using a large batch (17,000 mL) of seawater for dilution water, which had been prepared at the same

salinity as the effluent, using natural seawater, or hypersaline or artificial sea salts and deionized water. 

11.10.2.2 If the embryos have been incubating at 25°C, 30‰ salinity, and a 16-h photoperiod, for 5 to 6 days with

aeration and daily water renewals, approximately 24 h prior to hatching, the salinity of the seawater in the incubation

chamber may be reduced from 30‰ to the test salinity, if lower than 30‰. In addition to maintaining good water

quality, reducing the salinity and/or changing the water may also help to initiate hatching over the next 24 h. A few

larvae may hatch 24 h ahead of the majority. Remove these larvae and reserve them in a separate dish, maintaining the

same culture conditions. It is preferable to use only the larvae that hatch in the 24 h prior to starting the test. However,

if sufficient numbers of larvae do not hatch within the 24-h period, the larvae that hatch prior to 24 h are added to the

test organisms. The test organisms are then randomly selected for the test. 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 Label the test chambers with a marking pen. Use of color coded tape to identify each treatment and

replicate. A minimum of five effluent concentrations and a control are used for each test. Each treatment (including
70

controls) must have a minimum of four replicates. For exposure chambers, use 1000 mL beakers, non-toxic disposable

plasticware, or glass chambers with a sump area as illustrated in the inland silverside test method (see Section 13). 

11.10.2.4 Prepare the test solutions and add to the test chambers. 

11.10.2.5 The test is started by randomly placing larvae from the common pool into each test chamber until each

chamber contains a minimum of 10 larvae, for a total of a minimum of 40 for each concentration (see Appendix A). 

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.

11.10.2.6 The chambers may be placed on a light table to facilitate counting the larvae.

11.10.2.7 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. 

11.10.3 LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE

11.10.3.1 The light quality and intensity should be at ambient laboratory levels, which is approximately 10-20

μE/m2

/s, or 50 to 100 foot candles (ft-c), with a photoperiod of 16 h light and 8 h darkness. The water temperature in

the test chambers should be maintained at 25 ± 1°C. The test salinity should be in the range of 20 to 30‰ to

accommodate receiving waters that may fall within this range. Conduct of this test at salinities less than 20‰ may

cause an unacceptably low growth response and thereby invalidate the test. The salinity should vary by no more than ±

2‰ among the chambers on a given day. If effluent and receiving water tests are conducted concurrently, the salinities

of these tests should be similar.

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 a

satisfactory DO. The DO should be measured on new solutions at the start of the test (Day 0) and before daily renewal

of test solutions on subsequent days. The DO 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

treatments and the control should be aerated. The aeration rate should not exceed 100 bubbles/min, using a pipet with a

1-2 mm orifice, such as a 1-mL KIMAX® serological pipet, or equivalent. Care should be taken to ensure that

turbulence resulting from aeration does not cause undue stress on the fish. 

11.10.5 FEEDING 

11.10.5.1 Artemia nauplii are prepared as described above.

11.10.5.2 Sheepshead minnow larvae are fed newly-hatched (less than 24-h old) Artemia nauplii once a day from

hatch day 0 through day 6; larvae are not fed on day 7. Feed 0.10 g nauplii per test chamber on days 0-2, and 0.15 g

nauplii per test chamber on days 3-6. Equal amounts of Artemia nauplii must be added to each replicate test chamber

to minimize the variability of larval weight. Sufficient numbers of nauplii should be fed to ensure that some remain

alive overnight in the test chambers. An adequate but not excessive amount should be provided to each replicate on a

daily basis. Feeding excessive amounts of nauplii will result in a depletion in DO to a lower than acceptable level

(below 4.0 mg/L). Siphon as much of the uneaten Artemia nauplii as possible from each chamber daily to ensure that

the larvae principally eat newly hatched nauplii.

11.10.5.3 On days 0-2, weigh 4 g wet weight or pipette 4 mL of concentrated, rinsed Artemia nauplii for a test with

five treatments and a control. Resuspend the 4 g Artemia in 80 mL of natural or artificial seawater in a 100 mL beaker. 

Aerate or swirl Artemia to maintain a thoroughly mixed suspension of nauplii. Dispense 2 mL Artemia suspension by

pipette or adjustable syringe to each test chamber. Collect only enough Artemia in the pipette or syringe for one test
71

chamber or settling of Artemia may occur, resulting in unequal amounts of Artemia being distributed to the replicate

test chambers.

11.10.5.4 On days 3-6, weigh 6 g wet weight or pipette 6 mL Artemia suspension for a test with five treatments and a

control. Resuspend the 6 g Artemia in 80 mL of natural or artificial seawater in a 100 mL beaker. Aerate or swirl as 2

mL is dispensed to each test chamber.

11.10.5.5 If the survival rate in any test replicate on any day falls below 50%, reduce the volume of Artemia added to

that test chamber by one-half (i.e., from 2 mL to 1 mL) and continue feeding one-half the volume through day 6. 

Record the time of feeding on data sheets (Figure 2).

11.10.6 DAILY CLEANING OF TEST CHAMBERS 

11.10.6.1 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. As much of the uneaten Artemia as possible should

be siphoned from each chamber to ensure that the larvae principally eat newly hatched nauplii. Alternately, a large

pipet (50 mL), fitted with a safety pipet filler or rubber bulb, can be used. Because of their small size during the first

few days of the tests, larvae are easily drawn into the siphon tube when cleaning the test chambers. By placing the test

chambers on a light box, inadvertent removal of live 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 live larvae caught in the

siphon can be retrieved and returned to the appropriate test chamber. Any incidence of removal of live larvae from the

test chambers by the siphon during cleaning, and subsequent return to the chambers, should be noted in the test records. 

11.10.7 OBSERVATIONS DURING THE TEST

11.10.7.1 Routine Chemical and Physical Determinations 

1.10.7.1.1 DO is measured at the beginning and end of each 24-h exposure period in one test chamber at each test

concentration and in the control.

11.10.7.1.2 Temperature, pH, and salinity are measured at the end of each 24-h exposure period in one test chamber at

each test concentration and in the control. Temperature should also be monitored continuously, 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.

11.10.7.1.3 The pH is measured in the effluent sample each day. 

11.10.7.1.4 Record all the measurements on the data sheet (Figure 2). 
72

Test Dates: Species:Type Effluent: Field Lab Test

Effluent Tested:

CONCENTRATION:

REPLICATE: REPLICATE: REPLICATE: 

DAYS 0 1 2 3 4 5 6 7 0 1 23 4 5 6 7 0 1 23 4 5 6 7 0 1 

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVADRY WT

CONCENTRATION:

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVADRY WT

CONCENTRATION:

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

#LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVADRY WT

TIME

FED

COMMENTS:

Figure 2. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Daily record of larval survival and t
73

Test Dates: Species:Type Effluent: Field Lab Test

Effluent Tested:

CONCENTRATION:

REPLICATE: REPLICATE: REPLICATE: 

DAYS 0 1 2 3 4 5 6 7 0 1 23 4 5 6 7 0 1 23 4 5 6 7 0 1 

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVADRY WT

CONCENTRATION:

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVADRY WT

CONCENTRATION:

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

#LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVAE/DRY WT

MEANWEIGHT/LARVAE (mg)± SD

# LARVADRY WT

TIME

FED

COMMENTS:

Figure 2. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Daily record of larval survival and t

(From USEPA, 1987b).
74

11.10.7.2 Routine Biological Observations 

11.10.7.2.1 The number of live larvae in each test chamber are recorded daily (Figure 2), and the dead larvae are

discarded. 

11.10.7.2.2 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 the above operations. 

11.10.8 TEST SOLUTION RENEWAL 

11.10.8.1 The test solutions are renewed daily using freshly prepared solution, immediately after cleaning the test

chambers. For on-site toxicity studies, fresh effluent and receiving water samples used in toxicity tests should be

collected daily, and no more than 24 h should elapse between collection of the sample and use in the test (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 must be collected, preferably on days one, three, and five. Maintain the samples at

0-6°C 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 (750 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 7-d 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 4% formalin or 70% ethanol. 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, siphon or pour live larvae onto a 500 μm mesh screen in a large beaker

to retain the larvae and allow Artemia and debris to be rinsed away. Rinse the larvae with deionized water to wash

away salts that might contribute to the dry weight. Sacrifice the larvae in an ice bath of deionized water. 

11.10.9.3 Small aluminum weighing pans can be used to dry and weigh the larvae. Mark for identification an

appropriate number of small aluminum weighing pans (one per replicate). Weigh to the nearest 0.01 mg, and record

the weights (Figure 3).
75

Test Dates: Species: 

Pan

No.

Conc.

&

Rep.

Initial

Wt.

(mg)

Final

Wt.

(mg)

Diff.

(mg)

No.

Larvae

Av. Wt./

Larvae

(mg)

Figure 3. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. Dry

weights of larvae (from USEPA 1987b).
76

11.10.9.4 Immediately prior to drying, rinse the preserved larvae in distilled (or deionized) water. The rinsed larvae

from each test chamber are transferred to a tared weighing pan and dried at 60°C for 24 h or at 105°C for a minimum

of 6 h. Immediately upon removal from the drying oven, the weighing pans are placed in a desiccator until weighed, to

prevent the absorption of moisture from the air. Weigh to the nearest 0.01 mg all weighing pans containing dried

larvae and subtract the tare weight to determine the dry weight of larvae in each replicate. Record the weights (Figure

3). 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 (Figure 3). For the controls, also calculate the mean weight per

surviving fish in the test chamber to evaluate if weights met test acceptable criteria (see Subsection 11.12). Complete

the summary data sheet (Figure 4) after calculating the average measurements and statistically analyzing the dry

weights and percent survival. Average dry weights should be expressed to the nearest 0.001 mg.

11.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA 

11.11.1 A summary of test conditions and test acceptability criteria is listed in Table 3.

11.12 ACCEPTABILITY OF TEST RESULTS 

11.12.1 The tests are acceptable if (l) the average survival of control larvae equals or exceeds 80%, and (2) the

average dry weight per surviving unpreserved control larvae is equal to or greater than 0.60 mg, or (3) the average dry

weight per surviving preserved control larvae is equal to or greater than 0.50 mg. The above minimum weights

presume that the age of the larvae at the start of the test is less than or equal to 24 h. 

11.13 DATA ANALYSIS

11.13.1 GENERAL 

11.13.1.1 Tabulate and summarize the data. A sample set of survival and growth response data is listed in Table 4. 

11.13.1.2 The endpoints of toxicity tests using the sheepshead minnow 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 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,

program listings, 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. 
77

Test Dates: Species: 

Effluent Tested: 

TREATMENT

NO. LIVE

LARVAE

SURVIVAL

(%)

MEAN DRY WT/

LARVAE (MG)

± SD

SIGNIF. DIFF.

FROM CONTROL

(o)

MEAN

TEMPERATURE

(°C)

± SD

MEAN SALINITY

‰

± SD

AVE DISSOLVED

OXYGEN

(MG/L) ± SD

COMMENTS:

Figure 4. Data form for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test. 

Summary of test results (from USEPA, 1987b).
78

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE

SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL SURVIVAL AND

GROWTH TEST WITH EFFLUENTS AND RECEIVING WATERS (TEST METHOD 1004.0)1

1. Test type: Static renewal (required) 

2. Salinity: 20‰ to 32‰ (± 2‰ of the selected test salinity) 

(recommended) 

3. Temperature: 25 ± 1°C (recommended) 

Test temperatures must not deviate (i.e., maximum minus

minimum termperature) by more than 3°C during the test

(required) 

4. Light quality: Ambient laboratory illumination (recommended) 

5. Light intensity: 10-20 μE/m2

/s (50-100 ft-c) (ambient laboratory levels)

(recommended) 

6. Photoperiod: 16 h light, 8 h darkness (recommended) 

7. Test chamber size: 600 mL - 1 L beakers or equivalent (recommended) 

8. Test solution volume: 500-750 mL/replicate (loading and DO restrictions must be met)

(recommended) 

9. Renewal of test solutions: Daily (required) 

10. Age of test organisms Newly hatched larvae (less than 24 h old; less than or equal to 

24-h range in age) (required) 

11. No. larvae per test chamber: 10 (required minimum)

12. No. replicate chambers per concentration 4 (required minimum)

13. No. larvae per concentration: 40 (required minimum)

14. Source of food: Newly hatched Artemia nauplii, (less than 24-h old) (required) 

15. Feeding regime: Feed once a day 0.10 g wet weight Artemia nauplii per replicate

on Days 0-2; Feed 0.15 g wet weight Artemia nauplii per

replicate on Days 3-6 (recommended) 

16. Cleaning: Siphon daily, immediately before test solution renewal and

feeding (required) 

17. Aeration: None, unless DO falls below 4.0 mg/L, then aerate all

chambers. Rate should be less than 100 bubbles/minimum

(recommended) 

1

 For the purposes of reviewing WET test data submitted under NPDES permits, each test condition listed above is

identified as required or recommended (see Subsection 10.2 for more information on test review). Additional

requirements may be provided in individual permits, such as specifying a given test condition where several options

are given in the method. 
79

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE

SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL SURVIVAL AND

GROWTH TEST WITH EFFLUENTS AND RECEIVING WATERS (TEST METHOD 1004.0)

(CONTINUED) 

18. Dilution water: Uncontaminated source of natural seawater; deionized water

mixed with hypersaline brine or artificial sea salts (HW

MARINEMIX®, FORTY FATHOMS®, GP2 or equivalent)

(available options)

19. Test concentrations: Effluent: 5 and a control (required) 

Receiving Waters: 100% receiving water (or minimum of 5)

and a control (recommended) 

20. Dilution factor: Effluents: $ 0.5 (recommended) 

Receiving waters: None, or $ 0.5 (recommended) 

21. Test duration: 7 days (required) 

22. Endpoints: Survival and growth (weight) (required) 

23. Test acceptability criteria: 80% or greater survival in controls; average dry weight per

surviving organism in control chambers must be 0.60 mg or

greater, if unpreserved, or 0.50 mg or greater after no more than

7 days in 4% formalin or 70% ethanol (required) 

24. Sampling requirements: 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 (e.g., 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) (required) 

25. Sample volume required: 6 L per day (recommended) 
80

TABLE 4. SUMMARY OF SURVIVAL AND GROWTH DATA FOR SHEEPSHEAD MINNOW,

CYPRINODON VARIEGATUS, LARVAE EXPOSED TO AN EFFLUENT FOR SEVEN DAYS1

Effl.

Conc.

(%)

Proportion of Survival in

 Replicate Chambers 

A B C D

Mean

Prop.

Surv

Avg Dry Wgt (mg) in

 Replicate Chambers 

A B C D

Mean

Dry Wgt

(mg)

0.0 1.0 1.0 1.0 1.0 1.00 1.29 1.32 1.59 1.27 1.368

6.25 1.0 1.0 0.9 1.0 0.98 1.27 1.00 0.97 0.97 1.053

12.5 1.0 1.0 1.0 1.0 1.00 1.32 1.37 1.35 1.34 1.345

25.0 1.0 1.0 1.0 0.8 0.95 1.29 1.33 1.20 0.94 1.190

50.0 0.8 0.8 0.7 0.6 0.73 1.62 0.56 0.46 0.46 0.525

100.0 0.0 0.0 0.0 0.0 0.00 --- --- --- --- ---

1 Four replicates of 10 larvae each. 

11.13.2 EXAMPLE OF ANALYSIS OF SHEEPHEAD MINNOW, CYPRINODON VARIEGATUS 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 endpoint. 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 IC, EC, and LC endpoint. 

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. 

11.13.2.4 Probit Analysis (Finney, 1971; see Appendix H) 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 H-K). 
81

Figure 5. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus, larval

survival data by hypothesis testing.
82

Figure 6. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus, larval

survival data by point estimation.
83

11.13.2.5 Example of Analysis of Survival Data 

11.13.2.5.1 This example uses the survival data from the Sheepshead Minnow Larval Survival and Growth Test. 

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 effluent concentration and control are listed in Table 5. A plot of the survival proportions is

provided in Figure 7. Since there was 100% mortality in all four replicates for the 100% concentration, it was not

included in the statistical analysis and was considered a qualitative mortality effect.

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 6. 

11.13.2.6.2 Calculate the denominator, D, of the statistic:

D ' j

n

i'1

(Xi

&X)

2

Where: Xi = the ith centered observation 

 

X ̄ = the overall mean of the centered observations 

n = the total number of centered observations 
84

TABLE 5. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, SURVIVAL DATA 

Replicate Control

Effluent Concentration (%)

6.25 12.5 25.0 50.0

RAW A

B

C

D

1.0

1.0

1.0

1.0

1.0

1.0

0.9

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

0.8

0.8

0.8

0.7

0.6

ARC SINE

TRANSFORMED

A

B

C

D

1.412

1.412

1.412

1.412

1.412

1.412

1.249

1.412

1.412

1.412

1.412

1.412

1.412

1.412

1.412

1.107

1.107

1.107

0.991

0.886

Mean ( ) Y ̄ i

Si

2

i

1.412

0.0 

1 

1.371

0.007

2 

1.412

0.0 

3 

1.336

0.023

4 

1.023

0.011

5 

TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE 

Replicate Control

Effluent Concentration (%)

6.25 12.5 25.0 50.0

A

B

C

D

0.0

0.0

0.0

0.0

 0.041

 0.041

-0.122

 0.041

0.0

0.0

0.0

0.0

 0.076

 0.076

 0.076

-0.229

 0.084

 0.084

-0.032

-0.137

11.13.2.6.3 For this set of data,

n = 20 

 

X ̄ ' 1

20 (&0.001) ' 0.000

D = 0.1236 

86

W ' 1

D [j

k

i'1

ai(X (n&i%1) & X (i))]2

W ' 1

0.1236 (0.3178)2 ' 0.8171

11.13.2.6.4 Order the centered observations from smallest to largest 

 X(1) # X(2) # ... # X(n)

where X(i) denotes the ith ordered observation. The ordered observations for this example are listed in Table 7. 

11.13.2.6.5 From Table 4, Appendix B, for the number of observations, n, obtain the coefficients a1, a2, ... a k where

k is n/2 if n is even and (n-1)/2 if n is odd. For the data in this example, n = 20 and k = 10. The ai

 values are listed

in Table 8. 

11.13.2.6.6 Compute the test statistic, W, as follows: 

The differences X(n-i+1) - X(i) are listed in Table 8. For the data in this example, 

TABLE 7. ORDERED CENTERED OBSERVATIONS FOR THE SHAPIRO-WILK'S EXAMPLE 

i X(i) i X(i)

1 -0.229 11 0.0 

2 -0.137 12 0.0 

3 -0.122 13 0.041 

4 -0.032 14 0.041 

5 0.0 15 0.041 

6 0.0 16 0.076 

7 0.0 17 0.076 

8 0.0 18 0.076 

9 0.0 19 0.084 

10 0.0 20 0.084 

11.13.2.6.7 The decision rule for this test is to compare W as calculated in Subsection 11.13.2.6.6 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 = 0.817 is less than the critical value, conclude that the data are not normally

distributed.
87

11.13.2.6.8 Since the data do not meet the assumption of normality, Steel's Many-one Rank Test will be used to

analyze the survival data. 

TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE

i ai X(n-i+1) - X(i)

1 0.4734 0.313 X(20) - X(1)

2 0.3211 0.221 X(19) - X(2)

3 0.2565 0.198 X(18) - X(3)

4 0.2085 0.108 X(17) - X(4)

5 0.1686 0.076 X(16) - X(5)

6 0.1334 0.041 X(15) - X(6)

7 0.1013 0.041 X(14) - X(7)

8 0.0711 0.041 X(13) - X(8)

9 0.0422 0.0 X(12) - X(9)

10 0.0140 0.0 X(11) - X(10)

11.13.2.7 Steel's Many-one Rank Test 

11.13.2.7.1 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. 

11.13.2.7.2 An example of assigning ranks to the combined data for the control and 6.25% effluent concentration

is given in Table 9. This ranking procedure is repeated for each control/concentration combination. The complete

set of rankings is summarized in Table 10. The ranks are next summed for each effluent concentration, as shown in

Table 11.

11.13.2.7.3 For this example, determine if the survival in any of the effluent concentrations is significantly lower

than the survival in the control. 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 survival at each of the various effluent

concentrations with some "minimum" or critical rank sum, at or below which the survival would be considered

significantly lower than the control. At a significance level of 0.05, the minimum rank sum in a test with four

concentrations (excluding the control) and four replicates is 10 (see Table 5, Appendix E). 

11.13.2.7.4 Since the rank sum for the 50% effluent concentration is equal to the critical value, the proportion

surviving in the 50% concentration is considered significantly less than that in the control. Since no other rank

sums are less than or equal to the critical value, no other concentrations have a significantly lower proportion

surviving than the control. Hence, the NOEC and the LOEC are the 25% and 50% concentrations, respectively. 

11.13.2.8 Calculation of the LC50

11.13.2.8.1 The data used for the calculation of the LC50 is summarized in Table 12. For estimating the LC50, the

data for the 100% effluent concentration with 100% mortality is included. 

11.13.2.8.2 Because there are at least two partial mortalities in this set of test data, calculation of the LC50 using

Probit Analysis is recommended. For this set of data, however, the test for heterogeneity of variance was

significant. Probit Analysis is not appropriate in this case. Inspection of the data reveals that, once the data is

smoothed and adjusted, the proportion mortality in the lowest effluent concentration will not be zero although the

proportion mortality in the highest effluent concentration will be one. Therefore, the Spearman-Karber Method is

appropriate for this data.

 
88

TABLE 9. ASSIGNING RANKS TO THE CONTROL AND 6.25% EFFLUENT CONCENTRATION FOR

STEEL'S MANY-ONE RANK TEST 

Rank Transformed Effluent

Proportion Concentration

Surviving (%)

1 1.249 6.25 

5 1.412 6.25 

5 1.412 6.25 

5 1.412 6.25

5 1.412 Control

5 1.412 Control

5 1.412 Control 

5 1.412 Control

TABLE 10. TABLE OF RANKS

 Effluent Concentration (%) 

Replicate Control 6.25 12.5 25.0 50.0 

A 1.412 (5,4.5,5,6.5) 1.412 (5) 1.412 (4.5) 1.412 (5) 1.107 (3.5) 

B 1.412 (5,4.5,5,6.5) 1.412 (5) 1.412 (4.5) 1.412 (5) 1.107 (3.5)

C 1.412 (5,4.5,5,6.5) 1.249 (1) 1.412 (4.5) 1.412 (5) 0.991 (2) 

D 1.412 (5,4.5,5,6.5) 1.412 (5) 1.412 (4.5) 1.107 (1) 0.886 (1) 

TABLE 11. RANK SUMS 

Effluent Concentration (%) Rank Sum

6.25

12.5

25.0

50.0

16

18

16

10
89

11.13.2.8.3 Before the LC50 can be calculated the data must be smoothed and adjusted. For the data in this

example, because the observed proportion mortality for the 12.5% effluent concentration is less than the observed

response proportion for the 6.25% effluent concentration, the observed responses for the control and these two

groups must be averaged:

p s

o ' p s1 ' p s2 ' 0.00%0.025%0.00

3 ' 0.0253 ' 0.0083

Where: ps

i

 = the smoothed observed mortality proportion for effluent concentration i

11.13.2.8.3.1 Because the rest of the responses are monotonic, additional smoothing is not necessary. The

smoothed observed proportion mortalities are shown in Table 12.

11.13.2.8.4 Because the smoothed observed proportion mortality for the control is now greater than zero, the data

in each effluent concentration must be adjusted using Abbott's formula (Finney, 1971). The adjustment takes the

form:

Where: ps

0 = the smoothed observed proportion mortality for the control

pa

i

 = (ps

i

 - ps

0) / (1 - ps0)

ps

i = the smoothed observed proportion mortality for effluent concentration i

11.13.2.8.4.1 For the data in this example, the data for each effluent concentration must be adjusted for control

mortality using Abbott's formula, as follows:

p a

0 ' p a1 ' p a2 ' p s

1 &p s0

1&p s

0

' 0.0083&0.0083

1&0.0083 ' 0.000.9917 ' 0.0

p a

3 ' p s

3 &p s0

1&p s

0

' 0.05&0.0083

1&0.0083 ' 0.04170.9917 ' 0.042

p a

4 ' p s

4 &p s0

1&p s

0

' 0.275&0.0083

1&0.0083 ' 0.26670.9917 ' 0.269

p a

5 ' p s

5 &p s0

1&p s

0

' 1.000&0.0083

1&0.0083 ' 0.99170.9917 ' 1.000

The smoothed, adjusted response proportions for the effluent concentrations are shown in Table 12. 
90

m ' j

k&1

i'1

(p a

i %1) (Xi

%Xi%1)

2

TABLE 12. DATA FOR EXAMPLE OF SPEARMAN-KARBER ANALYSIS

Smoothed,

Effluent Number of Smoothed Adjusted

Concentration Number of Organisms Mortality Mortality Mortality 

% Deaths Exposed Proportion Proportion Proportion

Control 0 40 0.000 0.0083 0.000

 6.25 1 40 0.025 0.0083 0.000

12.5 0 40 0.000 0.0083 0.000

25.0 2 40 0.050 0.0500 0.042

50.0 11 40 0.275 0.2750 0.269

100.0 40 40 1.000 1.0000 1.000

11.13.2.8.5 Calculate the log10 of the estimated LC50, m, as follows:

Where: pa

i = the smoothed adjusted proportion mortality at concentration i

Xi = the log10 of concentration i

k = the number of effluent concentrations tested, not including the control

11.13.2.8.5.1 For this example, the log10 of the estimated LC50, m, is calculated as follows:

m = [(0.000 - 0.000) (0.7959 + 1.0969)]/2 +

[(0.042 - 0.000) (1.0969 + 1.3979)]/2 +

[(0.269 - 0.042) (1.3979 + 1.6990)]/2 +

[(1.000 - 0.269) (1.6990 + 2.0000)]/2

= 1.755873 

11.13.2.8.6 Calculate the estimated variance of m as follows:

V(m) ' j

k&1

i'2

p a

i (1&p ai )(Xi%1%Xi&1)

2

4(ni&1)

Where: Xi = the log10 of concentration i

ni = the number of organisms tested at effluent concentration i

pa

i = the smoothed adjusted observed proportion mortality at effluent concentration i

k = the number of effluent concentrations tested, not including the control

11.13.2.8.6.1 For this example, the estimated variance of m, V(m), is calculated as follows:

V(m) = (0.000)(1.000)(1.3979 - 0.7959)2

/4(39) +

(0.042)(0.958)(1.6990 - 1.0969)2

/4(39)+

 (0.269)(0.731)(2.0000 - 1.3979)2

/4(39)

= 0.0005505
91

11.13.2.8.7 Calculate the 95% confidence interval for m: m ± 2.0 %&&V&(&m&)

11.13.2.8.7.1 For this example, the 95% confidence interval for m is calculated as follows:

1.755873 ± 2 0.0005505 ' (1.754772, 1.756974)

11.13.2.8.8 The estimated LC50 and a 95% confidence interval for the estimated LC50 can be found by taking

base10 antilogs of the above values.

11.13.2.8.8.1 For this example, the estimated LC50 is calculated as follows:

LC50 = antilog(m) = antilog(1.755873) = 57.0%.

11.13.2.8.8.2 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(1.754772) = 56.9%

upper limit: antilog(1.756974) = 57.1%

11.13.3 EXAMPLE OF ANALYSIS OF SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, GROWTH

DATA 

11.13.3.1 Formal statistical analysis of the growth data is outlined in Figure 8. The response used in the statistical

analysis is mean weight per original organism for each replicate. Because this measurement is based on the number

of original organisms exposed (rather than the number surviving), the measured response is a combined survival and

growth endpoint that can be termed biomass. The IC25 and IC50 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 an NOEC and LOEC 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 testing 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, Steels'

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. The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the nonparametric alternative. For

detailed information on the Bonferroni adjustment, see Appendix D.

11.13.3.4 The data, mean and variance of the observations at each concentration including the control are listed in

Table 13. A plot of the mean weights for each treatment is provided in Figure 9. Since there is no survival in the

100% concentration, it is not considered in the growth analysis. Additionally, since there is significant mortality in

the 50% effluent concentration, its effect on growth is not considered.
92

Figure 8. Flowchart for statistical analysis of the sheepshead minnow, Cyprinodon variegatus, larval growth data.
93

D ' j

n

i'1

(Xi&X)

2

TABLE 13. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, GROWTH DATA

 Effluent Concentration (%)

Replicate Control 6.25 12.5 25.0 50.0 100.0 

A 1.29 1.27 1.32 1.29 - -

B 1.32 1.00 1.37 1.33 - - 

C 1.59 0.97 1.35 1.20 - - 

D 1.27 0.97 1.34 0.94 - -

Mean 1.368 1.053 1.345 1.190 - - (Y ̄ i

)

S2

i 0.0224 0.0212 0.0004 0.0307 - -

i 123 45 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 14.

TABLE 14. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

 Effluent Concentration (%)

Replicate Control 6.25 12.5 25.0

A -0.078 0.217 -0.025 0.100

B -0.048 -0.053 0.025 0.140

C 0.222 -0.083 0.005 0.010

D -0.098 0.083 -0.005 -0.250

11.13.3.5.2 Calculate the denominator, D, of the test statistic:

Where: Xi = 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 = 16 
94

X ̄ ' 1

16 (&0.004) ' 0.00024 ' 0.00

D = 0.2245

11.13.3.5.3 Order the centered observations from smallest to largest: 

X(1) # X(2) # ... # X(n)

Where X(i) is the ith ordered observation. These ordered observations are listed in Table 15.

TABLE 15. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

i X(i) i X(i)

1 -0.250 9 -0.005

2 -0.098 10 0.005

3 -0.083 11 0.010

4 -0.083 12 0.025

5 -0.078 13 0.100

6 -0.053 14 0.140

7 -0.048 15 0.217

8 -0.025 16 0.222
95

Figure 9. Plot of weight data from sheepshead minnow, Cyprinodon variegatus, larval survival and growth test.
96

W ' 1

D [j

k

i'1

ai (X (n&i%1)&X (i))]

2

W ' 1

0.2245 (0.4588)2 ' 0.938

11.13.3.5.4 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-1)/2 if n is odd. For the data in this example, n = 16 and k = 8. The ai values

are listed in Table 16.

TABLE 16. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE 

i ai X(n-i+1) - X(i)

1 0.5056 0.472 X(16) - X(1)

2 0.3290 0.315 X(15) - X(2)

3 0.2521 0.223 X(14) - X(3)

4 0.1939 0.183 X(13) - X(4)

5 0.1447 0.103 X(12) - X(5)

6 0.1005 0.063 X(11) - X(6)

7 0.0593 0.053 X(10) - X(7)

8 0.0196 0.020 X(9) - X(8)

11.13.3.5.5 Compute the test statistic, W, as follows: 

The differences X(n-i+1) - X(i) are listed in Table 16. 

For this set of data:

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 16 observations (n) is 0.844. Since W = 0.938 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 effluent

concentrations including the control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as

follows:
97

S2 '

('

P

i'1

ViS 2

i )

'

P

i'1

Vi

C ' 1%[3(p&1)]&1['

P

i'1

1/Vi

&('

P

i'1

Vi)

&1

]

B '

[('

P

i'1

Vi) ln S2 & '

P

i'1

Vi ln S 2

i ]

C

Where: Vi = degrees of freedom for each effluent concentration and control, V i

 = (n i - (1)) 

ni = the number of replicates for concentration i 

p = number of levels of effluent concentration including the control

ln = loge

i = 1, 2, ..., p where p is the number of concentrations including the control

 

11.13.3.6.2 For the data in this example (see Table 14), all effluent concentrations including the control have

the same number of replicates (ni

 = 4 for all i). Thus, V i = 3 for all i. 

11.13.3.6.3 Bartlett's statistic is therefore: 

B ' [(12)ln(0.0187) & 3'

P

i'1

ln(S 2

i )]/1.139

= [12(-3.979) - 3(-18.876)]/1.139 

= 8.882/1.139 

= 7.798 

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 three

degrees of freedom, is 11.345. Since B = 7.798 is less than the critical value of 11.345, conclude that the

variances are not different. 
98

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 17.

TABLE 17. ANOVA TABLE

Source df Sum of Squares Mean Square (MS) 

(SS) (SS/df)

Between p - 1 SSB S 2

B ' SSB/(p&1)

Within N - p SSW S 2

W ' SSW/(N&p)

Total N - 1 SST

Where: p = number of concentration levels including the control

N = total number of observations n1 + n2 ... + n p

ni = number of observations in concentration i 

 Between Sum of Squares SSB ' '

P

i'1

T 2

i /ni

&G 2/N

 Total Sum of Squares SST ' '

P

i'1

'

ni

j'1

Y 2

ij &G 2

/N

 Within Sum of Squares SSW ' SST&SSB

 G = the grand total of all sample observations, G ' '

P

i'1

Ti

 Ti = the total of the replicate measurements for concentration i

 Yij = the jth observation for concentration i (represents the mean dry weight of the mysids for

concentration i in test chamber j) 

11.13.3.7.2 For the data in this example: 

 n1 = n2 = n3 = n4 = 4 

N = 16 

T1 = Y11 + Y12 + Y13 + Y14 = 5.47 
99

SSB ' '

P

i'1

T 2

i /ni

&G 2

/N

SSW ' SST&SSB

 T2 = Y21 + Y22 + Y23 + Y24 = 4.21 

 T3 = Y31 + Y32 + Y33 + Y34 = 5.38 

T4 = Y41 + Y42 + Y43 + Y44 = 4.76 

 G = T1 + T2 + T3 + T4 = 19.82 

 

 

= 1 (99.247) - (19.82)

2

 = 0.260 

 4 16 

 SST ' '

P

i'1

'

ni

j'1

Y 2

ij &G 2

/N

 = 25.036 - (19.82)

2

 = 0.484 

 16

 

 = 0.484 - 0.260 = 0.224 

 

 SB

2

 = SSB/(p-1) = 0.260/(4-1) = 0.087 

 

 SW

2

 = SSW/(N-p) = 0.224/(16-4) = 0.019 

11.13.3.7.3 Summarize these calculations in the ANOVA table (Table 18).
100

TABLE 18. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE 

Source df Sum of Squares Mean Square(MS) 

(SS) (SS/df) 

Between 3 0.260 0.087 

Within 12 0.224 0.019 

Total 15 0.484 

11.13.3.7.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control

combination as follows:

t

Y Y

Sn n i

i

w i

= −

+

( )

(/ ) (/ )

1

1 1 1

Where: &Yi = mean dry weight for effluent concentration i 

 

&Y1 = mean dry weight for the control 

SW = square root of the within mean square 

n1 = number of replicates for the control 

ni = number of replicates for concentration i. 

11.13.3.7.5 Table 19 includes the calculated t values for each concentration and control combination. In this

example, comparing the 6.25% concentration with the control, the calculation is as follows:

TABLE 19. CALCULATED T VALUES

Effluent Concentration (%) i ti

6.25 2 3.228

12.5 3 0.236

25.0 4 1.824
101

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, 12 degrees of freedom for error and three concentrations (excluding the control) the critical value is

2.29. The mean weight for concentration i is considered significantly less than the mean weight for the control if

ti

 is greater than the critical value. Since t 2 is greater than 2.29, the 6.25% concentration has significantly lower

growth than the control. However, the 12.5% and 25% concentrations do not exhibit this effect. Hence the

NOEC and the LOEC for growth cannot be calculated. 

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 Sw (1/n1)%(1/n)

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) 

n1 = the number of replicates in the control. 

11.13.3.7.8 In this example:

MSD ' 2.29(0.10) (1/4)%(1/4)

= 2.29 (0.138)(0.707) 

= 0.223

11.13.3.7.9 Therefore, for this set of data, the minimum difference that can be detected as statistically

significant is 0.223 mg.

11.13.3.7.10 This represents a 16% reduction in mean weight from the control. 

11.13.3.8 Calculation of the ICp

11.13.3.8.1 The growth data from Table 4 are utilized in this example. As seen from Table 4 and Figure 7, the

observed means are not monotonically non-increasing with respect to concentration (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 trends). Therefore, the means are smoothed prior to

calculating the IC. In the following discussion, the observed means are represented by &Y i

 and the smoothed
102

means by Mi.

11.13.3.8.2 Starting with the control mean, &Y 1 = 1.368 and &Y 2 = 1.053, we see that &Y 1 >&Y 2. Set M1 = &Y 1. 

Comparing &Y 2 to &Y 3, &Y 2<&Y3.

11.13.3.8.3 Calculate the smoothed means:

 M2 = M3 = (&Y 2 +&Y 3)/2 = 1.199

11.13.3.8.4 Since &Y 6 = 0 < &Y 5 = 0.525 <&Y 4 = 1.190 <&Y 4 = 1.345, set M3 = 1.199, M4 = 1.190, M5 = 0.525, and

set M6 = 0.

11.13.3.8.5 Table 20 contains the response means and smoothed means and Figure 10 gives a plot of the

smoothed response curve.

TABLE 20. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, MEAN GROWTH RESPONSE

AFTER SMOOTHING

Effluent

Conc. (%) i

Response Means

(mg) Yi

Smoothed

Means

 (mg) Mi

Control

6.25

12.50

25.00

50.00

100.00

1

2

3

4

5

6

1.368 

1.053 

1.345 

1.189 

0.525 

0.0 

1.368 

1.199 

1.199 

1.189 

0.525 

0.0 
103

Figure 10. Plot of raw data, observed means, and smoothed means for the sheepshead minnow, Cyprinodon varieagatus, growth data fr

20.
104

ICp ' Cj%[M1(1&p/100)&Mj]

(C(j%1)&Cj)

(M(j%1)&Mj

)

ICp'Cj

%[M1(1&p/100)&Mj]

(C(j%1)&Cj)

(M(j%1)&Mj

)

 11.13.3.8.6 An IC25 and 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 1.026 mg, where M1(1-p/100) = 1.368(1-

25/100). A 50% reduction in mean dry weight, compared to the controls, would result in a mean dry weight of

0.684 mg. Examining the smoothed means and their associated concentrations (Table 4), the response, 1.026 mg,

is bracketed by C4 = 25.0% effluent and C5 = 50.0% effluent. The response (0.684 mg) is bracketed by

C4 = 25.0% effluent and C 5 = 50% effluent.

11.13.3.8.7 Using the equation from Section 4.2 of Appendix M, the estimate of the IC25 is calculated as follows:

 

 IC25 = 25.0 + [1.368(1 - 25/100) - 1.189] (50.00 - 25.00)

 (0.525 - 1.189)

 = 31.2%

11.13.3.8.8 Using the equation from Section 4.2 of Appendix L, the estimate of the IC50 is calculated as follows:

IC50 = 50.0 + [1.368(1-50/100) - 0525] (100.00-50.00)

(0.0 - 0.525)

 = 44.0%

11.13.3.8.9 When the ICPIN program was used to analyze this set of data, requesting 80 resamples, the estimate of

the IC25 was 31.1512%. The empirical 95% confidence interval for the true mean was 22.0420% and 36.3613%. 

The computer program output for the IC25 for this data set is shown in Figure 11.

11.13.3.8.10 When the ICPIN program was used to analyze this set of data for the IC50, requesting 80 resamples,

the estimate of the IC50 was 44.0230%. The empirical 95% confidence interval for the true mean was 39.1011% and

49.0679%. The computer program output is shown in Figure 12.
105

Conc. ID 1 2 3 4 5 6

-------------------------------------------------------------------------------------------------------------------------------

Conc. Tested 0 6.25 12.5 25 50 100

-------------------------------------------------------------------------------------------------------------------------------

Response 1 1.29 1.27 1.32 1.29 .62 0

Response 2 1.32 1 1.37 1.33 .560 0

Response 3 1.59 .972 1.35 1.2 .46 0

Response 4 1.27 .97 1.34 .936 .46 0

-------------------------------------------------------------------------------------------------------------------------------

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: Effluent

Test Start Date: Test Ending Date: 

Test Species: Cyprinodon variegatus

Test Duration: 7-d

DATA FILE: sheep.icp

OUTPUT FILE: sheep.i25

---------------------------------------------------------------------------------------------------------------------------------------

Conc. Number Concentration Response Std. Pooled

 ID Replicates % Means Dev. Response Means

----------------------------------------------------------------------------------------------------------------------------------------

 1 4 0.000 1.368 0.150 1.368

 2 4 6.250 1.053 0.145 1.199

 3 4 12.500 1.345 0.021 1.199

 4 4 25.000 1.189 0.177 1.189

 5 4 50.000 0.525 0.079 0.525

 6 4 100.000 0.000 0.000 0.000

--------------------------------------------------------------------------------------------------------------------------------------

The Linear Interpolation Estimate: 31.1512 Entered P Value: 25

---------------------------------------------------------------------------------------------------------------------------------------

Number of Resamplings: 80

The Bootstrap Estimates Mean: 30.6175 Standard Deviation: 2.9490

Original Confidence Limits: Lower: 25.4579 Upper: 34.4075

Expanded Confidence Limits: Lower: 22.0420 Upper: 36.3613

Resampling time in Seconds: 1.70 Random Seed: -2137496326

Figure 11. ICPIN program output for the IC25.
106

----------------------------------------------------------------------------------------------------------------------------------

Conc. ID 1 2 3 4 5 6

----------------------------------------------------------------------------------------------------------------------------------

Conc. Tested 0 6.25 12.5 25 50 100

----------------------------------------------------------------------------------------------------------------------------------

Response 1 1.29 1.27 1.32 1.29 .62 0

Response 2 1.32 1 1.37 1.33 .560 0

Response 3 1.59 .972 1.35 1.2 .46 0

Response 4 1.27 .97 1.34 .936 .46 0

-------------------------------------------------------------------------------------------------------------------------------

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: Effluent

Test Start Date: Test Ending Date: 

Test Species: Cyprinodon variegatus

Test Duration: 7-d

DATA FILE: sheep.icp

OUTPUT FILE: sheep.i50

---------------------------------------------------------------------------------------------------------------------------------------

Conc. Number Concentration Response Std. Pooled

 ID Replicates % Means Dev. Response Means

----------------------------------------------------------------------------------------------------------------------------------------

 1 4 0.000 1.368 0.150 1.368

 2 4 6.250 1.053 0.145 1.199

 3 4 12.500 1.345 0.021 1.199

 4 4 25.000 1.189 0.177 1.189

 5 4 50.000 0.525 0.079 0.525

 6 4 100.000 0.000 0.000 0.000

--------------------------------------------------------------------------------------------------------------------------------------

The Linear Interpolation Estimate: 44.0230 Entered P Value: 50

---------------------------------------------------------------------------------------------------------------------------------------

Number of Resamplings: 80

The Bootstrap Estimates Mean: 44.3444 Standard Deviation: 1.7372

Original Confidence Limits: Lower: 40.9468 Upper: 47.1760

Expanded Confidence Limits: Lower: 39.1011 Upper: 49.0679

Resampling time in Seconds: 1.70 Random Seed: -156164614

Figure 12. ICPIN program output for the IC50.
107

11.14 PRECISION AND ACCURACY

11.14.1 PRECISION – Data on single-laboratory and multilaboratory precision are described below (Subsections

11.14.1.1 and 11.14.1.2). Single-laboratory precision is a measure of the reproducibility of test results when tests are

conducted using a specific method under reasonably constant conditions in the same laboratory. Single-laboratory

precision is synonymous with the terms within-laboratory precision and intralaboratory precision. Multilaboratory

precision is a measure of the reproducibility of test results from different laboratories using the same test method and

analyzing the same test material. Multilaboratory precision is synonymous with the term interlaboratory precision. 

Interlaboratory precision, as used in this document, includes both within-laboratory and between-laboratory

components of variability. In recent multilaboratory studies, these two components of interlaboratory precision have

been displayed separately (termed within-laboratory and between-laboratory variability) and combined (termed total

interlaboratory variability). The total interlaboratory variability that is reported from these studies is synonymous

with interlaboratory variability reported from other studies where individual variability components are not

separated.

11.14.1.1 Single-Laboratory Precision

11.14.1.1.1 Data on the single-laboratory precision of the Sheepshead Minnow Larval Survival and Growth Test

using FORTY FATHOMS® artificial seawater, natural seawater, and GP2 with copper sulfate, sodium dodecyl

sulfate, and hexavalent chromium, as reference toxicants, are given in Tables 21-26. The IC25, IC50, or LC50 data

(coefficient of variation), indicating acceptable precision for the reference toxicants (copper, sodium dodecyl sulfate,

and hexavalent chromium), are also listed in these Tables.

11.14.1.1.2 EPA evaluated within-laboratory precision of the Sheepshead Minnow, Cyprinodon variegatus, Larval

Survival and Growth Test using a database of routine reference toxicant test results from five laboratories (USEPA,

2000b). The database consisted of 57 reference toxicant tests conducted in 5 laboratories using reference toxicants

including: cadmium and potassium chloride. Among the 5 laboratories, the median within-laboratory CV calculated

for routine reference toxicant tests was 13% for the IC25 growth endpoint. In 25% of laboratories, the withinlaboratory CV was less than 9%; and in 75% of laboratories, the within-laboratory CV was less than 14%. 

11.14.1.2 Multilaboratory Precision

11.14.1.2.1 Data from a study of multilaboratory test precision, involving a total of seven tests by four participating

laboratories, are listed in Table 27. The laboratories reported very similar results, indicating good interlaboratory

precision. The coefficient of variation (IC25) was 44.2% and (IC50) was 56.9%, indicating acceptable precision. 

11.14.1.2.2 In 2000, EPA conducted an interlaboratory variability study of the Sheepshead Minnow, Cyprinodon

variegatus, Larval Survival and Growth Test (USEPA, 2001a; USEPA, 2001b). In this study, each of 7 participant

laboratories tested 4 blind test samples that included blank, effluent, reference toxicant, and receiving water sample

types. The blank sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater, the effluent sample

was a municipal wastewater spiked with KCl, the receiving water sample was a natural seawater spiked with KCl,

and the reference toxicant sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater spiked with

KCl. Of the 28 Sheepshead Minnow Larval Survival and Growth Tests conducted in this study, 100% were

successfully completed and met the required test acceptability criteria. Of 7 tests that were conducted on blank

samples, none showed false positive results for the survival endpoint or the growth endpoint. Results from the

reference toxicant, effluent, and receiving water sample types were used to calculate the precision of the method. 

Table 28 shows the precision of the IC25 for each of these sample types. Averaged across sample types, the total

interlaboratory variability (expressed as a CV%) was 10.5% for IC25 results. Table 29 shows the frequency

distribution of survival and growth NOEC endpoints for each sample type. For the survival endpoint, NOEC values

spanned two concentrations for the reference toxicant sample type and one concentration for the effluent and
108

receiving water sample types. The percentage of values within one concentration of the median was 100% for each

of the sample types. For the growth endpoint, NOEC values spanned one concentration for the reference toxicant

sample type and two concentrations for the effluent and receiving water sample types. The percentage of values

within one concentration of the median was 100% for each of the sample types. 

11.14.2 ACCURACY

11.14.2.1 The accuracy of toxicity tests cannot be determined.
109

TABLE 21. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW, CYPRINODON 

VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST PERFORMED IN FORTY

FATHOMS® ARTIFICIAL SEAWATER, USING LARVAE FROM FISH MAINTAINED AND

SPAWNED IN FORTY FATHOMS® ARTIFICIAL SEAWATER, USING COPPER (CU)

SULFATE AS A REFERENCE TOXICANT1,2,3,4,5

Most

Test NOEC IC25 IC50 Sensitive

Number (μg/L) (μg/L) (μg/L) Endpoint6

1 50 113.3 152.3 S

2 <507 54.3 97.5 G

3 < 507 41.8 71.4 G

4 50 63.2 90.8 S

5 <507 57.7 99.8 S

6 50 48.3 132.5 G

7 50 79.6 159.7 G

8 50 123.5 236.4 G

n: 5 8 8

Mean: NA 72.7 130.0

CV(%): NA 41.82 40.87

1 Data from USEPA (1988a) and USEPA (1991a).

2 Tests performed by Donald J. Klemm, Bioassessment and Ecotoxicology Branch, EMSL, Cincinnati, OH.

3 All tests were performed using FORTY FATHOMS® synthetic seawater. Three replicate exposure chambers, each

with 15 larvae, were used for the control and each copper concentration. Copper concentrations used in Tests 1-6

were: 50, 100, 200, 400, and 800 mg/L. Copper concentrations in Tests 7-8 were: 25, 50, 100, 200 and 400 mg/L. 

4 Adults collected in the field. 

5 For a discussion of the precision of data from chronic toxicity test see Section 4, Quality Assurance.

6 Endpoints: G=growth; S=survival.

7 Lowest concentration tested was 50 μg/L (NOEC Range: < 50* - 50 μg/L). 
110

TABLE 22. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW, CYPRINODON

VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST PERFORMED IN FORTY

FATHOMS® ARTIFICIAL SEAWATER, USING LARVAE FROM FISH MAINTAINED AND

SPAWNED IN FORTY FATHOMS® ARTIFICIAL SEAWATER, USING SODIUM DODECYL

SULFATE (SDS) AS A REFERENCE TOXICANT1,2,3,4,5,6

Most

Test NOEC IC25 IC50 Sensitive

Number (mg/L) (mg/L) (mg/L) Endpoint7

1 1.0 1.2799 1.5598 S

2 1.0 1.4087 1.8835 S

3 1.0 2.3051 2.8367 S

4 0.5 1.9855 2.6237 G

5 1.0 1.1901 1.4267 S

6 0.5 1.1041 1.4264 G

n: 6 6 6

Mean: NA 1.5456 1.9595

CV(%): NA 31.44 31.82

 

1 Data from USEPA (1988a) and USEPA (1991a). 

2 Tests performed by Donald J. Klemm, Bioassessment and Ecotoxicology Branch, EMSL, Cincinnati, OH. 

3 All tests were performed using FORTY FATHOMS® synthetic seawater. Three replicate exposure chambers,

each with 15 larvae, were used for the control and each SDS concentration. SDS concentrations in Tests 1-2

were: 1.0, 1.9, 3.9, 7.7, and 15.5 mg/L. SDS concentrations in Tests 3-6 were: 0.2, 0.5, 1.0, 1.9, and 3.9 mg/L. 

4 Adults collected in the field. 

5 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 

6 NOEC Range: 0.5 -1.0 mg/L (this represents a difference of one exposure concentration). 

7 Endpoints: G=growth; S=survival 
111

TABLE 23. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW, CYPRINODON 

VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST PERFORMED IN NATURAL

SEAWATER, USING LARVAE FROM FISH MAINTAINED AND SPAWNED IN NATURAL

SEAWATER, USING COPPER (CU) SULFATE AS A REFERENCE TOXICANT1,2,3,4,5,6

Most

Test NOEC IC25 IC50 Sensitive

Number (μg/L) (μg/L) (μg/L) Endpoint7

1 125 320.3 437.5 S

2 31 182.3 323.0 G

3 125 333.4 484.4 S

4 125 228.4 343.8 S

5 125 437.5 NC8 S

 n: 5 5 4

Mean: NA 300.4 396.9

CV(%): NA 33.0 19.2

1 Data from USEPA (1988a) and USEPA (1991a).

2 Tests performed by George Morrison and Elise Torello, ERL-N, USEPA, Narragansett, RI. 

3 Three replicate exposure chambers, each with 10-15 larvae, were used for the control and each copper concentration.

Copper concentrations were: 31, 63, 125, 250, and 500 μg/L. 

4

 NOEC Range: 31 - 125 μg/L (this represents a difference of two exposure concentrations).

5 Adults collected in the field. 

6 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 

7 Endpoints: G=growth; S=survival.

8 NC = No linear interpolation estimate could be calculated from the data, since none of the group response means

were less than 50 percent of the control response mean.
112

TABLE 24. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW, CYPRINODON 

VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST PERFORMED IN NATURAL

SEAWATER, USING LARVAE FROM FISH MAINTAINED AND SPAWNED IN NATURAL

SEAWATER, USING SODIUM DODECYL SULFATE (SDS) AS A REFERENCE

TOXICANT1,2,3,4,5,6

Most

Test NOEC IC25 IC50 Sensitive

Number (mg/L) (mg/L) (mg/L) Endpoint7

1 2.5 2.9 3.6 S

2 1.3 NC18 NC29 G

3 1.3 1.9 2.4 S

4 1.3 2.4 NC2 G

5 1.3 1.5 1.8 S

n: 5 4 3

Mean: NA 2.2 2.6

CV(%): NA 27.6 35.3

1 Data from USEPA (1988a) and USEPA (1991a).

2 Tests performed by George Morrison and Elise Torello, ERL-N, USEPA, Narragansett, RI. 

3 Three replicate exposure chambers, each with 10-15 larvae, were used for the control and each SDS concentration. 

SDS concentrations were: 0.3, 0.6, 1.3, 2.5, and 5.0 mg/L. 

4 NOEC Range: 1.3 - 2.5 mg/L (this represents a difference of one exposure concentration).

5 Adults collected in the field.

6 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.

7 Endpoints: G=growth; S=survival.

8 NC1 = No linear interpolation estimate could be calculated from the data, since none of the group response means

were less than 75 percent of the control response mean.

9 NC2 = No linear interpolation estimate could be calculated from the data, since none of the group response means

were less than 50 percent of the control response mean. 
113

TABLE 25. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW, CYPRINODON

VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST PERFORMED IN FORTY

FATHOMS® ARTIFICIAL SEAWATER, USING LARVAE FROM FISH MAINTAINED AND

SPAWNED IN FORTY FATHOMS® ARTIFICIAL SEAWATER, AND HEXAVALENT

CHROMIUM AS THE REFERENCE TOXICANT1,2,3,4,5

Most

Test NOEC IC25 IC50 Sensitive

Number (mg/L) (mg/L) (mg/L) Endpoint6

1 2.0 5.8 11.4 G

2 1.0 2.9 9.9 G

3 4.0 6.9 11.5 G

4 2.0 2.4 9.2 G

5 1.0 3.1 10.8 G

 n: 5 5 5

Mean: NA 4.2 10.6

CV(%): NA 47.6 9.7

1 Tests performed by Donald Klemm, Bioassessment and Ecotoxicology Branch, EMSL, Cincinnati, OH.

2 All tests were performed using Forty Fathoms® synthetic seawater. Three replicate exposure chambers, each with 15

larvae, were used for the control and each hexavalent chromium concentration. Hexavalent chromium concentrations

used in Tests 1-5 were: 1.0, 2.0, 4.0, 8.0, 16.0, and 32.0 mg/L. 

3 NOEC Range: 1.0 - 4.0 mg/L (this represents a difference of four exposure concentrations)

4 Adults collected in the field. 

5 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 

6 Endpoints: G=growth; S=survival.
114

TABLE 26 COMPARISON OF LARVAL SURVIVAL (LC50) AND GROWTH (IC50) VALUES FOR THE

SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EXPOSED TO SODIUM DODECYL

SULFATE (SDS) AND COPPER (CU) SULFATE IN GP2 ARTIFICIAL SEAWATER MEDIUM

OR NATURAL SEAWATER1,2,3,4

 

 Survival Growth 

SDS (mg/L) GP2 NSW GP2 NSW

7.49 8.13 7.39 8.41

8.70 8.87 8.63 8.51

8.38 8.85 8.48 9.33

Mean 8.19 8.62 8.17 8.75

CV (%) 7.7 4.9 8.3 5.8

Copper(μg/L) GP2 NSW GP2 NSW

455 412 341 333

467 485 496 529

390 528 467 776

Mean 437 475 435 546

CV (%) 9.4 12.3 18.9 40.7

1 Tests performed by George Morrison and Glen Modica, ERL-N, USEPA, Narragansett, RI. 

2 Three replicate exposure chambers, each with 10-15 larvae, were used for the control and each SDS concentration. 

SDS concentrations were: 0.3, 0.6, 1.3, 2.5, and 5.0 mg/L. 

3 Adults collected in the field. 

4 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 
115

TABLE 27. DATA FROM INTERLABORATORY STUDY OF THE SHEEPSHEAD MINNOW,

CYPRINODON VARIEGATUS, LARVAL SURVIVAL AND GROWTH TEST USING AN

INDUSTRIAL EFFLUENT AS A REFERENCE TOXICANT1,2,3

 Most Sensitive Endpoint4 

Test NOEC IC25 IC50

Number (%) (%) (%)

Laboratory A 1 3.2 (S,G) 7.4 (S) 7.4 (G)

2 3.2 (S,G) 7.6 (S) 14.3 (G)

Laboratory B 1 3.2 (S,G) 5.7 (G) 9.7 (G)

2 3.2 (S,G) 5.7 (G) 8.8 (G)

Laboratory C 1 1.0 (S) 4.7 (S) 7.2 (S)

Laboratory D 1 3.2 (S,G) 7.4 (G) 24.7 (G)

2 1.0 (G) 5.2 (S) 7.2 (S)

 n: 7 7 7

 Mean: NA 5.5 11.3

 CV(%): NA 44.2 56.9

1 Data from USEPA (1987b), USEPA (1988a), and USEPA (1991a).

2 Effluent concentrations were: 0.32, 1.0, 3.2, 10.0, and 32.0%.

3 NOEC Range: 1.0 - 3.2% (this represents a difference of one exposure concentration).

4 Endpoints: G=growth; S=survival. 
116

TABLE 28. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPES1

Test Endpoint Sample Type CV (%)2

IC25 Reference toxicant 18.4

Effluent 6.12

Receiving water 7.15

Average 10.5

1 From EPA’s WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).

2 CVs were calculated based on the total interlaboratory variability (including both within-laboratory and betweenlaboratory components of variability). Individual within-laboratory and between-laboratory components of variability

could not be calculated since the study design did not provide within-laboratory replication for this sample type.

TABLE 29. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR VARIOUS SAMPLE

TYPES1

Test Endpoint Sample Type

Median

NOEC

Value

% of Results at

the Median

% of Results

±12

% of Results

$23

Survival NOEC Reference toxicant 25% 57.1 42.9 0.00

Effluent 25% 100 0.00 0.00

Receiving water 25% 100 0.00 0.00

Growth 

NOEC

Reference toxicant 25% 100 0.00 0.00

Effluent 12.5% 57.1 42.9 0.00

Receiving water 12.5% 71.4 28.6 0.00

1 From EPA’s WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b).

2 Percent of values at one concentration interval above or below the median. Adding this percentage to the percent of

values at the median yields the percent of values within one concentration interval of the median.

3 Percent of values two or more concentration intervals above or below the median.
117

SECTION 12

TEST METHOD

SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS

EMBRYO-LARVAL SURVIVAL AND TERATOGENICITY TEST

METHOD 1005.0

12.1 SCOPE AND APPLICATION

12.1.1 This method, adapted in part from USEPA (1981) and USEPA (1987b), estimates the chronic toxicity of

effluents and receiving waters to the sheepshead minnow, Cyprinodon variegatus, using embryos and larvae in a

nine-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 acute toxicity for desired exposure periods

(i.e., 24-h, 48-h, 96-h LC50s).

12.1.3 Detection limits of the toxicity of an effluent or chemical 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

volatile and highly degradable toxicants present in the source may not be detected in the test. 

12.1.5 This test 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 Sheepshead minnow, Cyprinodon variegatus, embryos and larvae are exposed in a static renewal system to

different concentrations of effluent or to receiving water starting shortly after fertilization of the eggs through four

days posthatch. 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). 

12.3.2 Adverse effects of low dissolved oxygen concentrations (DO), high concentrations of suspended and/or

dissolved solids, and extremes of pH may mask the effect of toxic substances. 

12.3.3 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). 

12.3.4 Pathogenic and/or predatory organisms in the dilution water and effluent may affect test organism survival,

and confound test results. 
118

12.3.5 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-dependent

toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also increases (see Subsection

8.8.6), so upward pH drift may increase sample toxicity. For metals, toxicity may increase or decrease with

increasing pH. Lead and copper were found to be more acutely toxic at pH 6.5 than at pH 8.0 or 8.5, while nickel

and zinc were more toxic at pH 8.5 than at pH 6.5 (USEPA, 1992). In situations where sample toxicity is confirmed

to be artifactual and due to pH drift (as determined by parallel testing as described in Subsection 12.3.5.1), the

regulatory authority may allow for control of sample pH during testing using procedures outlined in Subsection

12.3.5.2. It should be noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large

(more than 1 pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold

for toxicity. 

12.3.5.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one with

controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to drift during the

test. In the controlled-pH treatment, the pH is maintained using the procedures described in Subsection 12.3.5.2. 

The pH to be maintained in the controlled-pH treatment (or target pH) will depend on the objective of the test. If

the objective of the WET test is to determine the toxicity of the effluent in the receiving water, the pH should be

maintained at the pH of the receiving water (measured at the edge of the regulatory mixing zone). If the objective

of the WET test is to determine the absolute toxicity of the effluent, the pH should be maintained at the pH of the

sample after adjusting the sample salinity for use in marine testing. 

12.3.5.1.1 During parallel testing, the pH must be measured in each treatment at the beginning (i.e., initial pH) and

end (i.e., final pH) of each 24-h exposure period. For each treatment, the mean initial pH (e.g., averaging the initial

pH measured each day for a given treatment) and the mean final pH (e.g., averaging the final pH measured each day

for a given treatment) must be reported. pH measurements taken during the test must confirm that pH was

effectively maintained at the target pH in the controlled-pH treatment. For each treatment, the mean initial pH and

the mean final pH should be within ± 0.3 pH units of the target pH. Test procedures for conducting toxicity

identification evaluations (TIEs) also recommend maintaining pH within ± 0.3 pH units in pH-controlled tests

(USEPA, 1996).

12.3.5.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing. Total

ammonia concentrations greater than 5 mg/L in the 100% effluent are an indicator that toxicity observed in the test

may be due to ammonia (USEPA, 1992). 

12.3.5.1.3 Results from both of the parallel tests (pH-controlled and uncontrolled treatments) must be reported to

the regulatory authority. If the uncontrolled test meets test acceptability criteria and shows no toxicity at the

permitted instream waste concentration, then the results from this test should be used for determining compliance. 

If the uncontrolled test shows toxicity at the permitted instream waste concentration, then the results from the pHcontrolled test should be used for determining compliance, provided that this test meets test acceptability criteria

and pH was properly controlled (see Subsection 12.3.5.1.1). 

12.3.5.1.4 To confirm that toxicity observed in the uncontrolled test was artifactual and due to pH drift, the results

of the controlled and uncontrolled-pH tests are compared. If toxicity is removed or reduced in the pH-controlled

treatment, artifactual toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of

artifactual toxicity is representative of a given effluent, the regulatory authority may require additional information

or additional parallel testing before pH control (as described in Subsection 12.3.5.2) is applied routinely to

subsequent testing of the effluent. 

12.3.5.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-controlled

atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding 1N NaOH or 1N HCl

(see Subsection 8.8.9). The addition of acids and bases should be minimized to reduce the amount of additional

ions (Na or Cl) added to the sample. pH is then controlled using the CO2-controlled atmosphere technique. This

may be accomplished by placing test solutions and test organisms in closed headspace test chambers, and then

injecting a predetermined volume of CO2 into the headspace of each test chamber (USEPA, 1991b; USEPA, 1992);
119

or by placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and air (USEPA, 1996). 

Prior experimentation will be needed to determine the appropriate CO2/air ratio or the appropriate volume of CO2 to

inject. This volume will depend upon the sample pH, sample volume, container volume, and sample constituents. 

If more than 5% CO2 is needed, adjust the solutions with acids (1N HCl) and then flush the headspace with no more

than 5% CO2 (USEPA, 1992). If the objective of the WET test is to determine the toxicity of the effluent in the

receiving water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH of the receiving

water (measured at the edge of the regulatory mixing zone). If the objective of the WET test is to determine the

absolute toxicity of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH

of the sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and Mount

(1992) provide techniques and guidance for controlling test pH using a CO2-controlled atmosphere. In pHcontrolled testing, control treatments must be subjected to all manipulations that sample treatments are subjected to. 

These manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-controlled

testing, the pH also must be measured in each treatment at the beginning and end of each 24-h exposure period to

confirm that pH was effectively controlled at the target pH level. 

12.4 SAFETY

12.4.1 See Section 3, Health and Safety. 

12.5 APPARATUS AND EQUIPMENT

12.5.1 Facilities for holding and acclimating test organisms. 

12.5.2 Sheepshead minnow culture unit -- see Subsection 12.6.12 below. To perform toxicity tests on-site or in the

laboratory, sufficient numbers of newly fertilized eggs must be available, preferably from an in-house sheepshead

minnow culture unit. If necessary, embryos can be obtained from outside sources if shipped in well oxygenated

water in insulated containers. 

12.5.2.1 A test using 15 embryos per test vessel and four replicates per concentration, will require 360

newly-fertilized embryos at the start of the test. A test with a minimum of 10 embryos per test vessel and three

replicates per concentration, and with five effluent concentrations and a control, will require a minimum of 180

embryos at the start of the test.

12.5.3 Brine Shrimp, Artemia, Culture Unit -- for feeding sheepshead minnow larvae in the continuous culture unit

(see Subsection 12.6.12 below). 

12.5.4 Samplers -- automatic sampler, preferably with sample cooling capability, that can collect a 24-h composite

sample of 5 L, and maintain sample temperature at 4EC. 

12.5.5 Environmental Chamber or Equivalent Facility with Temperature Control (25 ± 1EC). 

12.5.6 Water Purification System -- Millipore Milli-Q®, deionized water (DI) or equivalent. 

12.5.7 Balance -- analytical, capable of accurately weighing to 0.00001 g. Note: An analytical balance is not

needed for this test but is needed for other specified toxicity test methods with growth endpoints.

12.5.8 Reference Weights, Class S -- for checking the performance of the balance. The reference weights should

bracket the expected weights of reagents, and the expected weights of the weighing pans and the weights of the

weighing pans plus larvae. 

12.5.9 Air Pump -- for oil free air supply. 
120

12.5.10 Air Lines, and Air Stones -- for aerating water containing embryos, larvae, or supplying air to test solution

with low DO 

12.5.11 Meters, pH and DO -- for routine physical and chemical measurements. 

12.5.12 Standard or Micro-Winkler Apparatus -- for determining DO (optional). 

12.5.13 Dissecting microscope -- for examining embryos and larvae. 

12.5.14 Light box -- for counting and observing embryos and larvae. 

12.5.15 Refractometer -- for determining salinity. 

12.5.16 Thermometers, glass or electronic, laboratory grade -- for measuring water temperatures. 

12.5.17 Thermometers, bulb-thermograph or electronic-chart type -- for continuously recording temperature. 

12.5.18 Thermometer, National Bureau of Standards Certified (see USEPA Method 170.1, USEPA, 1979b) -- to

calibrate laboratory thermometers. 

12.5.19 Test Chambers -- four (minimum of three), borosilicate glass or non-toxic plastic labware per test

concentration. Care must be taken to avoid inadvertently removing embryos or larvae when test solutions are

decanted from the chambers. To avoid potential contanimation 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). The

covers are removed only for observation and removal of dead organisms. 

12.5.20 Beakers -- six Class A, borosilicate glass or non-toxic plasticware, 1000 mL for making test solutions. 

12.5.21 Wash Bottles -- for deionized water, for washing embryos from substrates and containers, and for rinsing

small glassware and instrument electrodes and probes. 

12.5.22 Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-toxic plastic labware,

10-1000 mL for making test solutions. 

12.5.23 Pipets, volumetric -- Class A, 1-100 mL. 

12.5.24 Pipets, automatic -- adjustable, 1-100 mL. 

12.5.25 Pipets, serological -- 1-10 mL, graduated. 

12.5.26 Pipet bulbs and fillers -- PROPIPET®, or equivalent. 

12.5.27 Droppers and glass tubing with fire polished aperatures, 4 mm ID -- for transferring embryos and larvae. 

12.5.28 Siphon with bulb and clamp -- for cleaning test chambers. 

12.5.29 NITEX® or stainless steel mesh sieves, (#150 μm, 500 μm, and 3-5 mm) -- for collecting Artemia nauplii

and fish embryos, and for spawning baskets, respectively. 
121

12.6 REAGENTS AND CONSUMABLE MATERIALS

12.6.1 Sample containers -- for sample shipment and storage (see Section 8, Effluent and Receiving Water

Sampling, Sample Handling, and Sample Preparation for Toxicity Tests). 

12.6.2 Data sheets (One set per test) -- for data recording (see Figure 1). 

12.6.3 Tape, colored -- for labelling test chambers. 

12.6.4 Markers, waterproof -- for marking containers, etc. 

12.6.5 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument manufacturer) -- for Standards and

Calibration Check (see USEPA Method 150.1, USEPA, 1979b). 

12.6.6 Membranes and filling solutions for dissolved oxygen probe (see USEPA Method 360.1, USEPA, 1979b),

or reagents -- for modified Winkler analysis. 

12.6.7 Laboratory quality assurance samples and standards -- for calibration of the above methods. 

12.6.8 Reference toxicant solutions -- see Section 4, Quality Assurance. 

12.6.9 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). 

12.6.10 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.

12.6.10.1 Saline test and dilution water -- The salinity of the test water must be in the range of 5 to 32‰. The

salinity should vary no more than ±2‰ among chambers on a given day. If effluent and receiving water tests are

conducted concurrently, the salinities of the water should be similar.

12.6.10.2 The overwhelming majority of industrial and sewage treatment effluents entering marine and estuarine

systems contain little or no measurable salts. Exposure of sheepshead minnow embryos to these effluents will

require adjustments in the salinity of the test solutions. It is important to maintain a constant salinity across all

treatments. In addition, it may be desirable to match the test salinity with that of the receiving water. Two methods

are available to adjust salinities -- a hypersaline brine derived from natural seawater or artificial sea salts.
122

Test Dates: Species: 

Type Effluent: Field: Lab: Test: 

Effluent Tested: 

Original pH: Salinity: DO: 

 CONCENTRATION:

 Replicate I:

DAY 0 1 2 3 4 5 6 7 8 9

#Live/Dead

 Embryo-Larvae

Terata

Temp. (EC)

Salinity (ppt)

DO (mg/L)

pH

 

CONCENTRATION:

 Replicate II:

DAY 0 1 2 3 4 5 6 7 8 9

#Live/Dead

 Embryo-Larvae

Terata

Temp. (EC)

Salinity (ppt)

DO (mg/L)

pH

Comments:

Note: Final endpoint for this test is total mortality (combined total number of dead embryos, dead larvae, and

deformed larvae) (see Subsection 12.10.9 and 12.13).

Figure 1. Data form for sheepshead minnow, Cyprinodon variegatus, embryo-larval survival/teratogenicity test. 

Daily record of embryo-larval survival/terata and test conditions.
123

 CONCENTRATION:

 Replicate III:

DAY 0 1 2 3 4 5 6 7 8 9

#Live/Dead

 Embryo-Larvae

Terata

Temp. (EC)

Salinity (ppt)

DO (mg/L)

pH

 CONCENTRATION:

 Replicate IV:

DAY 0 1 2 3 4 5 6 7 8 9

#Live/Dead

 Embryo-Larvae

Terata

Temp. (EC)

Salinity (ppt)

DO (mg/L)

pH

Comments:

Note: Final endpoint for this test is total mortality (combined total number of dead embryos, dead larvae, and

deformed larvae) (see Subsection 12.10.9 and 12.13). 

Figure 1. Data form for sheepshead minnow, Cyprinodon variegatus, embryo-larval survival/teratogenicity test. 

Daily record of embryo-larval survival/terata and test conditions (CONTINUED).
124

12.6.10.3 Hypersaline brine (HSB): HSB has several advantages that make it desirable for use in toxicity testing. 

It can be made from any high quality, filtered seawater by evaporation, and can be added to the effluent or to

deionized water to increase the salinity. HSB derived from natural seawater contains the necessary trace metals,

biogenic colloids, and some of the microbial components necessary for adequate growth, survival, and/or

reproduction of marine and estuarine organisms, and may be stored for prolonged periods without any apparent

degradation. However if 100% HSB is used as a diluent, the maximum concentration of effluent that can be tested

using HSB is limited to 80% at 20‰ salinity, and 70% at 30‰ salinity.

12.6.10.3.1 The ideal container for making HSB from natural seawater is one that (1) has a high surface to volume

ratio, (2) is made of a non-corrosive material, and (3) is easily cleaned (fiberglass containers are ideal). Special care

should be used to prevent any toxic materials from coming in contact with the seawater being used to generate the

brine. If a heater is immersed directly into the seawater, ensure that the heater materials do not corrode or leach any

substances that would contaminate the brine. One successful method used is a thermostatically controlled heat

exchanger made from fiberglass. If aeration is used, use only oil-free air compressors to prevent contamination. 

12.6.10.3.2 Before adding seawater to the brine generator, thoroughly clean the generator, aeration supply tube,

heater, and any other materials that will be in direct contact with the brine. A good quality biodegradable detergent

should be used, followed by several (at least three) thorough deionized water rinses. 

12.6.10.3.3 High quality (and preferably high salinity) seawater should be filtered to at least l0 μm before placing

into the brine generator. Water should be collected on an incoming tide to minimize the possibility of

contamination. 

12.6.10.3.4 The temperature of the seawater is increased slowly to 40EC. The water should be aerated to prevent

temperature stratification and to increase water evaporation. The brine should be checked daily (depending on

volume being generated) to ensure that salinity does not exceed 100‰ and that the temperature does not exceed

40EC. Additional seawater may be added to the brine to obtain the volume of brine required. 

12.6.10.3.5 After the required salinity is attained, the HSB should be filtered a second time through a 1 μm filter

and poured directly into portable containers (20 L) cubitainers or polycarbonate water cooler jugs are suitable. The

containers should be capped and labelled with the date the brine was generated and its salinity. Containers of HSB

should be stored in the dark and maintained at room temperature until used.

12.6.10.3.6 If a source of HSB is available, test solutions can be made by following the directions below. 

Thoroughly mix together the deionized water and brine before mixing in the effluent. 

12.6.10.3.7 Divide the salinity of the HSB by the expected test salinity to determine the proportion of deionized

water to brine. For example, if the salinity of the HSB is 100‰ and the test is to be conducted at 20‰, 100‰

divided by 20‰ = 5.0. The proportion of brine is one part in five (one part brine to four parts deionized water). To

make 1 L of seawater at 20‰ salinity from a HSB of 100‰, divide 1 L (1000 mL) by 5.0. The result, 200 mL, is

the quantity of HSB needed to make 1 L of sea water. The difference, 800 mL, is the quantity of deionized water

required. 

12.6.10.3.8 Table 1 illustrates the composition of test solutions at 20‰ if they are prepared by serial dilution of

effluent with 20‰ salinity seawater. 

12.6.10.4 Artificial sea salts: HW MARINEMIX® brand sea salts have been used successfully at the USEPA,

Region 6, Houston laboratory to culture sheepshead minnows and perform the embryo-larval survival and

teratogenicity test. EMSL-Cincinnati has found FORTY FATHOMS® artificial sea salts to be suitable for culturing

sheepshead minnows and for performing the larval survival and growth test and embryo-larval test. Artificial sea

salts may be used for culturing sheepshead minnows and for the embryo larval test if the criteria for acceptability of

test data are satisfied (see Subsection 12.11). 
125

12.6.10.4.1 Synthetic sea salts are packaged in plastic bags and mixed with deionized water or equivalent. The

instructions on the package of sea salts should be followed carefully, and salts should be mixed in a separate

container -- not the culture tank. The deionized water used in hydration should be in the temperature range of

21-26EC. Seawater made from artificial sea salts is conditioned (Spotte, 1973; Spotte et al., 1984; Bower, 1983)

before it is used for culturing or testing. After adding the water, place an airstone in the container, cover, and aerate

the solution mildly for at least 24 h before use. 

12.6.11 BRINE SHRIMP, ARTEMIA, CULTURE -- for feeding cultures. 

12.6.11.1 Newly-hatched Artemia nauplii are used as food in the sheepshead minnow culture, and a brine shrimp

culture unit should be prepared (USEPA, 2002a). 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.

12.6.11.2 Each new batch of Artemia cysts must be evaluated for size (Vanhaecke and Sorgeloos, 1980, and

Vanhaecke et al., 1980) and nutritional suitability (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. 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 organic chlorine pesticides exceeds 0.15 μg/g wet weight or the total

concentration of organochlorine pesticides plus PCBs exceeds 0.30 μg/g wet weight. (For analytical methods see

USEPA, 1982).
126

TABLE 1. PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 20‰, USING 20‰

NATURAL OR ARTIFICAL SEAWATER, HYPERSALINE BRINE, OR ARTIFICAL SEA

SALTS

 Solutions To Be Combined 

Effluent

Solution

Effluent

Conc.

(%)

Volume of

Effluent

Solution

Volume of Diluent

Seawater (20‰)

1 1001,2 4000 mL - - - 

2 50 2000 mL Solution 1 + 2000 mL 

3 25 2000 mL Solution 2 + 2000 mL 

4 12.5 2000 mL Solution 3 + 2000 mL 

5 6.25 2000 mL Solution 4 + 2000 mL 

Control 0.0 2000 mL 

Total 10000 mL 

1 This illustration assumes: (1) the use of 400 mL of test solution in each of four replicates and 400 mL for

chemical analysis (total of 2000 mL) for the control and five concentrations of effluent (2) an effluent dilution

factor of 0.5, and (3) the effluent lacks appreciable salinity. A sufficient initial volume (4000 mL) of effluent is

prepared by adjusting the salinity to the desired level. In this example, the salinity is adjusted by adding

artificial sea salts to the 100% effluent, and preparing a serial dilution using 20‰ seawater (natural seawater,

hypersaline brine, or artificial seawater). The salinity of the initial 4000 mL of 100% effluent is adjusted to

20‰ by adding 80 g of dry artificial sea salts (HW MARINEMIX or FORTY FATHOMS®), and mixing for 1

h. Test concentrations are then made by mixing appropriate volumes of salinity-adjusted effluent and 20‰

salinity dilution water to provide 4000 mL of solution for each concentration. If hypersaline brine alone

(100‰) is used to adjust the salinity of the effluent, the highest concentration of effluent that could be achieved

would be 80% at 20‰ salinity, and 70% at 30‰ salinity. 

2 The same procedures would be followed in preparing test concentrations at other salinities between 20‰ and

30‰: (1) The salinity of the bulk (initial) effluent sample would be adjusted to the appropriate salinity using

artificial sea salts or hypersaline brine, and (2) the remaining effluent concentrations would be prepared by

serial dilution, using a large batch (10 L) of seawater for dilution water, which had been prepared at the same

salinity as the effluent, using natural seawater, hypersaline and deionized water. 

12.6.11.3 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 of deionized water, to a 2 L separatory funnel, or equivalent. 

2. Add 10 mL Artemia cysts to the separatory funnel and aerate for 24 h at 27EC. (Hatching time varies

with incubation temperature and the geographic strain of Artemia used (see USEPA, 1985d, USEPA,

2002a; 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 five to 10 minutes. To prevent mortality, do

not leave the concentrated nauplii at the bottom of the funnel more than 10 minutes without aeration. 
127

4. Drain the nauplii into a beaker or funnel fitted with a #150 μm NITEX® or stainless steel screen, and

rinse with seawater or equivalent before use. 

12.6.11.4 Testing Artemia nauplii as food for toxicity test organisms. 

12.6.11.4.1 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 sheepshead minnow larvae. 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. 

12.6.11.4.2 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. 

12.6.11.4.3 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. 

12.6.11.4.4 The average seven-day survival of larvae should be 80% or greater, and (2) the average dry weight of

larvae should be 0.60 mg or greater, if dried and weighed immediately after the test, or (3) the average dry weight of

larvae should be 0.50 mg or greater, if the larvae are preserved in 4% formalin before drying and weighing. The

above minimum weights presume that the age of the larvae at the start of the test is not greater than 24 h.

12.6.12 TEST ORGANISMS, SHEEPSHEAD MINNOWS, CYPRINODON VARIEGATUS

12.6.12.1 Brood stock 

12.6.12.1.1 Adult sheepshead minnows for use as brood stock may be obtained by seine in Gulf of Mexico and

Atlantic coast estuaries, from commercial sources, or from young fish raised to maturity in the laboratory. Feral

brood stocks and first generation laboratory fish are preferred, to minimize inbreeding. 

12.6.12.1.2 To detect disease and to allow time for acute mortality due to the stress of capture, field-caught adults

are observed in the laboratory a minimum of two weeks before using as a source of gametes. Injured or diseased

fish are discarded. 

12.6.12.1.3 Sheepshead minnows can be continuously cultured in the laboratory from eggs to adults. The larvae,

juvenile, and adult fish should be kept in appropriate size rearing tanks, maintained at ambient laboratory

temperature. The larvae should be fed sufficient newly hatched Artemia nauplii daily to assure that live nauplii are

always present. Juveniles are fed frozen adult brine shrimp and a commercial flake food, such as TETRA SM-80®

or MARDEL AQUARIAN® Tropical Fish Flakes, or equivalent. Adult fish are fed flake food three or four times

daily, supplemented with frozen adult brine shrimp. 

12.6.12.1.3.1 Sheepshead minnows reach sexual maturity in three-to-five months after hatch, and have an average

standard length of approximately 27 mm for females and 34 mm for males. At this time, the males begin to exhibit

sexual dimorphism and initiate territorial behavior. When the fish reach sexual maturity and are to be used for

natural spawning, the temperature should be controlled at 18-20EC. 

12.6.12.1.4 Adults can be maintained in natural or artificial seawater in a flow-through or recirculating, aerated

system consisting of an all-glass aquarium, or equivalent. 

12.6.12.1.5 The system is equipped with an undergravel or outside biological filter of shells (see Spotte, 1973;

Bower, 1983) for conditioning the biological filter, or a cartridge filter, such as a MAGNUM® Filter, or an EHEIM®

Filter, or equivalent, at a salinity of 20-30‰ and a photoperiod of 16 h light/8 h dark. 
128

12.6.12.2 Obtaining Embryos for Toxicity Tests 

12.6.12.2.1 Embryos can be shipped to the laboratory from an outside source or obtained from adults held in the

laboratory. Ripe eggs can be obtained either by natural spawning or by intraperitoneal injection of the females with

human chorionic gonadotrophin (HCG) hormone. If the culturing system for adults is temperature controlled,

natural spawning can be induced. Natural spawning is preferred because repeated spawnings can be obtained from

the same brood stock, whereas with hormone injection, the brood stock is sacrificed in obtaining gametes. 

12.6.12.2.2 It should be emphasized that the injection and hatching schedules given below are to be used only as

guidelines. Response to the hormone varies from stock to stock and with temperature. Time to hatch and percent

hatch also vary among stocks and among batches of embryos obtained from the same stock, and are dependent on

temperature, DO, and salinity. 

12.6.12.2.3 Forced Spawning 

12.6.12.2.3.1 HCG is reconstituted with sterile saline or Ringer's solution immediately before use. The standard

HCG vial contains 1,000 IU to be reconstituted in 10 mL of saline. Freeze-dried HCG which comes with

premeasured and sterilized saline is the easiest to use. Use of a 50 IU dose requires injection of 0.05 mL of

reconstituted hormone solution. Reconstituted HCG may be used for several weeks if kept in the refrigerator. 

12.6.12.2.3.2 Each female is injected intraperitoneally with 50 IU HCG on two consecutive days, starting at least

four days prior to the beginning of a test. Two days following the second injection, eggs are stripped from the

females and mixed with sperm derived from excised macerated testes. At least 10 females and five males are used

per test to ensure that there is a sufficient number of viable embryos. 

12.6.12.2.3.3 HCG is injected into the peritoneal cavity, just below the skin, using as small a needle as possible. A

50 IU dose is recommended for females approximately 27 mm in standard length. A larger or smaller dose may be

used for fish which are significantly larger or smaller than 27 mm. With injections made on days one and two,

females which are held at 25EC should be ready for stripping on Day 4. Ripe females should show pronounced

abdominal swelling, and release at least a few eggs in response to a gentle squeeze. Injected females should be

isolated from males. It may be helpful if fish that are to be injected are maintained at 20EC before injection, and the

temperature raised to 25EC on the day of the first injection. 

12.6.12.2.3.4 Prepare the testes immediately before stripping the eggs from the females. Remove the testes from

three to five males. The testes are paired, dark grey organs along the dorsal midline of the abdominal cavity. If the

head of the male is cut off and pulled away from the rest of the fish, most of the internal organs can be pulled out of

the body cavity, leaving the testes behind. The testes are placed in a few mL of seawater until the eggs are ready. 

12.6.12.2.3.5 Strip the eggs from the females, into a dish containing 50-100 mL of seawater, by firmly squeezing

the abdomen. Sacrifice the females and remove the ovaries if all the ripe eggs do not flow out freely. Break up any

clumps of ripe eggs and remove clumps of ovarian tissue and underripe eggs. Ripe eggs are spherical,

approximately 1 mm in diameter, and almost clear.

12.6.12.2.3.6 While being held over the dish containing the eggs, the testes are macerated in a fold of NITEX®

screen (250-500 μm mesh) dampened with seawater. The testes are then rinsed with seawater to remove the sperm

from tissue, and the remaining sperm and testes are washed into the dish. Let the eggs and milt stand together for

10-15 minutes, swirling occasionally. 

12.6.12.2.3.7 Pour the contents of the dish into a beaker, and insert an airstone. Aerate gently, such that the water

moves slowly over the eggs, and incubate at 25EC for 60-90 minutes. After incubation, wash the eggs on a NITEX®

screen and resuspend them in clean seawater. 
129

12.6.12.2.4 Natural Spawning 

12.6.12.2.4.1 Short-term (Demand) Embryo Production 

12.6.12.2.4.1.1 Adult fish should be maintained at 18-20EC in a temperature controlled system. To obtain

embryos for a test, adult fish (generally, at least eight to 10 females and three males) are transferred to a spawning

chamber, with a photoperiod of 16 h light/8 h dark and a temperature of 25EC, two days before the beginning of the

test. The spawning chambers are approximately 20 x 35 x 22 cm high (USEPA, 1978), and consist of a basket of

3-5 mm NITEX® mesh, made to fit into a 57-L (15 gal) aquarium. Spawning generally will begin within 24 h or

less. The embryos will fall through the bottom of the basket and onto a collecting screen (250-500 μm mesh) or

tray below the basket. The collecting tray should be checked for embryos the next morning. The number of eggs

produced is highly variable. The number of spawning units required to provide the embryos needed to perform a

toxicity test is determined by experience. If the trays do not contain sufficient embryos after the first 24 h, discard

the embryos, replace the trays, and collect the embryos for another 24 h or less. To help keep the embryos clean,

the adults are fed while the screens are removed. 

12.6.12.2.4.1.2 The embryos are collected in a tray placed on the bottom of the tank. The collecting tray consists

of 250-500 μm NITEX® screen attached to a rigid plastic frame. The collecting trays with newly-spawned, embryos

are removed from the spawning tank, and the embryos are collected from the screens by washing them with a wash

bottle or removing them with a fine brush. The embryos from several spawning units may be pooled in a single

container to provide a sufficient number to conduct the test(s). The embryos are transferred into a petri dish or

equivalent, filled with fresh culture water, and are examined using a dissecting microscope or other suitable

magnifying device. Damaged and infertile eggs are discarded (see Figure 2). It is strongly recommended that the

embryos be obtained from fish cultured in-house, rather than from outside sources, to eliminate the uncertainty of

damage caused by shipping and handling that may not be observable, but which might affect the results of the test. 

12.6.12.2.4.1.3 After sufficient embryos are collected for the test, the adult fish are returned to the (18-20EC)

culture tanks. 

12.6.12.2.4.2 Sustained Natural Embryo Production 

12.6.12.2.4.2.1 Sustained (long-term), daily, embryo production can be achieved by maintaining mature fish in

tanks, such as a (285 L or 75 gal) LIVING STREAM® tank, at a temperature of 23-25EC. Embryos are produced

daily, and when needed, embryo "collectors" are placed on the bottom of the tank on the afternoon preceding the

start of the test. The next morning, the embryo collectors are removed and the embryos are washed into a shallow

glass culture dish using artificial seawater. 

12.6.12.2.4.2.2 Four embryo collectors, approximately 20 cm x 45 cm, will approximately cover the bottom of the

285 L tank. The collectors are fabricated from plastic fluorescent light fixture diffusors (grids), with cells

approximately 14 mm deep X 14 mm square. A screen consisting of 500 μm mesh is attached to one side (bottom)

of the grid with silicone adhesive. The depth and small size of the grid protects the embryos from predation by the

adult fish. 

12.6.12.2.4.2.3 The brood stock is replaced annually with feral stock. 

12.6.12.2.5 Test Organisms 

12.6.12.2.5.1 Embryos spawned over a less than 24-h period, are used for the test. These embryos may be used

immediately to start a test or may be placed in a suitable container and transported for use at a remote location. 

When overnight transportation is required, embryos should be obtained when they are no more than 8-h old. This

permits the tests at the remote site to be started with less than 24-h old embryos. Embryos should be transported or

shipped in clean, insulated containers, in well aerated or oxygenated fresh seawater or aged artificial sea water of

correct salinity, and should be protected from extremes of temperature and any other stressful conditions during
130

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, should be less than 2EC. Instantaneous

changes of pH, dissolved ions, osmotic strength, and DO should also be kept to a minimum. 

12.6.12.2.5.2 The number of embryos needed to start the test will depend on the number of tests to be conducted

and the objectives. If the test is conducted with four replicate test chambers (minimum of three) at each toxicant

concentration and in the control, with 15 embryos (minimum of 10) in each test chamber, and the combined

mortality of embryos prior to the start of the test is less than 20%, 400 viable embryos are required for the test. 

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. At estuarine and marine sites, samples

are usually collected at mid-depth. Receiving water toxicity is determined with samples used directly as collected or

with samples passed through a 60 μm NITEX® filter and compared without dilution, against a control. Using four

replicate chambers per test, each containing 400-500 mL, and 400 mL for chemical analysis, would require

approximately 2.0-2.5 L or more of sample per test per day. 

12.10.1.2 Effluents 

12.10.1.2.1 The selection of the effluent test concentration 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 ±100%, and allows for

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 dilution factors are increased beyond 0.5 and

declines rapidly if smaller dilution factors are used. Therefore, USEPA recommends the use of the $0.5 dilution

factor. If 100‰ salinity HSB is used as a diluent, the maximum concentration of effluent that can be tested will be

80% at 20‰ and 70% at 30‰ salinity.

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 1-2

h of the test, additional dilutions at the lower range of effluent concentrations should be added.

12.10.1.2.3 The volume of effluent required to initiate the test and for daily renewal of four replicates (minimum of

three) per concentration for five concentrations of effluent and a control, each containing 400 mL of test solution, is

approximately 4 L. Prepare enough test solution (approximately 3000 mL) at each effluent concentration to refill

the test chambers and provide at least 400 mL additional volume for chemical analyses. 
131

12.10.1.2.4 Maintain the effluent at 0-6EC. Plastic containers such as 8-20 L cubitainers have proven successful

for effluent collection and storage. 

12.10.1.2.5 Just prior to test initiation (approximately 1 h), the temperature of a sufficient quantity of the sample(s)

to make the test solutions should be adjusted to the test temperature (25 ± 1EC) and maintained at that temperature

during the addition of dilution water. 

12.10.1.2.6 Higher effluent concentrations (i.e., 25%, 50%, and 100%) may require aeration to maintain adequate

dissolved oxygen concentrations. However, if one solution is aerated, all concentrations must be aerated. Aerate

effluent as it warms and continue to gently aerate test solutions in the test chambers for the duration of the test. 

12.10.1.2.7 Effluent dilutions should be prepared for all replicates in each treatment in one beaker to minimize

variability among the replicates. The test chambers are labelled with the test concentration and replicate number. 

Dispense into the appropriate effluent dilution chamber.

12.10.1.3 Dilution Water

12.10.1.3.1 Dilution water may be uncontaminated natural seawater (receiving water), HSB prepared from natural

seawater, or artifical seawater prepared from FORTY FATHOMS® or GP2 sea salts (see Table 3 in Section 7,

Dilution Water). Other artificial sea salts may be used for culturing sheepshead minnows if the control criteria for

acceptability of test data are satisfied.

12.10.2 START OF THE TEST

12.10.2.1 Tests should begin as soon as possible, preferably within 24 h after sample collection. For on-site

toxicity studies, no more than 24 h should elapse between collection of the effluent and use in an embryo-larval

study. The maximum holding time following retrieval of the sample from the sampling device should not exceed 36

h for off-site toxicity studies 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). 

12.10.2.2 Label the test chambers with a marking pen. 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

concentration (including controls) is to have four replicates (minimum of three). Use 500 mL beakers,

crystallization dishes, nontoxic disposable plastic labware, or equivalent for test chambers. 

12.10.2.3 Prepare the test solutions (see Table 1) and add to the test chambers. 

12.10.2.4 Gently agitate and mix the embryos to be used in the test in a large container so that eggs from different

spawns are evenly dispersed. 

12.10.2.5 The test is started by randomly placing embryos from the common pool, using a small bore (2 mm), fire

polished, glass tube calibrated to contain approximately the desired number of embryos, into each of four replicate

test chamber, until each chamber contains 15 embryos (minimum of 10), for a total of 60 embryos (minimum of 30)

for each concentration (four replicates recommended, three minimum) (see Appendix A). The amount of water

added to the chambers when transferring the embryos 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 undamaged embryos. It may be more convenient and efficient to

transfer embryos to intermediate containers of dilution water for examination and counting. After the embryos have

been examined and counted in the intermediate container, assign them to the appropriate test chamber and transfer

them with a minimum of dilution water. 
132

12.10.2.7 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. 

12.10.3 LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE

12.10.3.1 The light quality and intensity should be at ambient laboratory levels, approximately 10-20 μE/m2

/s, or

50-100 foot candles (ft-c), with a photoperiod of 16 h of light and 8 h of darkness. The test water temperature

should be maintained at 25 ± 1EC. The salinity should be 5 to 32‰ ± 2‰ to accommodate receiving waters that

may fall within this range. The salinity should vary no more than ±2‰ among the chambers on a given day. If

effluent and receiving water tests are conducted concurrently, the salinities of these tests should be similar.

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. The DO should not fall below 4.0 mg/L (see Section 8, Effluent and Receiving Water Sampling,

Sample Holding, and Sample Preparation for Toxicity Tests). If it is necessary to aerate, all treatments and the

control should be aerated. The aeration rate should not exceed 100 bubbles/min, using a pipet with a 1-2 mm

orifice, such as a 1 mL KIMAX® Serological Pipet, or equivalent. Care should be taken to ensure that turbulence

resulting from the aeration does not cause undue physical stress to the fish. 

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 at each test concentration and

in the control. 

12.10.6.1.2 Temperature, pH, and salinity are measured at the end of each 24-h exposure period in 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 chambers at least at the end of the test to determine

the temperature variation in the environmental chambers. 

12.10.6.1.3 The pH is measured in the effluent sample each day before new test solutions are made.

12.10.6.1.4 Record all measurements on the data sheet (Figure 1).

12.10.6.2 Routine Biological Observations

12.10.6.2.1 At the end of the first 24 h of exposure, before renewing the test solutions, examine and count the

embryos. Remove the dead embryos (milky colored and opaque) and record the number. If the rate of mortality or

fungal infection exceeds 20% in the control chambers, or if excessive nonconcentration related mortality occurs,

terminate the test and start a new test with new embryos. If the above mortality conditions do not occur, continue

the test for the full nine days. 

12.10.6.2.2 At 25EC, hatching begins on about the sixth day. After hatching begins, count the number of dead and

live embryos and the number of hatched, dead, live, and deformed and/or debilitated larvae, daily (see Figure 2 for

illustrations of morphological development of embryo and larva). Deformed larvae are those with gross

morphological abnormalities such as curved spines, lack of appendages, lack of fusiform shape (non-distinct mass),
133

a colored beating heart in an opaque mass, lack of mobility, abnormal swimming, or other characteristics that

preclude survival. Remove dead embryos and dead and deformed larvae as previously discussed and record the

numbers for all test observations (see Figure 2). 

12.10.6.2.3 Protect the embryos and larvae from unnecessary disturbance during the test by carefully carrying out

the daily test observations, solution renewals, and removal of dead organisms. Make sure 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 tank causes a problem.

12.10.8 TEST SOLUTION RENEWAL

12.10.8.1 The test solutions are renewed daily using freshly prepared solution, immediately after cleaning the test

chambers. For on-site toxicity studies, fresh effluent and receiving water samples used in toxicity tests should be

collected daily, and no more than 24 h should elapse between collection of the sample and use in the test (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 must be collected, preferably on days 1, 3, and 5. Maintain the

samples at 0-6EC until used.
134

Figure 2 Embryonic development of sheepshead minnow, Cyprinodon variegatus: A. Mature unfertilized egg,

showing attachment filaments and micropyle, X33; B. Blastodisc fully developed; C,D. Blastodisc, 8

cells; E. Blastoderm, 16 cells; F. Blastoderm, late cleavage stage; G. Blastoderm with germ ring formed,

embryonic shield developing; H. Blastoderm covers over 3⁄4 of yolk, yolk noticeably constricted; I.

Early embryo. From Kuntz (1916).
135

Figure 2. Embryonic development of sheepshead minnow, Cyprinodon variegatus: J. Embryo 48 h after

fertilization, now segmented throughout, pigment on yolk sac and body, otoliths formed; K. 

Posterior portion of embryo free from yolk and moves freely within egg membrane, 72 h after

fertilization; L. Newly hatched fish, actual length 4 mm; M. Larval fish five days after hatching,

actual length 5 mm; N. Young fish 9 mm in length; O. Young fish 12 mm in length

(CONTINUED). From Kuntz (1916).
136

12.10.8.2 The test solutions are adjusted to the correct salinity and renewed daily using freshly collected samples. 

During the daily renewal process, 7-10 mm of water is left in the chamber to ensure that the embryos and larvae remain

submerged during the renewal process. New test solution (400 mL) should be added slowly by pouring down the side

of the test chamber to avoid exposing the embryos and larvae to excessive turbulence. 

12.10.8.3 Prepare test solutions daily, making a minimum of five concentrations and a control. If concurrent effluent

and receiving water testing occurs, the effluent test salinity should closely approximate that of the receiving water test. 

If an effluent is tested alone, select a salinity which approximately matches the salinity of the receiving waters. Table 1

illustrates the quantities of effluent, seawater, deionized water, and artificial sea salts needed to prepare 3 L of test

solution at each effluent concentration for tests conducted at 20‰ salinity. 

12.10.9 TERMINATION OF THE TEST

12.10.9.l The test is terminated after nine days of exposure, or four days post-hatch, whichever comes first. Count the

number of surviving, dead, and deformed and/or debilitated larvae, and record the numbers of each. The deformed

larvae are treated as dead. Keep a separate record of the total number of deformed larvae for use in reporting the

teratogenicity of the test solution. 

12.11 ACCEPTABILITY OF TEST RESULTS

12.11.1 For the test results to be acceptable, survival in the controls must be at least 80% or better. 

12.12 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

12.12.1 A summary of test conditions and test acceptability criteria is listed in Table 2. 

12.13 DATA ANALYSIS

12.13.1 General

12.13.1.1 Tabulate and summarize the data. 

12.13.1.2 The endpoints of this toxicity test are based on total mortality, combined number of dead embryos, dead

larvae, and deformed larvae. The EC endpoints are calculated using Probit Analysis (Finney, 1971). LOEC and

NOEC values, for total mortality, are obtained using a hypothesis test approach such as Dunnett's Procedure (Dunnett,

1955) or Steel's Many-one Rank Test (Steel, 1959; Miller, 1981). See the Appendices for examples of the manual

computations, program listings, and examples of data input and program output. 
137

TABLE 2. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE

SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EMBRYO-LARVAL SURVIVAL

AND TERATOGENICITY TEST WITH EFFLUENTS AND RECEIVING WATERS (TEST

METHOD 1005.0)1

1. Test type:

2. Salinity:

3. Temperature:

4. Light quality:

5. Light intensity:

6. Photoperiod:

7. Test chamber size:

8. Test solution volume:

9. Renewal of test solutions:

10. Age of test organisms:

11. No. of embryos per chamber:

12. No. replicate test chambers

per concentration:

13. No. embryos per concentration:

14. Feeding regime:

15. Aeration:

16. Dilution water:

Static renewal (required) 

5‰ to 32‰ (±2‰ of the selected test salinity)

(recommended) 

25 ± 1EC (recommended) 

Test temperatures must not deviate (i.e., maximum minus

minimum temperature) by more than 3EC during the test

(required) 

Ambient laboratory light (recommended) 

10-20 μE/m2

/s, or 50-100 ft-c (ambient laboratory levels)

(recommended) 

16 h light, 8 h darkness (recommended) 

400-500 mL (recommended) 

250-400 mL per replicate (loading and DO restrictions must

be met) (recommended) 

Daily (required)

Less than 24 h old (required) 

15 (recommended) 

10 (required minimum)

4 (recommended)

3 (required minimum) 

60 (recommended)

30 (required minimum)

Feeding not required

None unless DO falls below 4.0 mg/L (recommended) 

Uncontaminated source of natural seawater; deionized water

mixed with hypersaline brine or artificial sea salts (HW

MARINEMIX®, FORTY FATHOMS®, GP2, or equivalent)

(available options)

1

 For the purposes of reviewing WET test data submitted under NPDES permits, each test condition listed above is

identified as required or recommended (see Subsection 10.2 for more information on test review). Additional

requirements may be provided in individual permits, such as specifying a given test condition where several options

are given in the method. 
TABLE 2. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE

SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EMBRYO-LARVAL SURVIVAL

AND TERATOGENICITY TEST WITH EFFLUENTS AND RECEIVING WATERS (TEST

METHOD 1005.0)1

138

17. Test concentrations:

18. Dilution factor:

19. Test duration:

20. Endpoints:

21. Test acceptability criteria:

22. Sampling requirements:

23. Sample volume required:

Effluents: 5 and a control (required minimum) 

Receiving waters: 100% receiving water (or minimum of 5)

and a control (recommended) 

Effluent: $0.5 (recommended) 

Receiving waters: None, or $0.5 (recommended) 

9 days (required) 

Percent hatch; percent larvae dead or with debilitating

morphological and/or behavior abnormalities such as: gross

deformities; curved spine; disoriented, abnormal swimming

behavior; surviving normal larvae from original embryos

(required)

80% or greater survival in controls (required)

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 (e.g.,

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)

(required)

5 L per day (recommended) 

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 SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EMBRYOLARVAL SURVIVAL AND TERATOGENICITY DATA 

12.13.2.1 Formal statistical analysis of the total mortality data is outlined in Figure 3. 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% mortality in all of the test chambers are excluded from the statistical analysis of the NOEC and LOEC,

but included in the estimation of the EC endpoints. 

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-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
139

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.

12.13.2.4 Probit Analysis (Finney, 1971; see Appendix H) 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 H-K).

12.13.2.5 Example of Analysis of Survival Data

12.13.2.5.1 The data for this example are listed in Table 3. 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 described in Appendix B. The raw and transformed data, means and variences of the

transformed observations at each SDS concentration and control are listed in Table 3. A plot of the data is provided in

Figure 4. Since there is 100% mortality in all replicates for the 8.0 mg/L concentration, it is not included in this

statistical analysis and is considered a qualitative mortality effect.
140

Figure 3. Flowchart for statistical analysis of sheepshead minnow, Cyprinodon variegatus, embryo-larval

survival and teratogenicity test. Survival and terata data.
141

TABLE 3. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, EMBRYO-LARVAL TOTAL

MORTALITY DATA

SDS Concentration (mg/L)

Replicate Control 0.5 1.0 2.0 4.0 8.0

RAW

ARC SINE

TRANSFORMED

Mean ('Yi

)

S2

i

i

A

B

C

D

A

B

C

D

0.1

0.0

0.1

0.0

0.322

0.159

0.322

0.159

0.241

0.009

1

0.0

0.2

0.2

0.1

0.159

0.464

0.464

0.322

0.352

0.021

2

0.0

0.1

0.1

0.2

0.159

0.322

0.322

0.464

0.317

0.016

3

0.3

0.1

0.2

0.4

0.580

0.322

0.464

0.685

0.513

0.024

4

0.9

0.7

0.8

0.8

1.249

0.991

1.107

1.107

1.114

0.011

5

1.0

1.0

1.0

1.0

–

–

–

–

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.

TABLE 4: CENTERED OBSERVATIONS FOR SHAPIRO-WILK’S EXAMPLE

SDS Concentration (mg/L)

Replicate Control 0.5 1.0 2.0 4.0 8.0

A

B

C

D

0.081

-0.082

0.081

-0.082

-0.193

0.112

0.112

-0.030

-0.158

0.005

0.005

0.147

0.067

-0.191

-0.049

0.172

0.135

-0.123

-0.007

-0.007

–

–

–

–
142

Figure 4. Plot of Sheepshead minnow, Cyprinodon variegatus, total mortality data from the embryo-larval test
143

D ' j

n

i'1

(Xi

&X)  ̄ 2

X ̄ ' 1

20 (&0.005) ' 0.000

X (1)#X (2)#...#X (n)

12.13.2.6.2 Calculate the denominator, D, of the statistic:

Where: Xi = the ith centered observation

X ̄ = the overall mean of the centered observations

n = the total number of centered observations

12.13.2.6.3 For this set of data, n = 20

D = 0.2428

12.13.2.6.4 Order the centered observations from smallest to largest

Where: X(i) = the ith ordered observation

The ordered observations for this example are listed in Table 5.

TABLE 5. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

i X(i) i X(i)

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

-0.193

-0.191

-0.158

-0.123

-0.082

-0.082

-0.049

-0.030

-0.007

-0.007

11

12

13

14

15

16

17

18

19

20

0.005

0.005

0.067

0.081

0.081

0.112

0.112

0.135

0.147

0.172

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-1)/2 if n is odd. For the data in this example, n = 20 and k = 10. The ai

 values are listed in

Table 6.
144

W ' 1

D [j

k

i'1

ai (X (n&i%1) & X (i))]2

W ' 1

0.2428 (0.4807)2 ' 0.952

B '

[(j

P

i'1

Vi) 1n S ̄2 & j

P

i'1

Vi 1n S 2

i ]

C

TABLE 6. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK’S EXAMPLE

i ai X(n-i+1) - X(i)

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

0.365

0.338

0.293

0.295

0.194

0.163

0.130

0.097

0.012

0.012

X(20) - X(1)

X(19) - X(2)

X(18) - X(3)

X(17) - X(4)

X(16) - X(5)

X(15) - X(6)

X(14) - X(7)

X(13) - X(8)

X(12) - X(9)

X(11) - X(10)

12.13.2.6.6 Compute the test statistic, W, as follows:

The differences X(n-i+1) - X(i) are listed in Table 6. For the data in this example,

12.13.2.6.7 The decision rule for this test is to compare W as calculated in Section 12.13.2.6.6 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 = 0.952 is greater than the critical value, conclude that the data are normally distributed.

12.13.2.7 Test for Homogeneity of Variance

12.13.2.7.1 The test used to examine whether the variation in mean proportion mortality is the same across all toxicant

concentrations including the control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as follows:

Where: Vi = degrees of freedom for each copper concentration and control, Vi

 = (ni - 1)

p = number of concentration levels including the control

ln = loge
145

S ̄ 2 '

(j

p

i'1

ViSi)

2

j

p

i'1

Vi

C ' 1 % [3 (p&1)]&1 [j

p

i'1

i/vi & (j

p

i'1

vi)&1]

B '

(15) [ln (0.01262)]&3j

p

i'1

ln(S 2

i )

1.33

' 15 (&4.1227) & 3 (&20.9485)

1.33

' 0.886

i = 1, 2, ..., p where p is the number of concentrations including the control

ni = the number of replicates for concentration i

12.13.2.7.2 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.

12.13.2.7.3 For the data in this example, Vi

 = 3, p=5, 2 S ̄ = 0.0162, and C = 1.133. The calculated B value is:

12.13.2.7.4 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 = 0.886 is less than

the critical value of 13.277, conclude that the variances are not different. 

12.13.2.8 Dunnett's Procedure 

12.13.2.8.1 To obtain an estimate of the pooled variance for the Dunnett's Procedure, construct an ANOVA table as

described in Table 7.
146

SSB ' j

p

i'1

T 2

i /ni

&G 2

/N

SST ' j

p

i'1

j

ni

j'1

Y 2

ij&G 2

/N

SSW ' SST&SSB

TABLE 7. ANOVA TABLE

Source df Sum of Squares (SS)

Mean Square (MS)

(SS/df)

Between

Within

Total

p - 1

N - p

N - 1

SSB

SSW

SST

S = SSB/(p-1) 2

B

S = SSW/(N-p) 2

W

Where: p = number of concentration levels including the control

N = total number of observations n1 + n2 ... + np

ni = number of observations in concentration i 

Between Sum of Squares

Total Sum of Squares

 Within Sum of Squares

G = the grand total of all sample observations, G ' j

P

i'1

Ti

Ti = the total of the replicate measurements for concentration i

Yij = the jth observation for concentration i (represents the mean dry weight of the mysids for

concentration i in test chamber j)

12.13.2.8.2 For the data in this example:

n1 = n2 = n3 = n4 = n5 = 4 

N = 20
147

SSB ' j

p

i'1

T 2

i /ni

&G 2

/N

' 1

4 (28.561) & (10.143)2

20 ' 1.996

SST ' j

P

i'1

j

ni

j'1

Y 2

ij&G 2

/N

' 7.383 & (10.143)2

20 ' 1.996

SSW ' SST&SSB ' 2.239 & 1.996 ' 0.243

S 2

w ' SSB/(p&1) ' 1.996/(5&1) ' 0.499

S 2

w ' SSW/(N&p) ' 0.243/(20&5) ' 0.016

t

i ' (Y ̄1&Y ̄ i

)

Sw (1/n1) % (1/ni

)

T1 = Y11 + Y12 + Y13 + Y14 = 0.962 

T2 = Y21 + Y22 + Y23 + Y24 = 1.409 

T3 = Y31 + Y32 + Y33 + Y34 = 1.267 

T4 = Y41 + Y42 + Y43 + Y44 = 2.051 

T5 = Y51 + Y52 + Y53 + Y54 = 4.454 

G = T1 + T2 + T3 + T4 = 10.143

12.13.2.8.3 Summarize these calculations in the ANOVA table (Table 8).

TABLE 8. ANOVA TABLE FOR DUNNETT’S PROCEDURE EXAMPLE

Source df

Sum of Squares

(SS)

Mean Square (MS)

(SS/df)

Between

Within

Total

4 

15 

19 

1.996

0.243

2.239

0.499

0.016

12.13.2.8.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control

combination as follows: 
148

t

2 ' 0.352 & 0.241

[0.1265 (1/4) % (1/4)]

'1.241

MSD ' d Sw (1/n1) % (1/n)

Where: = mean proportion surviving for concentration i Y ̄ i

Y ̄ = mean proportion surviving for the control 1

Sw = square root of the within mean square

n1 = number of replicates for the control

ni = number of replicates for concentration i

Since we are looking for an increased response in percent of total mortality over control, the control mean is subtracted

from the mean at a concentration. 

12.13.2.8.5 Table 9 includes the calculated t values for each concentration and control combination. In this example,

comparing the 0.5 mg/L concentration with the control the calculation is as follows:

12.13.2.8.6 Since the purpose of this test is to detect a significant increase in total mortality, 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 proportion of total mortality for concentration "i" is considered significantly less than the mean proportion of

total mortality for the control if ti

 is greater than the critical value. Therefore, the 2.0 mg/L and the 4.0 mg/L

concentrations have significantly higher mean proportions of total mortality than the control. Hence the NOEC is 1.0

mg/L and the LOEC is 2.0 mg/L.

TABLE 9. CALCULATED T VALUES

SDS Concentration (mg/L) i ti

0.5

1.0

2.0

4.0

2

3

4

5

1.241

0.850

3.041

9.760

12.13.2.8.7 To quantify the sensitivity of the test, the minimum significant difference (MSD) that can be detected

statistically may be calculated.

Where: d = the critical value for Dunnett's procedure

SW = the square root of the within mean square
149

MSD ' 2.36 (0.1265) (1/4) % (1/4)

' 2.36 (0.1265) (0.7071)

' 0.211

n1 = the number of replicates in the control

n = The common number of replicates at each concentration (this assumes equal replication at each

concentration)

12.13.2.8.8 In this example:

12.13.2.8.9 The MSD (0.450) is in transformed units. To determine the MSD in terms of percent survival, carry out

the following conversion.

1. Add the MSD to the transformed control mean.

0.241 + 0.211 = 0.452 

2. Obtain the untransformed values for the control mean and the sum calculated in 1.

[ Sine (0.241) ]2

 = 0.057

[ Sine (0.452) ]2

 = 0.191

3. The untransformed MSD (MSDu) is determined by subtracting the untransformed values from

Step 2. 

MSDu = 0.191 - 0.057 = 0.134

12.13.2.8.10 Therefore, for this set of data, the minimum difference in mean proportion of total mortality between the

control and any SDS concentration that can be detected as statistically significant is 0.134. 

12.13.2.8.11 This represents a 268% increase in mortality from the control.

12.13.2.9 Calculation of the LC50 

12.13.2.9.1 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 H.
150

TABLE 10. DATA FOR PROBIT ANALYSIS

SDS Concentration (mg/L)

Control 0.5 1.0 2.0 4.0 8.0

Number Dead

Number Exposed

2 

40 

5 

40 

4 

40 

10 

40 

32

40

40

40

12.13.2.9.2 For this example, the chi-square test for heterogeneity was not significant. Thus Probit Analysis appears

appropriate for this set of data.

12.13.2.9.3 Figure 5 shows the output data for the Probit Analysis of the data from Table 10 using the USEPA Probit

Program.
151

USEPA PROBIT ANALYSIS PROGRAM

USED FOR CALCULATING LC/EC VALUES

Version 1.5

Probit Analysis of Sheepshead Minnow Embryo-Larval Survival and Teratogenicity Data

Conc.

Number

Exposed

Number

Resp.

Observed

Proportion

Responding

Proportion

Responding

Adjusted for

Controls

Control

0.5000

1.0000

2.0000

4.0000

8.0000

40

40

40

40

40

40

2 

5 

4 

10 

32 

40 

0.5000

0.1250

0.1000

0.2500

0.8000

1.0000

0.0000

0.0369

0.0094

0.1745

0.7799

1.0000

Chi - Square for Heterogeneity (calculated) = 0.782

Chi - Square for Heterogeneity (tabular value) = 7.815

Probit Analysis of Sheephead Minnow Embryo-Larval Survival and Teratogenicity Data

Estimated LC/EC Values and Confidence Limits

Point

Exposure

Conc.

Lower 95%

Confidence

Upper

Limits

LC/EC 1.00 

LC/EC 50.00 

1.187

2.912

0.643

2.432

1.601

3.361

Figure 5. Output for USEPA Probit Analysis Program, Version 1.5
152

12.14 PRECISION AND ACCURACY

12.14.1 PRECISION

12.14.1.1 Single-Laboratory Precision 

12.14.1.1.1 Data on the single-laboratory precision of the Sheepshead Minnow Embryo-larval Survival and

Teratogenicity test are available for eight tests with copper sulfate and five tests with sodium dodecyl sulfate (USEPA,

1989a). The data for the first five tests show that the same NOEC and LOEC, 240 μg Cu/L and 270 μg Cu/L,

respectively, were obtained in all five tests, which is the maximum level of precision that can be attained. Three

additional tests (6-8) were performed with narrower (20 μg) concentration intervals, to more precisely identify the

threshold concentration. The NOEC and LOEC for these tests are 200 μg and 220 μg Cu/L, respectively. For sodium

dodecyl sulfate, the NOEC's and LOEC's for all tests are 2.0 and 4.0 mg/L, respectively. The precision, expressed as

the coefficient of variation (CV%), is indicated in Tables 11-12. For copper (Cu), the coefficient of variation,

depending on the endpoint used, ranges from 2.5 to 6.1% which indicates excellent precision. For sodium dodecyl

sulfate (SDS), the coefficient of variation, depending on the endpoint used, ranges from 11.7 to 51.2%, indicating

acceptable precision. 

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.
153

TABLE 11. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW, CYPRINODON

VARIEGATUS, EMBRYO-LARVAL SURVIVAL AND TERATOGENICITY TEST 

PERFORMED IN HW MARINEMIX® ARTIFICIAL SEAWATER, USING EMBRYOS FROM

FISH MAINTAINED AND SPAWNED IN HW MARINEMIX® ARTIFICIAL SEAWATER

USING COPPER (CU) SULFATE AS REFERENCE TOXICANT1,2,3,4,5,6,7

Test

Number

EC1

(μg/L)

EC5

(μg/L)

EC10

(μg/L)

EC50

(μg/L)

NOEC

(μg/L)

1

2

3

4

5

6

7

8

n:

Mean:

CV (%):

173 

* 

* 

182 

171 

* 

* 

195 

4 

180 

6.1 

189 

* 

* 

197 

187 

* 

* 

203 

4 

194 

3.8 

198 

* 

* 

206 

197 

* 

* 

208 

4 

202 

2.8 

234 

* 

* 

240 

234 

* 

* 

226 

4 

233 

2.5 

240 

240 

240 

240 

240 

< 200 

220 

220 

7 

NA 

NA 

1 Data from USEPA (1988a) and USEPA (1991a). 2 Tests performed by Terry Hollister, Aquatic Biologist, Houston Facility, Environmental Services Division,

Region 6, USEPA, Houston, Texas. 3 Cyprinodon variegatus embryos used in the tests were less than 20 h old when the tests began. Two replicate test

chambers were used for the control and each toxicant concentration. Ten embryos were randomly added to each

test chamber containing 250 mL of test or control water. Solutions were renewed daily. The temperature and

salinity of the test solutions were 24 ± 1EC and 20‰, respectively. 4 Copper test concentrations were prepared using copper sulfate. Copper concentrations for Tests 1-5 were: 180,

210, 240, 270, and 300 μg/L. Copper concentrations for Test 6 were: 220, 240, 260, 280, and 300 μg/L. Copper

concentrations for Tests 7-8 were: 200, 220, 240, 260, and 280 μg/L. Tests were conducted over a two-week

period. 5 Adults collected in the field. 6 NOEC Range: 200-240 μg/L (this represents a difference of two exposure concentrations). 7 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 

* = Data did not fit the Probit model.
154

TABLE 12. SINGLE-LABORATORY PRECISION OF THE SHEEPSHEAD MINNOW, CYPRINODON

VARIEGATUS, EMBRYO-LARVAL SURVIVAL AND TERATOGENICITY TEST

PERFORMED IN HW MARINEMIX® ARTIFICIAL SEAWATER, USING EMBRYOS FROM

FISH MAINTAINED AND SPAWNED IN HW MARINEMIX® ARTIFICIAL SEAWATER

USING SODIUM DODECYL SULFATE (SDS) AS REFERENCE TOXICANT1,2,3,4,5,6,7

Test

Number

EC1

(mg/L)

EC5

(mg/L)

EC10

(mg/L)

EC50

(mg/L)

NOEC

(mg/L)

1

2

3

4

5

n:

Mean:

CV (%):

1.7

*

0.4

1.9

1.3

4

1.3

51.2

2.0

*

0.7

2.2

1.7

4

1.6

41.6

2.2

*

0.9

2.4

1.9

4

1.9

35.0

3.1

*

2.5

3.3

3.0

4

2.9

11.7

2.0

4.0

2.0

2.0

2.0

5

NA

NA

1 Data from USEPA (1988a) and USEPA (1991a). 2 Tests performed by Terry Hollister, Aquatic Biologist, Houston Facility, Environmental Services Division,

Region 6, USEPA, Houston, Texas. 3 Cyprinodon variegatus embryos used in the tests were less than 20 h old when the tests began. Two replicate test

chambers were used for the control and each toxicant concentration. Ten embryos were randomly added to each

test chamber containing 250 mL of test or control water. Solutions were renewed daily. The temperature and

salinity of the test solutions were 24 ± 1EC and 20‰, respectively. 4 SDS concentrations for all tests were: 0.5, 1.0, 2.0, 4.0, and 8.0 mg/L. Tests were conducted over a three-week

period. 5 Adults collected in the field. 6 NOEC Range: 2.0-4.0 mg/L (this represents a difference of two exposure concentrations). 7 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 

* = Data did not fit the Probit model.
155

 SECTION 13

TEST METHOD

INLAND SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL AND GROWTH

METHOD 1006.0

13.1 SCOPE AND APPLICATION 

13.1.1 This method estimates the chronic toxicity of effluents and receiving waters to the inland silverside,

Menidia beryllina, using seven to 11-day old 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 species. 

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, 96-h LC50s).

13.1.3 Detection limits of the toxicity of an effluent or chemical 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

volatile and highly degradable toxicants present in the source may not be detected in the test. 

13.1.5 This test 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 Inland silverside, Menidia beryllina, seven to 11-day old 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

growth of the larvae.

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).

13.3.2 Adverse effects of low dissolved oxygen (DO) concentrations, high concentrations of suspended and/or

dissolved solids, and extremes of pH, may mask or confound the effects of toxic substances. 

13.3.3 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). 

13.3.4 Pathogenic and/or predatory organisms in the dilution water and effluent may affect test organism survival,

and confound test results. 

13.3.5 Food added during the test may sequester metals and other toxic substances and confound test results. 

13.3.6 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-dependent

toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also increases (see Subsection

8.8.6), so upward pH drift may increase sample toxicity. For metals, toxicity may increase or decrease with
156

increasing pH. Lead and copper were found to be more acutely toxic at pH 6.5 than at pH 8.0 or 8.5, while nickel

and zinc were more toxic at pH 8.5 than at pH 6.5 (USEPA, 1992). In situations where sample toxicity is confirmed

to be artifactual and due to pH drift (as determined by parallel testing as described in Subsection 13.3.6.1), the

regulatory authority may allow for control of sample pH during testing using procedures outlined in Subsection

13.3.6.2. It should be noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large

(more than 1 pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold

for toxicity. 

13.3.6.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one with

controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to drift during the

test. In the controlled-pH treatment, the pH is maintained using the procedures described in Subsection 13.3.6.2. 

The pH to be maintained in the controlled-pH treatment (or target pH) will depend on the objective of the test. If

the objective of the WET test is to determine the toxicity of the effluent in the receiving water, the pH should be

maintained at the pH of the receiving water (measured at the edge of the regulatory mixing zone). If the objective

of the WET test is to determine the absolute toxicity of the effluent, the pH should be maintained at the pH of the

sample after adjusting the sample salinity for use in marine testing. 

13.3.6.1.1 During parallel testing, the pH must be measured in each treatment at the beginning (i.e., initial pH) and

end (i.e., final pH) of each 24-h exposure period. For each treatment, the mean initial pH (e.g., averaging the initial

pH measured each day for a given treatment) and the mean final pH (e.g., averaging the final pH measured each day

for a given treatment) must be reported. pH measurements taken during the test must confirm that pH was

effectively maintained at the target pH in the controlled-pH treatment. For each treatment, the mean initial pH and

the mean final pH should be within ± 0.3 pH units of the target pH. Test procedures for conducting toxicity

identification evaluations (TIEs) also recommend maintaining pH within ± 0.3 pH units in pH-controlled tests

(USEPA, 1996).

13.3.6.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing. Total

ammonia concentrations greater than 5 mg/L in the 100% effluent are an indicator that toxicity observed in the test

may be due to ammonia (USEPA, 1992). 

13.3.6.1.3 Results from both of the parallel tests (pH-controlled and uncontrolled treatments) must be reported to

the regulatory authority. If the uncontrolled test meets test acceptability criteria and shows no toxicity at the

permitted instream waste concentration, then the results from this test should be used for determining compliance. 

If the uncontrolled test shows toxicity at the permitted instream waste concentration, then the results from the pHcontrolled test should be used for determining compliance, provided that this test meets test acceptability criteria

and pH was properly controlled (see Subsection 13.3.6.1.1). 

13.3.6.1.4 To confirm that toxicity observed in the uncontrolled test was artifactual and due to pH drift, the results

of the controlled and uncontrolled-pH tests are compared. If toxicity is removed or reduced in the pH-controlled

treatment, artifactual toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of

artifactual toxicity is representative of a given effluent, the regulatory authority may require additional information

or additional parallel testing before pH control (as described in Subsection 13.3.6.2) is applied routinely to

subsequent testing of the effluent. 

13.3.6.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-controlled

atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding 1N NaOH or 1N HCl

(see Subsection 8.8.9). The addition of acids and bases should be minimized to reduce the amount of additional

ions (Na or Cl) added to the sample. pH is then controlled using the CO2-controlled atmosphere technique. This

may be accomplished by placing test solutions and test organisms in closed headspace test chambers, and then

injecting a predetermined volume of CO2 into the headspace of each test chamber (USEPA, 1991b; USEPA, 1992);

or by placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and air (USEPA, 1996). 

Prior experimentation will be needed to determine the appropriate CO2/air ratio or the appropriate volume of CO2 to

inject. This volume will depend upon the sample pH, sample volume, container volume, and sample constituents. 
157

If more than 5% CO2 is needed, adjust the solutions with acids (1N HCl) and then flush the headspace with no more

than 5% CO2 (USEPA, 1992). If the objective of the WET test is to determine the toxicity of the effluent in the

receiving water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH of the receiving

water (measured at the edge of the regulatory mixing zone). If the objective of the WET test is to determine the

absolute toxicity of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH

of the sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and Mount

(1992) provide techniques and guidance for controlling test pH using a CO2-controlled atmosphere. In pHcontrolled testing, control treatments must be subjected to all manipulations that sample treatments are subjected to. 

These manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-controlled

testing, the pH also must be measured in each treatment at the beginning and end of each 24-h exposure period to

confirm that pH was effectively controlled at the target pH level. 

13.4 SAFETY

13.4.1 See Section 3, Health and Safety.

13.5 APPARATUS AND EQUIPMENT

13.5.1 Facilities for holding and acclimating test organisms. 

13.5.2 Brine shrimp, Artemia, Culture Unit -- see Subsection 13.6.16 below and Section 4, Quality Assurance. 

13.5.3 Menidia Beryllina Culture Unit -- see Subsection 13.6.17 below, Middaugh and Hemmer (1984), Middaugh

et al. (1986), USEPA (1987g) and USEPA (2002a) for detailed culture methods. This test requires from 180-360 7

to 11 day-old larvae. It is preferable to obtain the test organisms from an in-house culture unit. If it is not feasible

to culture fish in-house, embryos or larvae can be obtained from other sources by shipping them in well oxygenated

saline water in insulated containers. 

13.5.4 Samplers -- automatic sampler, preferably with sample cooling capability, that can collect a 24-h composite

sample of 5 L. 

13.5.5 Environmental chamber or equivalent facility with temperature control (25 ± 1EC). 

13.5.6 Water purification system -- Millipore Milli-Q®, deionized water (DI) or equivalent. 

13.5.7 Balance, analytical -- capable of accurately weighing to 0.00001 g.

13.5.8 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 weighing pans plus fish. 

13.5.9 Drying oven -- 50-105EC range, for drying larvae. 

13.5.10 Air pump -- for oil-free air supply. 

13.5.11 Air lines, plastic or pasteur pipettes, or air stones -- for gently aerating water containing the fragile larvae

or for supplying air to test solution with low DO 

13.5.12 Meters, pH and DO -- for routine physical and chemical measurements. 

13.5.13 Standard or micro-Winkler apparatus -- for calibrating DO (optional). 

13.5.14 Desiccator -- for holding dried larvae. 
158

13.5.15 Light box -- for counting and observing larvae. 

13.5.16 Refractometer -- for determining salinity. 

13.5.17 Thermometers, glass or electronic, laboratory grade -- for measuring water temperatures. 

13.5.18 Thermometers, bulb-thermograph or electronic chart type -- for continuously recording temperature. 

13.5.19 Thermometer, National Bureau of Standards Certified (see USEPA Method 170.1, USEPA, 1979b) -- to

calibrate laboratory thermometers. 

13.5.20 Test chambers -- four chambers per concentration. The chambers should be borosilicate glass or nontoxic

disposable plastic labware. To avoid potential contamination from the air and excessive evaporation of test

solutions during the test, the chambers should be covered during the test with safety glass plates or sheet plastic (6

mm thick). 

Figure 1. Glass chamber with sump area. Modified from Norberg and Mount (1985). From USEPA (1987c).

13.5.20.l Each test chamber for the inland silverside should contain a minimum of 750 mL of test solution. A

modified Norberg and Mount (1985) chamber (Figure 1), constructed of glass and silicone cement, has been used

successfully for this test. This type of chamber holds an adequate column of test solution and incorporates a sump

area from which test solutions can be siphoned and renewed without disturbing the fragile inland silverside larvae. 

Modifications for the chamber are as follows: 1) 200 μm mesh NITEX® screen instead of stainless steel screen; and

2) thin pieces of glass rods cemented with silicone to the NITEX® screen to reinforce the bottom and sides to
159

produce a sump area in one end of the chamber. Avoid excessive use of silicone, while still ensuring that the

chambers do not leak and the larvae cannot get trapped or escape into the sump area. Once constructed, check the

chambers for leaks and repair if necessary. Soak the chambers overnight in seawater (preferably in flowing water)

to cure the silicone cement before use. Other types of glass test chambers, such as the 1000 mL beakers used in the

short-term Sheepshead Minnow Larval Survival and Growth Test, may be used. It is recommended that each

chamber contain a minimum of 50 mL per larvae and allow adequate depth of test solution (5.0 cm). 

13.5.21 Beakers -- six Class A, borosilicate glass or non-toxic plasticware, 1000 mL for making test solutions. 

13.5.22 Mini-Winkler bottles -- for dissolved oxygen calibrations. 

13.5.23 Wash bottles -- for deionized water, for washing embryos from substrates and containers, and for rinsing

small glassware and instrument electrodes and probes. 

13.5.24 Crystallization dishes, beakers, culture dishes, or equivalent -- for incubating embryos. 

13.5.25 Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-toxic plastic labware,

10-1000 mL for making test solutions. 

13.5.26 Separatory funnels, 2 L -- Two - four for culturing Artemia. 

13.5.27 Pipets, volumetric -- Class A, 1-100 mL. 

13.5.28 Pipets, automatic -- adjustable, 1-100 mL. 

13.5.29 Pipets, serological -- 1-10 mL, graduated. 

13.5.30 Pipet bulbs and fillers -- PROPIPET®, or equivalent. 

13.5.31 Droppers, and glass tubing with fire polished edges, 4 mm ID -- for transferring larvae. 

13.5.32 Siphon with bulb and clamp -- for cleaning test chambers. 

13.5.33 Forceps -- for transferring dead larvae to weighing pans. 

13.5.34 NITEX® Mesh Sieves (# 150 μm, 500 μm, 3-5 mm) -- for collecting Artemia nauplii and fish larvae. 

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 data recording.

13.6.3 Tape, colored -- for labelling test chambers. 

13.6.4 Markers, waterproof -- for marking containers, etc. 

13.6.5 Vials, marked -- 24/test, containing 4% formalin or 70% ethanol, to preserve larvae (optional).

13.6.6 Weighing pans, aluminum -- 26/test (two extra). 
160

13.6.7 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument manufacturer) for standards and

calibration check (see USEPA Method 150.1, USEPA, 1979b). 

13.6.8 Membranes and filling solutions for DO probe (see USEPA Method 360.1, USEPA, 1979b), or reagents --

for modified Winkler analysis. 

13.6.9 Laboratory quality assurance samples and standards -- for the above methods. 

13.6.10 Reference toxicant solutions -- see Section 4, Quality Assurance.

13.6.11 Ethanol (70%) or formalin (4%) -- for use as a preservative for the fish larvae. 

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, receiving water, and dilution water -- see Section 7, Dilution Water; and Section 8, Effluent and

Surface Water Sampling, Sample Handling, and Sample Preparation for Toxicity Tests. 

13.6.13.1 Saline test and dilution water -- the salinity of the test water must be in the range of 5 to 32‰. The

salinity should vary by no more than ±2‰ among the chambers on a given day. If effluent and receiving water tests

are conducted concurrently, the salinities of these tests should be similar. 

13.6.13.2 The overwhelming majority of industrial and sewage treatment effluents entering marine and estuarine

systems contain little or no measurable salts. Exposure of Menidia beryllina larvae to these effluents will require

adjustments in the salinity of the test solutions. It is important to maintain a constant salinity across all treatments. 

In addition, it may be desirable to match the test salinity with that of the receiving water. Artificial sea salts or

hypersaline brine (100‰) derived from natural seawater may be used to adjust the salinities. 

13.6.13.3 Hypersaline brine (HSB): HSB has several advantages that make it desirable for use in toxicity testing. 

It can be made from any high quality, filtered seawater by evaporation, and can be added to the effluent or to

deionized water to increase the salinity. HSB derived from natural seawater contains the necessary trace metals,

biogenic colloids, and some of the microbial components necessary for adequate growth, survival, and/or

reproduction of marine and estuarine organisms, and may be stored for prolonged periods without any apparent

degradation. However, if 100% HSB is used as a diluent, the maximum concentration of effluent that can be tested

will be 70% at 30‰ salinity and 80% at 20‰ salinity.

13.6.13.3.1 The ideal container for making HSB from natural seawater is one that (1) has a high surface to volume

ratio, (2) is made of a noncorrosive material, and (3) is easily cleaned (fiberglass containers are ideal). Special care

should be used to prevent any toxic materials from coming in contact with the seawater being used to generate the

brine. If a heater is immersed directly into the seawater, ensure that the heater materials do not corrode or leach any

substances that would contaminate the brine. One successful method used is a thermostatically controlled heat

exchanger made from fiberglass. If aeration is used, use only oil free air compressors to prevent contamination. 

13.6.13.3.2 Before adding seawater to the brine generator, thoroughly clean the generator, aeration supply tube,

heater, and any other materials that will be in direct contact with the brine. A good quality biodegradable detergent

should be used, followed by several (at least three) thorough deionized water rinses.

13.6.13.3.3 High quality (and preferably high salinity) seawater should be filtered to at least 10 μm before placing

into the brine generator. Water should be collected on an incoming tide to minimize the possibility of

contamination. 

13.6.13.3.4 The temperature of the seawater is increased slowly to 40EC. The water should be aerated to prevent

temperature stratification and to increase water evaporation. The brine should be checked daily (depending on
161

volume being generated) to ensure that salinity does not exceed 100‰ and that the temperature does not exceed

40EC. Additional seawater may be added to the brine to obtain the volume of brine required. 

13.6.13.3.5 After the required salinity is attained, the HSB should be filtered a second time through a 1 μm filter

and poured directly into portable containers (20 L cubitainers or polycarbonate water cooler jugs are suitable). The

containers should be capped and labelled with the date the brine was generated and its salinity. Containers of HSB

should be stored in the dark and maintained at room temperature until used. 

13.6.13.3.6 If a source of HSB is available, test solutions can be made by following the directions below. 

Thoroughly mix together the deionized water and brine before mixing in the effluent. 

13.6.13.3.7 Divide the salinity of the HSB by the expected test salinity to determine the proportion of deionized

water to brine. For example, if the salinity of the HSB is 100‰ and the test is to be conducted at 20‰, 100‰

divided by 20‰ = 5.0. The proportion of brine is one part in five (one part brine to four parts deionized water). To

make 1 L of seawater at 20‰ salinity from a HSB of 100‰, divide 1 L (1000 mL) by 5.0. The result, 200 mL, is

the quantity of HSB needed to make 1 L of seawater. The difference, 800 mL, is the quantity of deionized water

required. 

13.6.13.3.8 Table 1 illustrates the composition of test solutions at 20‰ if they are made by combining effluent

(0‰), deionized water and HSB at 100‰ salinity. The volume (mL) of brine required is determined by using the

amount calculated above. In this case, 200 mL of brine is required for 1 L; therefore, 600 mL would be required for

3 L of solution. The volumes of HSB required are constant. The volumes of deionized water are determined by

subtracting the volumes of effluent and brine from the total volume of solution: 3,000 mL - mL effluent - mL HSB

= mL deionized water. 

13.6.13.4 Artificial sea salts: A modified GP2 artificial seawater formulation (Table 2) has been successfully used

to perform the inland silverside survival and growth test. The use of GP2 for holding and culturing of adults is not

recommended at this time.

13.6.13.4.1 The GP2 artificial sea salts (Table 2) should be mixed with deionized (DI) water or its equivalent in a

container other than the culture or testing tanks. The deionized water used for hydration should be between 21-

26EC. The artificial seawater must be conditioned (aerated) for 24-h before use as the testing medium. If the

solution is to be autoclaved, sodium bicarbonate is added after the solution has cooled. A stock solution of sodium

bicarbonate is made up by dissolving 33.6 gm NaHCO3 in 500 mL deionized water. Add 2.5 mL of this stock

solution for each liter of the GP2 artificial seawater.

13.6.14 ROTIFER CULTURE --for feeding cultures and test organisms

13.6.14.1 At hatching Menidia beryllina larvae are too small to ingest Artemia nauplii and must be fed rotifers,

Brachionus plicatilis. The rotifers can be maintained in continuous culture when fed algae (see Section 6 and

USEPA, 1987g). Rotifers are cultured in 10-15 L Pyrex® carboys (with a drain spigot near the bottom) at 25-28EC

and 25-35‰ salinity. Four 12 L culture carboys should be maintained simultaneously to optimize production. 

Clean carboys should be filled with autoclaved seawater. Alternatively, an immersion heater may be used to heat

saline water in the carboy to 70-80EC for 1-h. 
162

TABLE 1: PREPARATION OF 3 L SALINE WATER FROM DEIONIZED WATER AND A

HYPERSALINE BRINE OF 100‰ NEEDED FOR TEST SOLUTIONS AT 20‰ SALINITY

Effluent

Concentration

Volume of

Effluent

(0‰)

(mL)

Volume of

Deionized

Water

(mL)

Volume of

Hypersaline

Brine

(mL)

Total

Volume

(mL)

80 

40 

20 

10 

5 

Control 

2400

1200

600

300

150

0

0

1200

1800

2100

2250

2400

600

600

600

600

600

600

3000

3000

3000

3000

3000

3000

Total 4,650 9,750 3,600 18,000
163

TABLE 2. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2 ARTIFICIAL

SEAWATER FOR THE INLAND SILVERSIDE, MENIDIA BERYLLINA, TOXICITY

TEST1,2,3

Compound

Concentration

(g/L)

Amount (g)

Required for

20 L

NaCl

Na2SO4

KCl

KBr

Na2B4O7·10 H2O

MgCl2·6 H2O

CaCl2·2 H2O

SrCl2·6 H2O

NaHCO3

21.03

3.52

0.61

0.088

0.034

9.50

1.32

0.02

0.17

420.6

70.4

12.2

1.76

0.68

190.0

26.4

0.400

3.40

1 Modified GP2 from Spotte et al. (1984) 2 The constituent salts and concentrations were taken from USEPA (l990b). The salinity is 30.89 g/L. 3 GP2 can be diluted with deionized (DI) water to the desired test salinity.

13.6.14.2 When the water has cooled to 25-28°C, aerate and add a start-up sample of rotifers (50 rotifers/mL) and

food (about 1 L of a dense algal culture). The carboys should be checked daily to ensure that adequate food is

available and that the rotifer density is adequate. If the water appears clear, drain 1 L of culture water and replace it

with algae. Excess water can be removed through the spigot drain and filtered through a # 60 μm mesh screen. 

Rotifers collected on the screen should be returned to the culture. If a more precise measure of the rotifer

population is needed, rotifers collected from a known volume of water can be resuspended in a smaller volume,

killed with formalin and counted in a Sedgwick-Rafter cell. If the density exceeds 50 rotifers/mL, the amount of

food per day should be increased to 2 L of algae suspension. The optimum density of approximately 300-400

rotifers/mL may be reached in seven to 10 days and is sustainable for two to three weeks. At these densities, the

rotifers should be cropped daily. Keeping the carboys away from light will reduce the amount of algae attached to

the carboy walls. When detritus accumulates, populations of ciliates, nematodes, or harpacticoid copepods that may

have been inadvertently introduced can rapidly take over the culture. If this occurs, discard the cultures. 

13.6.15 ALGAL CULTURES -- for feeding rotifer cultures

13.6.15.1 Tetraselmus suecica or Chlorella sp. (see USEPA, 1987a) can be cultured in 20 L polycarbonate carboys

that are normally used for bottled drinking water. Filtered seawater is added to the carboys and then autoclaved

(110EC for 30 minutes). After cooling to room temperature, the carboys are placed in a temperature chamber

controlled at 18-20EC. One liter of T. suecica or Chlorella sp. starter culture and 100 mL of nutrients are added to

each carboy. 
164

13.6.15.2 Formula for algal culture nutrients. 

13.6.15.2.1 Add 180 g NaNO3, 12 g NaH2PO4, and 6.16 g EDTA to 12 L of deionized water. Mix with a magnetic

stirrer until all salts are dissolved (at least 1-h). 

13.6.15.2.2 Add 3.78 g FeCl3·6 H2O and stir again. The solution should be bright yellow. 

13.6.15.2.3 The algal culture is vigorously aerated via a pipette inserted through a foam stopper at the top of the

carboy. A dense algal culture should develop in 7 to 10 days and should be used by Day 14. Thus, start-up of

cultures should be made on a daily or every second day basis. Approximately 6 to 8 continuous cultures will meet

the feeding requirements of four 12 L rotifer cultures. When emptied, carboys are washed with soap and water and

rinsed thoroughly with deionized water before reuse.

13.6.16 BRINE SHRIMP, ARTEMIA, NAUPLII -- for feeding cultures and test organisms 

13.6.16.1 Newly hatched Artemia nauplii are used as food for inland silverside larvae in toxicity tests. Although

there are many commercial sources of brine shrimp cysts, the Brazilian or Colombian strains are being used because

the supplies examined have had low concentrations of chemical residues and produce nauplii of suitably small size. 

13.6.16.2 Each new batch of Artemia cysts must be evaluated for size (Vanhaecke and Sorgeloos, 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. 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 pesticides exceeds 0.15 μg/g wet weight or that the

total concentration of organochlorine pesticides plus PCBs does not exceed 0.30 μg/g wet weight. (For analytical

methods, see USEPA 1982). 

13.6.16.2.1 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 of deionized water, to a 2 L separatory funnel or equivalent. 

2. Add 10 mL Artemia cysts to the separatory funnel and aerate for 24 h at 27EC. (Hatching time varies

with incubation temperature and the geographic strain of Artemia used (see USEPA, 1985d; USEPA,

2002a; 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 10-15 minutes to prevent mortality, do not

leave the concentrated nauplii at the bottom of the funnel more than 10 minutes without aeration. 

4. Drain the nauplii into a beaker or funnel fitted with # 150 μm NITEX® or stainless steel screen, and

rinse with seawater or equivalent before use. 

13.6.16.3 Testing Artemia nauplii as food for toxicity test organisms. 

13.6.16.3.1 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 inland silverside larvae (see Subsection 13.11). 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 chambers each containing a minimum of 15 larvae, for each type of

food.

13.6.16.3.2 The feeding rate and frequency, test vessels and 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. 
165

13.6.16.3.3 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. 

13.6.16.4 Use of Artemia nauplii as food for inland silverside, Menidia beryllina, larvae. 

13.6.16.4.1 Menidia beryllina larvae begin feeding on newly hatched Artemia nauplii about five days after

hatching, and are fed Artemia nauplii daily throughout the 7-day larval survival and growth test. Survival of

Menidia beryllina larvae seven to nine days old is improved by feeding newly hatched (< 24-h old) Artemia nauplii. 

Equal amounts of Artemia nauplii must be fed to each replicate test chamber to minimize the variability of larval

weight. Sufficient numbers of nauplii should be fed to ensure that some remain alive overnight in the test chambers. 

An adequate but not excessive amount should be provided to each replicate on a daily basis. Feeding excessive

amounts of nauplii will result in a depletion in DO to below an acceptable level (below 4.0 mg/L). As much of the

uneaten Artemia nauplii as possible should be siphoned from each chamber prior to test solution renewal to ensure

that the larvae principally eat newly hatched nauplii. 

13.6.17 TEST ORGANISMS, INLAND SILVERSIDE, MENIDIA BERYLLINA

13.6.17.1 The inland silverside, Menidia beryllina, is one of three species in the atherinid family that are amenable

to laboratory culture; and one of four atherinid species used for chronic toxicity testing. Several atherinid species

have been utilized successfully for early life stage toxicity tests using field collected (Goodman et al., 1985) and

laboratory reared adults (Middaugh and Takita, 1983; Middaugh and Hemmer, 1984; and USEPA, 1987g). The

inland silverside, Menidia beryllina, populates a variety of habitats from Cape Cod, Massachusetts, to Florida and

west to Vera Cruz, Mexico (Johnson, 1975). It can tolerate a wide range of temperature, 2.9-32.5EC (Tagatz and

Dudley, 1961; Smith, 1971) and salinity, of 0-58‰ (Simmons, 1957; Renfro, 1960), having been reported from the

freshwaters of the Mississippi River drainage basin (Chernoff et al., 1981) to hypersaline lagoons (Simmons, 1957). 

Ecologically, Menidia spp. are important as major prey for many prominent commercial species (e.g., bluefish

(Pomatomus saltatrix), mackerel (Scomber scombrus), and striped bass (Morone saxatilis) (Bigelow and Schroeder,

1953). The inland silverside, Menidia beryllina, is a serial spawner, and will spawn under controlled laboratory

conditions. Spawning can be induced by diurnal interruption in the circulation of water in the culture tanks

(Middaugh et al., 1986; USEPA, 1987a). The eggs are demersal, approximately 0.75 mm in diameter (Hildebrand

and Schroeder, 1928), and adhere to vegetation in the wild, or to filter floss in laboratory culture tanks. The larvae

hatch in six to seven days when incubated at 25EC and maintained in seawater ranging from 5-30‰ (USEPA,

1987a). Newly hatched larvae are 3.5-4.0 mm in total length (Hildebrand, 1922).

13.6.17.2 Inland silverside, Menidia beryllina, adults (see USEPA, 1987g and USEPA, 2002a for detailed culture

methods) may be cultured in the laboratory or obtained from the Gulf of Mexico or Atlantic coast estuaries

throughout the year (Figure 2). Gravid females can be collected from low salinity waters along the Atlantic coast

during April to July, depending on the latitude. The most productive and protracted spawning stock can be obtained

from adults brought into the laboratory. Broodstocks, collected from local estuaries twice each year (in April and

October), will become sexually active after one to two months and will generally spawn for 4-6 months. 

13.6.17.3 The fish can be collected easily with a beach seine (3-6 mm mesh), but the seine should not be

completely landed onto the beach. Silversides are very sensitive to handling and should never be removed from the

water by net -- only by beaker or bucket. 

13.6.17.4 Samples may contain a mixture of inland silverside, Menidia beryllina, and Atlantic silverside, Menidia

menidia, on the Atlantic coast or inland silverside and tidewater silverside, Menidia peninsulae, on the Gulf Coast

(see USEPA, 1987g for additional information on morphological differences for identification). Johnson (1975)

and Chernoff et al. (1981) have attempted to differentiate these species. In the northeastern United States, M.

beryllina juveniles and adults are usually considerably smaller than M. menidia juveniles and adults (Bengtson,

1984), and can be separated easily in the field on that basis. 
166

13.6.17.5 Record the water temperature and salinity at each collection site. Aerate (portable air pump, battery

operated) the fish and transport to the laboratory as quickly as possible after collection. Upon arrival at the

laboratory, the fish and the water in which they were collected are transferred to a tank at least 0.9 m in diameter. A

filter system should be employed to maintain water quality (see USEPA, 1987g). Laboratory water is added to the

tank slowly, and the fish are acclimated at the rate of 2EC per day, to a final temperature of 25°C, and about 5‰

salinity per day, to a final salinity in the range of 20-32‰. The seawater in each tank should be brought to a

minimum volume of 150 L. A density of about 50 fish/tank is appropriate. Maintain a photoperiod of 16 h light/8 h

dark. Feed the adult fish flake food or frozen brine shrimp twice daily and Artemia nauplii once daily. Siphon the

detritus from the bottom of the tanks weekly. 

13.6.17.6 Larvae for a toxicity test can be obtained from the broodstock by spawning onto polyester aquarium

filter-fiber substrates, 15 cm long x 10 cm wide x 10 cm thick, which are suspended with a string 8-10 cm below the

surface of the water and in contact with the side of the holding tanks for 24-48 h, 14 days prior to the beginning of a

test. The floss should be gently aerated by placing it above an airstone, and weighted down with a heavy non-toxic

object. The embryos, which are light yellow in color, can be seen on the floss, and are round and hard to the touch

compared to the soft floss. 

13.6.17.7 Remove as much floss as possible from the embryos. The floss should be stretched and teased to prevent

the embryos from clumping. The embryos should be incubated at the test salinity and lightly aerated. At 25EC, the

embryos will hatch in about six to eight days. Larvae are fed about 500 rotifer larvae/day from hatch through four

days post-hatch. On Days 5 and 6, newly hatched (less than 12 h old) Artemia nauplii are mixed with the rotifers, to

provide a transition period. After Day 7, only nauplii are fed, and the age range for the nauplii can be increased

from 12 h old to 24 h old.

168

13.6.17.8 Silverside larvae are very sensitive to handling and shipping during the first week after hatching. For

this reason, if organisms must be shipped to the test laboratory, it may be impractical to use larvae less than 11 days

old because the sensitivity of younger organisms may result in excessive mortality during shipment. If organisms

are to be shipped to a test site, they should be shipped only as (1) early embryos, so that they hatch after arrival, or

(2) after they are known to be feeding well on Artemia nauplii (8-10 days of age). Larvae shipped at 8 - 10 days of

age would be 9 to 11 days old when the test is started. Larvae that are hatched and reared in the test laboratory can

be used at seven days of age.

13.6.17.9 If four replicates of 15 larvae are used at each effluent concentration and in the control, 360 larvae will

be needed for each test. 

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. At estuarine and marine sites, samples

are usually collected at mid-depth. Receiving water toxicity is determined with samples used directly as collected or

with samples passed through a 60 μm NITEX® filter and compared without dilution, against a control. Using four

replicate chambers per test, each containing 500-750 mL, and 400 mL for chemical analysis, would require

approximately 2.4-3.4 L or more of sample per day. 

13.10.1.2 Effluents 

13.10.1.2. 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 ±100%, and allows for

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 dilution factors are increased beyond 0.5 and

declines rapidly if smaller dilution factors are used. Therefore, USEPA recommends the use of the $0.5 dilution

factor. If 100% salinity HSB is used as a diluent, the maximum concentration of effluent that can be tested will be

80% at 20‰ salinity, and 70% at 30‰ salinity.

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 1-2

h of the test, additional dilutions at the lower range of effluent concentrations should be added. 

13.10.1.2.3 The volume of effluent required to initiate the test and for daily renewal of four replicates per

treatment for five concentrations of effluent and a control, each containing 750 mL of test solution, is approximately
169

5 L. Prepare enough test solution at each effluent concentration to provide 400 mL additional volume for chemical

analyses. 

13.10.1.2.4 Tests should begin as soon as possible after sample collection, preferably within 24 h. The maximum

holding time following retrieval of the sample from the sampling device should not exceed 36 h for off-site toxicity

studies unless permission is granted by the permitting authority. In no case should the test be started more than 72 h

after sample collection (see Section 8, Effluent and Receiving Water Sampling, Sample Handling, and Sample

Preparation for Toxicity Tests, Subsection 8.5.4).

13.10.1.2.5 Just prior to test initiation (approximately 1 h), the temperature of a sufficient quantity of the sample to

make the test solution should be adjusted to the test temperature (25 ± 1EC) and maintained at that temperature

during the addition of dilution waters. 

13.10.1.2.6 Effluent dilutions should be prepared for all replicates in each treatment in one beaker to minimize

variability among the replicates. The test chambers are labeled with the test concentration and replicate number. 

Dispense into the appropriate effluent dilution chamber.

13.10.1.3 Dilution Water

13.10.1.3.1 Dilution water may be uncontaminated natural seawater (receiving water), HSB prepared from natural

seawater, or artificial seawater prepared from FORTY FATHOMS® or GP2 sea salts (see Table 3 in Section 7,

Dilution Water). Other artificial sea salts may be used for culturing inland silverside minnows and for the larval

survival and growth test if the control criteria for acceptability of test data are satisfied. 

13.10.2 START OF THE TEST

13.10.2.1 Inland silverside larvae 7 to 11 days old can be used to start the survival and growth test. At this age, the

inland silverside feed on newly-hatched Artemia nauplii. At 25EC, tests with inland silverside larvae can be

performed at salinities ranging from 5 to 32‰. If the test salinity ranges from 16 to 32‰, the salinity for spawning,

incubation, and culture of the embryos and larvae should be maintained within this salinity range. If the test salinity

is in the range of 5 to 15‰, the embryos may be spawned at 30‰, but egg incubation and larval rearing should be

at the test salinity. If the specific salinity required for the test differs from the rearing salinity, adjustments of 5‰

daily should be made over the three days prior to start of test. 

13.10.2.2 One day Prior to Beginning of Test 

13.10.2.2.1 Set up the Artemia culture so that newly hatched nauplii will be available on the day the test begins. 

(see Section 7). 

13.10.2.2.2 Increase the temperature of water bath, room, or incubator to the required test temperature (25 ± 1EC). 

13.10.2.2.3 Label the test chambers with a marking pen. Use of color coded tape to identify each concentration

and replicate is helpful. A minimum of five effluent concentrations and a control should be selected for each test. 

Glass test chambers, such as crystallization dishes, beakers, or chambers with a sump area (Figure 1), with a

capacity for 500-750 mL of test solution, should be used. 

13.10.2.2.4 Randomize the position of test chambers in the temperature-controlled water bath, room, or incubator

at the beginning of the test, using a position chart. Assign numbers for the position of each test chamber using a

table of random numbers or similar process (see Appendix A for an example of randomization). Maintain the

chambers in this configuration throughout the test, using a position chart.

13.10.2.2.5 Because inland silverside larvae are very sensitive to handling, it is advisable to distribute them to their

respective test chambers which contain control water on the day before the test is to begin. Each test chamber
170

should contain a minimum of 10 larvae and it is required that there be four replicates minimum for each

concentration and control.

13.10.2.2.6 Seven to 11 day old larvae are active and difficult to capture and are subject to handling mortality. 

Carefully remove larvae (two to three at a time) by concentrating them in a corner of the aquarium or culture vessel,

and capture them with a wide-bore pipette, small petri dish, crystallization dish, 3-4 cm in diameter, or small pipette. 

They are active and will readily escape from a pipette. Randomly transfer the larvae (two to three at a time) into

each test chamber until the desired number (15) is attained. See Appendix A for an example of randomization. 

After the larvae are dispensed, use a light table to verify the number in each chamber. 

13.10.2.3 Before beginning the test remove and replace any dead larvae from each test chamber. The test is started

by removing approximately 90% of the clean seawater from each test chamber and replacing with the appropriate

test solution.

13.10.3 LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE

13.10.3.1 The light quality and intensity should be at ambient laboratory levels, which is approximately 10-20

μE/m2

/s, or 50-100 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 ± 1EC. The test salinity should be in the range of 5-

32‰, and the salinity should not vary by more than ±2‰ among the chambers on a given day. If effluent and

receiving water tests are conducted concurrently, the salinities of these tests should be similar. 

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 The DO should be measured on new solutions at the start of the test (Day 0) and before daily

renewal of test solutions on subsequent days. The DO 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 a 1-2 mm orifice such as a 1 mL KIMAX® serological pipet, or equivalent. Care should be taken

to ensure that turbulence resulting from aeration does not cause undue stress to the fish. 

13.10.5 FEEDING

13.10.5.l Artemia nauplii are prepared as described above. 

13.10.5.2 The test larvae are fed newly-hatched (less than 24 h old) Artemia nauplii once a day from Day 0

through Day 6; larvae are not fed on Day 7. Equal amounts of Artemia nauplii must be fed to each replicate test

chamber to minimize the variability of larval weight. Sufficient numbers of nauplii should be fed to ensure that

some remain alive overnight in the test chambers. An adequate, but not excessive amount of Artemia nauplii,

should be provided to each replicate on a daily basis. Feeding excessive amounts of Artemia nauplii will result in a

depletion in DO to below an acceptable level. Siphon as much of the uneaten Artemia nauplii as possible from each

chamber daily to ensure that the larvae principally eat newly hatched nauplii.

13.10.5.3 On Days 0-2, transfer 4 g wet weight or pipette 4 mL of concentrated, rinsed Artemia nauplii to seawater

in a 100 mL beaker, and bring to a volume of 80 mL. Aerate or swirl the suspension to equally distribute the nauplii

while withdrawing individual 2 mL portions of the Artemia nauplii suspension by pipette or adjustable syringe to

transfer to each replicate test chamber. Because the nauplii will settle and concentrate at the tip of the pipette during

the transfer, limit the volume of concentrate withdrawn each time to a 2 mL portion for one test chamber helps

ensure an equal distribution to the replicate chambers. Equal distribution of food to the replicates is critical for

successful tests. 
171

13.10.5.4 On Days 3-6, transfer 6 g wet weight or 6 mL of the Artemia nauplii concentrate to seawater in a 100 mL

beaker. Bring to a volume of 80 mL and dispense as described above.

13.10.5.5 If the larvae survival rate in any replicate on any day falls below 50%, reduce the volume of Artemia

nauplii suspension added to that test chamber by one-half (i.e., reduce from 2 mL to 1 mL) and continue feeding

one-half the volume through Day 6. Record the time of feeding on the data sheets. 

13.10.6 DAILY CLEANING OF TEST CHAMBERS 

13.10.6.1 Before the daily renewal of test solutions, uneaten and dead Artemia, 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 safety pipet

filler or rubber bulb, can be used. If the test chambers illustrated in Figure 1 are used, remove only as much of the

test solution from the chamber as is necessary to clean, and siphon the remainder of the test solution from the sump

area. Because of their small size during the first few days of the test, larvae are easily drawn into a siphon tube

when cleaning the test chambers. By placing the test chambers on a light box, inadvertent removal of larvae can be

greatly reduced because they can be more easily seen. If the water siphoned from the test chambers is collected in a

white plastic tray, the live larvae caught up in the siphon can be retrieved, and returned by pipette to the appropriate

test chamber and noted on data sheet. Any incidence of removal of live larvae from the test chambers by the siphon

during cleaning, and subsequent return to the chambers should be noted in the test records. 

13.10.7 OBSERVATIONS DURING THE TEST

13.10.7.1 Routine Chemical and Physical Determinations

13.10.7.l.l DO is measured at the beginning and end of each 24 h exposure period in one test chamber at all test

concentrations and in the control. 

13.10.7.1.2 Temperature, pH, and salinity are measured at the end of each 24 h exposure period in one test

chamber at all test concentrations 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 chambers at least the end of the test to determine the

temperature variation in the environmental chamber.

13.10.7.1.3 The pH is measured in the effluent sample each day before new test solutions are made.

13.10.7.1.4 Record all measurements on the data sheet (Figure 3)
172

Test Dates: Species:Type Effluent: Field Lab Test Effluent Tested:

CONCENTRATION:

REPLICATE: REPLICATE: REPLICATE: REPLICATE:

DAYS 0123456701234567012345670123456 7

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

CONCENTRATION:

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

CONCENTRATION:

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

TIME

FED

COMMENTS:

Figure 3. Data form for the inland silverside, Menida beryllina, larval survival and growth test. Daily record of larval survival and test conditions. 

(From USEPA, 1987c).
173

Test Dates: Species:Type Effluent: Field Lab Test Effluent Tested:

CONCENTRATION:

REPLICATE: REPLICATE: REPLICATE: REPLICATE:

DAYS 0123456701234567012345670123456 7

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

CONCENTRATION:

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

CONCENTRATION:

# LIVELARVAETEMP(EC)SALINITY(‰)DO(mg/L)#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

#LARVAE/DRY WT

MEAN WEIGHT/LARVAE (mg) ±SD

TIME

FED

COMMENTS:

Figure 3. Data form for inland silverside, Menida beryllina, larval survival and growth test. Daily record of larval survival and test conditions. 

(CONTINUED)(From USEPA, 1987c).
174

13.10.7.2 Routine Biological Observation

13.10.7.2.1 The number of live larvae in each test chamber are recorded daily (Figure 3), and the dead larvae are

discarded. 

13.10.7.2.2 Protect the larvae from unnecessary disturbances during the test by carrying out the daily test

observations, solution renewals, and removal of dead larvae. Make sure the larvae remain immersed at all times

during the performance of the above operations. 

13.10.8 TEST SOLUTION RENEWAL

13.10.8.1 The test solutions are renewed daily using freshly prepared solutions, immediately after cleaning the test

chambers. The water level in each chamber is lowered to a depth of 7-10 mm, leaving 10-15% of the test solution. 

New test solution is added slowly by refilling each chamber with the appropriate amount of test solution without

excessively disturbing the larvae. If the modified chamber is used (Figure 1), renewals should be poured into the

sump area using a narrow bore (approximately 9 mm ID) funnel. 

13.10.8.2 The effluent or receiving water used in the test is stored in an incubator or refrigerator at 0-6EC. Plastic

containers such as 8-20 L cubitainers have proven suitable for effluent collection and storage. For on-site toxicity

studies no more than 24 h should elapse between collection of the effluent and use in a toxicity test (see Section 8,

Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation for Toxicity Tests).

13.10.8.3 Approximately 1 h before test initiation, a sufficient quantity of effluent or receiving water sample is

warmed to 25 ± 1EC to prepare the test solutions. A sufficient quantity of effluent should be warmed to make the

daily test solutions. 

13.10.8.3.1 An illustration of the quantities of effluent and seawater needed to prepare test solution at the

appropriate salinity is provided in Table 2. 

13.10.9 TERMINATION OF THE TEST

13.10.9.1 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 in 4% formalin or 70% ethanol. Preserved organisms are dried and

weighed within seven days. For safety, formalin should be used under a hood. 

13.10.9.2 For immediate drying and weighing, siphon or pour live larvae onto a 500 μm mesh screen in a large

beaker to retain the larvae and allow Artemia to be rinsed away. Rinse the larvae with deionized water to remove

salts that might contribute to the dry weight. Sacrifice the larvae in an ice bath of deionized water. 

13.10.9. Small aluminum weighing pans can be used to dry and weigh larvae. An appropriate number of

aluminum weigh pans (one per replicate) are marked for identification and weighed to 0.01 mg, and the weights are

recorded (Figure 4) on the data sheets. 

13.10.9.4 Immediately prior to drying, rinse the preserved larvae in distilled (or deionized) water. The rinsed

larvae from each test chamber are transferred, using forceps, to a tared weighing pan and dried at 60EC for 24 h, or

at 105EC for a minimum of 6 h. Immediately upon removal from the drying oven, the weighing pans are placed in a

desiccator to cool and to prevent the adsorption of moisture from the air until weighed. Weigh all weighing pans

containing the dried larvae to 0.01 mg, subtract the tare weight to determine dry weight of larvae in each replicate. 

Record (Figure 4) the weights. Divide the dry weight by the number of original larvae per replicate to determine the

average dry weight, and record (Figures 4 and 5) on the data sheets. For the controls, also calculate the mean

weight per surviving fish in the test chamber to evaluate if weights met test acceptability criteria (see

Subsection 13.11). Complete the summary data sheet (Figure 5) after calculating the average measurements and
175

statistically analyzing the dry weights and percent survival for the entire test. Average weights should be expressed

to the nearest 0.001 mg.

13.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA 

13.11.1 A summary of test conditions and test acceptability criteria is listed in Table 3. 

13.12 ACCEPTABILITY OF TEST RESULTS 

13.12.1 Test results are acceptable if (1) the average survival of control larvae is equal to or greater than 80%, and

(2) where the test starts with seven-day old larvae, the average dry weight per surviving control larvae, when dried

immediately after test termination, is equal to or greater than 0.50 mg, or the average dry weight of the control

larvae preserved not more than seven days in 4% formalin or 70% ethanol equals or exceeds 0.43 mg. 

13.13 DATA ANALYSIS 

13.13.1 GENERAL

13.13.1.1 Tabulate and summarize the data. 

13.13.1.2 The endpoints of toxicity tests using the inland silverside 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 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.

13.13.1.3 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.
176

Test Dates: Species: 

Pan

No.

Conc.

&

Rep.

Initial

Wt.

(mg)

Final

Wt.

(mg)

Diff.

(mg)

No.

Larvae

Av. Wt./

Larvae

(mg)

Figure 4. Data form for the inland silverside, Menidia beryllina, larval survival and growth test. Dry weights of

larvae (from USEPA, 1987b).
177

Test Dates: Species: 

Effluent Tested: 

TREATMENT

NO. LIVE

LARVAE

SURVIVAL

(%)

MEAN DRY WT/

LARVAE (MG)

± SD

SIGNIF. DIFF.

FROM CONTROL

(o)

MEAN

TEMPERATURE

(EC)

± SD

MEAN SALINITY

‰

± SD

AVE. DISSOLVED

OXYGEN

(MG/L) ± SD

COMMENTS:

Figure 5. Data form for the inland silverside, Menidia beryllina, larval survival and growth test. Summary of

test results (from USEPA, 1987c).
178

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE

INLAND SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL AND GROWTH TEST

WITH EFFLUENTS AND RECEIVING WATERS (TEST METHOD 1006.0)1

 

1. Test type: Static renewal (required) 

2. Salinity: 5‰ to 32‰ (± 2‰ of the selected test salinity) 

(recommended) 

3. Temperature: 25 ± 1°C (recommended) 

Test temperatures must not deviate (i.e., maximum minus

minimum temperature) by more than 3°C during the test

(required) 

4. Light quality: Ambient laboratory illumination (recommended) 

5. Light intensity: l0-20 μE/m2

/s (50-100 ft-c) (Ambient laboratory levels) 

(recommended) 

6. Photoperiod: 16 h light, 8 h darkness (recommended) 

7. Test chamber size: 600 mL-1 L containers (recommended) 

8. Test solution volume: 500-750 mL/replicate (loading and DO restrictions must be 

met) (recommended) 

9. Renewal of test solutions: Daily (required) 

10. Age of test organisms: 7-11 days post hatch; less than or equal to 24-h range in age 

(required) 

11. No. larvae per test

chamber: 10 (required minimum)

12. No. replicate chambers

per concentration: 4 (required minimum)

13. No. larvae per concentration: 40 (required minimum)

14. Source of food: Newly hatched Artemia nauplii (survival of 7-9 days old 

Menidia beryllina larvae improved by feeding 24 h old 

Artemia) (required) 

15. Feeding regime: Feed 0.l0 g wet weight Artemia nauplii per replicate on days 

0-2; Feed 0.15 g wet weight Artemia nauplii per replicate on 

days 3-6 (recommended) 

16. Cleaning: Siphon daily, immediately before test solution renewal and 

feeding (required) 

 1

 For the purposes of reviewing WET test data submitted under NPDES permits, each test condition listed above is

identified as required or recommended (see Subsection 10.2 for more information on test review). Additional

requirements may be provided in individual permits, such as specifying a given test condition where several options

are given in the method.
179

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE

INLAND SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL AND GROWTH TEST

WITH EFFLUENTS AND RECEIVING WATERS (TEST METHOD 1006.0) (CONTINUED) 

 

17. Aeration: None, unless DO concentration falls below 4.0 mg/L, then 

aerate all chambers. Rate should be less than 100 

bubbles/minimum (recommended) 

18. Dilution water: Uncontaminated source of natural sea water, artificial

seawater; deionized water mixed with hypersaline brine or

artificial sea salts (HW MARINEMIX®, FORTY

FATHOMS®, GP2 or equivalent) (available options) 

19. Test concentrations: Effluent: 5 and a control (required) 

 Receiving Waters: 100% receiving water (or minimum of 5) 

and a control (recommended) 

 

20. Dilution factor: Effluents: $ 0.5 (recommended) 

Receiving waters: None, or $ 0.5 (recommended) 

21. Test duration: 7 days (required) 

22. Endpoints: Survival and growth (weight) (required) 

23. Test acceptability criteria: 80% or greater survival in controls, 0.50 mg average dry 

weight of control larvae where test starts with 7-days old

larvae and dried immediately after test termination, or 0.43 mg

or greater average dry weight per surviving control larvae,

preserved not more than 7 days in 4% formalin or 70%

ethanol (required) 

24. Sampling requirement: 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 (e.g., 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) (required) 

25. Sample volume required: 6 L per day (recommended) 

 

13.13.2 EXAMPLE OF ANALYSIS OF INLAND SILVERSIDE, MENIDIA BERYLLINA, SURVIVAL DATA 

13.13.2.1 Formal statistical analysis of the survival data is outlined in Figures 6 and 7. 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 endpoint. 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 IC, EC, and LC endpoint.

13.13.2.2 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
180

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 the 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. 

13.13.2.3 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. 

13.13.2.4 Probit Analysis (Finney, 1971; see Appendix H) 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 model, the Spearman-Karber

method, the Trimmed Spearman-Karber method, or the Graphical method may be used (see Appendices H-K).

13.13.2.5 Example of Analysis of Survival Data

13.13.2.5.1 This example uses the survival data from the inland silverside larval survival and growth test. The

proportion surviving in each replicate in this example must first be transformed by the arc sine transformation

procedure 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 4. A plot of the data is provided in Figure

8. Since there is 100% mortality in all three replicates for the 50% and 100% concentrations, they are not included

in this statistical analysis and are considered a qualitative mortality effect. 
181

Figure 6. Flowchart for statistical analysis of the inland silverside, Menidia beryllina, survival data by

hypothesis testing.
182

Figure 7. Flowchart for statistical analysis of the inland silverside, Menidia beryllina, survival data by point

estimation.
183

Figure 8. Plot of mean survival proportion of the inland silverside, Menidia beryllina, larvae.
184

TABLE 4. INLAND SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL DATA 

 Concentration 

Replicate Control 6.25 12.5 25.0 50.0 100.0

A 0.80 0.73 0.80 0.40 0.0 0.0

RAW B 0.87 0.80 0.33 0.53 0.0 0.0

C 0.93 0.87 0.60 0.07 0.0 0.0

ARC SINE A 1.107 1.024 1.107 0.685 - -

TRANS- B 1.202 1.107 0.612 0.815 - -

FORMED C 1.303 1.202 0.886 0.268 - -

Mean 1.204 1.111 0.868 0.589 (Y ̄ i

)

S 0.010 0.008 0.061 0.082 2

i

i 1 234

13.13.2.6 Test for Normality 

13.13.2.6.1 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 5.

TABLE 5. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE 

Effluent Concentration (%)

 Replicate Control 6.25 12.5 25.0 

A -0.097 -0.087 0.239 0.096 

B -0.002 -0.004 -0.256 0.226 

C 0.099 0.091 0.018 -0.321 
185

D ' j

n

i'1

(Xi&X ̄)

2

W ' 1

D [j

k

i'1

ai(X (n&1%1)&X (i))]2

13.13.2.6.2 Calculate the denominator, D, of the statistic: 

Where: Xi

 = the ith centered observation 

 

X ̄ = the overall mean of the centered observations 

n = the total number of centered observations 

13.13.2.6.3 For this set of data, n = 12 

X ̄ = 1 (0.002) = 0.0 

12

 D = 0.3214

13.13.2.6.4 Order the centered observations from smallest to largest: 

 

X(1)# X(2)# ...# X(n)

where X(i) denotes the ith ordered observation. The ordered observations for this example are listed in Table 6. 

TABLE 6. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE 

i X(i) i X(i)

1

2

3

4

5

6

-0.321

-0.256

-0.097

-0.087

-0.004

-0.002

7

8

9

10

11

12

0.018

0.091

0.096

0.099

0.226

0.239

13.13.2.6.5 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-1)/2 if n is odd. For the data in this example, n = 12 and k = 6. The ai

 values are listed in

Table 7. 

13.13.2.6.6 Compute the test statistic, W, as follows: 
186

B '

[(j

p

i'1

Vi) lnS ̄2&j

p

i'1

Vi lnS 2

i ]

C

The differences X(n-i+1) - X(i) are listed in Table 7. For the data in this example,

W = 1 (0.5513)2

 = 0.945 

 0.3214 

TABLE 7. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE

i ai X(n-i+1) - X(i)

1

2

3

4

5

6

0.5475

0.3325

0.2347

0.1586

0.0922

0.0303

0.560

0.482

0.196

0.183

0.095

0.020

X(12) - X(1)

X(11) - X(2)

X(10) - X(3)

X(9) - X(4)

X(8) - X(5)

X(7) - X(6)

13.13.2.6.7 The decision rule for this test is to compare W as calculated in Subsection 13.13.2.6.6 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 = 12

observations is 0.805. Since W = 0.945 is greater than the critical value, conclude that the data are normally

distributed. 

13.13.2.7 Test for Homogeneity of Variance

13.13.2.7.1 The test used to examine whether the variation in survival is the same across all effluent concentrations

including the control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as follows:

Where: Vi = degrees of freedom for each effluent concentration and control, Vi

 = (n i - 1) 

p = number of levels of effluent concentration including the control

ln = loge

i = 1, 2, ..., p where p is the number of concentrations including the control

ni = the number of replicates for concentration i. 

S ̄2 '

(j

p

i'1

ViS 2

i )

j

p

i'1

Vi
187

C ' 1%[3(p&1)]&1[j

p

i'1

1/Vi&(j

p

i'1

Vi)

&1

]

B ' [(8)ln(0.0402)&2j

p

i'1

ln(S 2

i )]/1.2083

13.13.2.7.2 For the data in this example (See Table 4), all effluent concentrations including the control have the

same number of replicates (ni

 = 3 for all i). Thus, Vi = 2 for all i. 

13.13.2.7.3 Bartlett's statistic is therefore: 

= [8(-3.21391) - 2(-14.731)]/1.2083 

= 3.7508/1.2083 

= 3.104 

13.13.2.7.4 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, at a significance level of 0.01 with three degrees

of freedom, is 11.345. Since B = 3.104 is less than the critical value of 11.345, conclude that the variances are not

different. 

13.13.2.8 Dunnett's Procedure 

13.13.2.8.1 To obtain an estimate of the pooled variance for the Dunnett's Procedure, construct an ANOVA table

as described in Table 8. 

TABLE 8. ANOVA TABLE

Source df Sum of Squares Mean Square (MS)

(SS) (SS/df)

Between p - 1 SSB = SSB/(p-1) S 2

B

Within N - p SSW = SSW/(N-p) S 2

W

Total N - 1 SST

Where: p = number of SDS concentration levels including the control

N = total number of observations n1 + n 2 ... + n p

ni = number of observations in concentration i 
188

SSB ' j Between Sum of Squares

p

i'1

T 2

i /ni

&G 2 /N

SST ' j Total Sum of Squares

p

i'1

j

nj

j'1

Y 2

ij &G 2 /N

SSW ' SST&SSB Within Sum of Squares

G = the grand total of all sample observations, G ' j

p

i'1

Ti

Ti = the total of the replicate measurements for concentration i

Yij = the jth observation for concentration i (represents the proportion surviving for toxicant 

concentration i in test chamber j) 

13.13.2.8.2 For the data in this example: 

n1 = n2 = n3 = n4 = 3 

N = 12 

T1 = Y11 + Y12 + Y13 = 3.612

T2 = Y21 + Y22 + Y23 = 3.333 

T3 = Y31 + Y32 + Y33 = 2.605 

T4 = Y41 + Y42 + Y43 = 1.768 

G = T1 + T2 + T3 + T4 = 11.318 

SSB ' j

p

i'1

T 2

i /ni

&G 2 /N

= 1 (34.067) - (11.318)2 = 0.681 

 3 12 

SST ' j

p

i'1

j

nj

j'1

Y 2

ij &G 2 /N

= 11.677 - (11.318)2 = 1.002 

12

SSW ' SST&SSB= 1.002 - 0.681 = 0.321 

S = SSB/(p-1) = 0.681/(4-1) = 0.227 2

B

S = SSW/(N-p) = 0.321/(12-4) = 0.040 2

W

13.13.2.8.3 Summarize these calculations in the ANOVA table (Table 9).
189

ti ' (Y ̄

i

&Y ̄

i

)

Sw (1/n1)%(1/ni

)

t

2 ' (1.204 & 1.111)

[0.020 (1/3) % (1/3)]

' 0.570

TABLE 9. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE 

Source df Sum of Squares Mean Square(MS) 

 (SS) (SS/df) 

Between 3 0.681 0.227 

Within 8 0.321 0.040 

Total 11 1.002 

13.13.2.8.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control

combination as follows: 

Where: = mean proportion surviving for effluent concentration i Y ̄ i

Y ̄ = mean proportion surviving for the control 1

SW = square root of the within mean square 

n1 = number of replicates for the control 

ni

 = number of replicates for concentration i. 

13.13.2.8.5 Table 10 includes the calculated t values for each concentration and control combination. In this

example, comparing the 1.0% concentration with the control the calculation is as follows: 
190

MSD ' d Sw (1/n1)%(1/n)

TABLE 10. CALCULATED T VALUES

Effluent Concentration (%) i ti

6.25 2 0.570

12.5 3 2.058

25.0 4 3.766

13.13.2.8.6 Since the purpose of this test is to detect a significant reduction in survival, 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, eight degrees of freedom for error and three concentrations (excluding the control) the critical value is 2.42. 

The mean proportion surviving for concentration i is considered significantly less than the mean proportion

surviving for the control if ti

 is greater than the critical value. Therefore, only the 25.0% concentration has a

significantly lower mean proportion surviving than the control. Hence the NOEC is 12.5% and the LOEC is 25.0%.

13.13.2.8.7 To quantify the sensitivity of the test, the minimum significant difference (MSD) that can be detected

statistically may be calculated. 

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) 

n1 = the number of replicates in the control. 

13.13.2.8.8 In this example: 

MSD ' 2.42(0.20) (1/3)%(1/3)

= 2.42 (0.20) (0.817) 

= 0.395 

13.13.2.8.9 The MSD (0.395) 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. 

1.204 - 0.395 = 0.809 
191

2. Obtain the untransformed values for the control mean and the difference calculated in step 1. 

 [ Sine (1.204) ]2

 = 0.871

[ Sine (0.809) ]2

 = 0.524 

3. The untransformed MSD (MSDu) is determined by subtracting the untransformed values 

from step 2. 

MSDu = 0.871 - 0.524 = 0.347 

13.13.2.8.10 Therefore, for this set of data, the minimum difference in mean proportion surviving between the

control and any effluent concentration that can be detected as statistically significant is 0.347. 

13.13.2.8.11 This represents a 40% decrease in survival from the control. 

13.13.2.9 Calculation of the LC50 

13.13.2.9.1 The data used for the Probit Analysis is summarized in Table 11. To perform the Probit Analysis, run

the USEPA Probit Analysis Program. An example of the program input and output is supplied in Appendix H.

TABLE 11. DATA FOR PROBIT ANALYSIS 

 Effluent Concentration (%) 

Control 6.25 12.5 25.0 50.0 100.0

Number Dead 6 9 19 45 45 45

Number Exposed 45 45 45 45 45 45

13.13.2.9.2 For this example, the chi-square test for heterogeneity was not significant. Thus Probit Analysis

appears to be appropriate for this set of data. 

13.13.2.9.3 Figure 9 shows the output data for the Probit Analysis of the data from Table 11 using the USEPA

Probit Program.

13.13.3 ANALYSIS OF INLAND SILVERSIDE, MENIDIA BERYLLINA, GROWTH DATA

13.13.3.1 Formal statistical analysis of the growth data is outlined in Figure 10. The response used in the

statistical analysis is mean weight per original organism for each replicate. Because this measurement is based on

the number of original organisms exposed (rather than the number surviving), the measured response is a combined

survival and growth endpoint that can be termed biomass. The IC25 and IC50 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 an NOEC and LOEC for growth. Concentrations above the NOEC for survival are

excluded from the hypothesis test for growth 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. The test for normality is the Shapiro-Wilk's Test and Bartlett's
192

Test is used to test for homogeneity of variance. If either of these test 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. 

13.13.3.3 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. The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the nonparametric alternative. For

detailed information on the Bonferroni adjustment, see Appendix D.

Probit Analysis of Inland Silverside Larval Survival Data

Conc. Number

Exposed

Number

Resp.

Observed

Proportion

Responding

Proportion

Responding

Adjusted for

Controls

Control 

6.2500 

12.5000 

25.0000 

50.0000 

100.0000 

45 

45 

45 

45 

 45 

45 

6 

9 

19 

30 

45 

 45 

0.1333 

0.2000 

0.4222 

0.6667 

1.0000 

1.0000 

0.0000 

0.0488 

0.3130 

0.6037 

1.0000 

1.0000 

Chi - Square for Heterogeneity (calculated) = 4.149 

Chi - Square for Heterogeneity (tabular value) = 7.815 

Probit Analysis of Inland Silverside Larval Survival Data

Estimated LC/EC Values and Confidence Limits

Point Exposure

Conc.

Lower Upper

95% Confidence Limits

LC/EC 1.00

LC/EC 50.00

4.980 

18.302 

2.023 

13.886 

7.789 

22.175 

Figure 9. Output for USEPA Probit Analysis Program, Version 1.5. 
193

Figure 10. Flowchart for statistical analysis of the inland silverside, Menida beryllina, growth data.
194

13.13.3.4 The data, mean and variance of the growth observations at each concentration including the control are

listed in Table 12. A plot of the data is provided in Figure 11. Since there was no survival in the 50% and 100%

concentrations, these are not considered in the growth analysis. Additionally, since there is significant mortality in

the 25% effluent concentration, its effect on growth is not considered.

TABLE 12. INLAND SILVERSIDE, MENIDIA BERYLLINA, GROWTH DATA

 Effluent Concentration % 

Replicate Control 6.25 12.5 25.0 50.0 100.0

A 0.751 0.737 0.722 0.196 - -

B 0.849 0.922 0.285 0.312 - -

C 0.907 0.927 0.718 0.079 - -

Mean 0.836 0.862 0.575 0.196 - - (Y ̄ i

)

S 0.0062 0.0117 0.0631 0.0136 - - 2

i

i 1 2 3456

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

observations within a concentration from each observation in that concentration. The centered observations are

summarized in Table 13.

TABLE 13. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

 Effluent Concentration (%) 

Replicate Control 6.25 12.5

A -0.085 -0.125 0.147 

B 0.013 0.060 -0.290

C 0.071 0.065 0.143
195

Figure 11. Plot of mean weights of inland silverside, Menidia beryllina, larval survival and growth test.
196

D ' j

n

i'1

(Xi&X ̄)

2

W ' 1

D [j

k

i'1

ai(X (n&i%1)&X (i))]2

13.13.3.5.2 Calculate the denominator, D, of the test statistic: 

Where: Xi

 = 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 = 9 

X ̄ = 1 (-0.002) = 0.000 

9

D = 0.162

13.13.3.5.3 Order the centered observations from smallest to largest: 

X(1)# X(2) #... # X(n)

Where X(i) is the ith ordered observation. These ordered observations are listed in Table 14. 

TABLE 14. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE 

i X(i) i X(i)

1 -0.290 6 0.065

2 -0.125 7 0.071

3 -0.085 8 0.143 

4 0.013 9 0.147 

5 0.060

13.13.3.5.4 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-1)/2 if n is odd. For the data in this example, n = 9 and k = 4. The ai

 values are listed in

Table 15. 

13.13.3.5.5 Compute the test statistic, W, as follows: 
197

B '

[(j

p

i'1

Vi) lnS ̄2&j

p

i'1

Vi lnS 2

i ]

C

The differences X(n-i+1) - X(i) are listed in Table 15. For this set of data: 

W = 1 (0.3800)2

 = 0.89

0.162

TABLE 15. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE 

i ai X(n-i+1) - X(i)

1 0.5888 0.437 X(9) - X(1)

2 0.3244 0.268 X(8) - X(2)

3 0.1976 0.156 X(7) - X(3)

4 0.0947 0.052 X(6) - X(4)

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 nine observations (n) is 0.764. Since W = 0.964 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 mean dry weight is the same across all effluent

concentrations including the control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as follows:

 

Where: Vi = degrees of freedom for each effluent concentration and control, Vi

 = (ni - 1) 

p = number of levels of effluent concentration including the control 

i = 1,2, ..., p where p is the number of concentrations including the control

ln = loge

ni = number of replicates for concentration i
198

S ̄2 '

(j

p

i'1

ViS 2

i )

j

p

i'1

Vi

C ' 1%[3(p&1)]&1[j

p

i'1

1/Vi

&(j

p

i'1

Vi)

&1

]

B ' [(6)ln(0.027)&2j

p

i'1

ln(S 2

i )/1.222

13.13.3.6.2 For the data in this example, (See Table 13) all effluent concentrations including the control have the

same number of replicates (ni

 = 3 for all i). Thus, Vi = 2 for all i. 

13.13.3.6.3 Bartlett's statistic is therefore: 

= [6(-3.612)-2(-12.290)]/1.222 

= 2.909/1.222 

= 2.38

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 2 degrees of

freedom, is 9.210. Since B = 2.38 is less than the critical value of 9.210, 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 16. 
199

SSB ' j

p

i'1

T 2

i /ni

&G 2 /N

SST ' j

p

i'1

j

nj

j'1

Y 2

ij &G 2 /N

SSW ' SST&SSB

TABLE 16. ANOVA TABLE

Source df Sum of Squares Mean Square (MS)

(SS) (SS/df)

Between p - 1 SSB = SSB/(p-1)

Within N - p SSW = SSW/(N-p)

Total N - 1 SST

Where: p = number of effluent concentrations including the control 

N = total number of observations n1 + n2 ... + np

ni = number of observations in concentration i 

Between Sum of Squares

Total Sum of Squares

Within Sum of Squares

G = the grand total of all sample observations, 

Ti

 = the total of the replicate measurements for concentration i

Yij = the jth observation for concentration i (represents the mean dry weight of the fish for toxicant 

 concentration i in test chamber j) 

13.13.3.7.2 For the data in this example: 

 n1 = n2 = n3 = 3 

 N = 9 

 T1 = Y11 + Y12 + Y13 = 0.751 + 0.849 + 0.907 = 2.507 

 T2 = Y21 + Y22 + Y23 = 0.727 + 0.922 + 0.927 = 2.576 

 T3 = Y31 + Y32 + Y33 = 0.722 + 0.285 + 0.718 = 1.725 

 G = T1 + T2 + T3 = 6.808 
200

ti ' (Y1 & Yi

)

Sw (1/n1 % 1/ni

)

= 1 (15.896) - (6.808)2 = 0.1488 

3 9

 = 5.463 - (6.808)2

 = 0.3131 

9

= 0.3131 - 0.1488 = 0.1643 

 = SSB/(p-1) = 0.1488/(3-1) = 0.0744

 = SSW/(N-p) = 0.1643/(9-3) = 0.0274

13.13.3.7.3 Summarize these calculations in the ANOVA table (Table 17).

TABLE 17. ANOVA TABLE FOR DUNNETT’S PROCEDURE EXAMPLE

Source df Sum of Squares Mean Square(MS) 

 (SS) (SS/df) 

Between 2 0.1488 0.0744 

Within 6 0.1643 0.0274 

Total 8 0.3131 

13.13.3.7.4 To perform the individual comparisons, calculate the t statistic for each concentration and control

combination as follows: 

Where: Yi

 = mean dry weight for effluent concentration i 

 

Y1 = mean dry weight for the control 

SW = square root of the within mean square 

n1 = number of replicates for the control 

ni

 = number of replicates for concentration i. 
201

t

2 ' (0.836&0.859)

[0.1655 (1/3)%(1/3)]

' &0.120

MSD ' dSw (1/n1)%(1/n)

MSD ' 2.34(0.1655) (1/3)%(1/3)

13.13.3.7.5 Table 18 includes the calculated t values for each concentration and control combination. In this

example, comparing the 6.25% concentration with the control the calculation is as follows:

TABLE 18. CALCULATED T VALUES

Effluent Concentration (ppb) i ti

6.25 2 -0.120

 12.5 3 1.931

13.13.3.7.6 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, six degrees of freedom for error and two concentrations (excluding the control) the critical value is 2.34. The

mean weight for concentration i is considered significantly less than mean weight for the control if ti

 is greater than

the critical value. Therefore, all effluent concentrations in this example do not have significantly lower mean weights

than the control. Hence the NOEC and the LOEC for growth cannot be calculated.

13.13.3.7.7 To quantify the sensitivity of the test, the minimum significant difference (MSD) that can be detected

statistically may be calculated. 

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) 

n1 = the number of replicates in the control. 

13.13.3.7.8 In this example: 

= 2.34 (0.1655)(0.8165) 

= 0.316 
202

13.13.3.7.9 Therefore, for this set of data, the minimum difference that can be detected as statistically significant is

0.316 mg. 

13.13.3.7.10 This represents a 37.8% reduction in mean weight from the control. 

13.13.3.8 Calculation of the ICp

13.13.3.8.1 The growth data from Tables 4 and 12 are utilized in this example. As seen in Table 19 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 reponses

between concentrations do not follow a linear trend). Therefore, the means are smoothed prior to calculating the IC. 

In the following discussion, the observed means are represented by and the smoothed means by Mi Y ̄ . i

13.13.3.8.2 Starting with the control mean, = 0.836 and = 0.859, we see that < . Set Mi = Yi Y ̄ . 1 Y ̄2 Y ̄1 Y ̄2

13.13.3.8.3 Calculate the smoothed means:

M1 = M2 = ( + )/2 = 0.847 Y ̄1 Y ̄2

13.13.3.8.4 Since = 0 < = 0.196 < = 0.575 < M2, set M3 = 0.575, M4 = 0.196, and M5 Y ̄ = 0. 5 Y ̄4 Y ̄3

13.13.3.8.5 Table 19 contains the response means and the smoothed means and Figure 12 gives a plot of the

smoothed response curve.

TABLE 19. INLAND SILVERSIDE MEAN GROWTH RESPONSE AFTER SMOOTHING

Response Smoothed

Effluent Means, Means, 

Conc. Yi Mi

(%) i (mg) (mg) 

Control 1 0.836 0.847

6.25 2 0.859 0.847

12.50 3 0.575 0.575

25.00 4 0.196 0.196

50.00 5 0.00 0.0

13.13.3.8.6 An IC25 and 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 0.627 mg, where M1(1-p/100) = 1.847(1-25/100). A

50% reduction in mean dry weight, compared to the controls, would result in a mean weight of 0.418 mg. Examining

the smoothed means and their associated concentrations (Table 20), the response, 0.627 mg, is bracketed by C2 =

6.25% effluent and C3 = 25.0% effluent. The response (0.418) is bracketed by C3 = 12.5% and by C4 = 25% effluent.
203

ICp ' Cj%[m1 (1&p/100)&Mj]

(Cj%1&Cj)

(mj%1&Mj

)

13.13.3.8.7 Using the equation from Section 4.2 of Appendix L, the estimate of the IC25 is calculated as follows:

IC25 = 6.25 + [0.847(1 - 25/100) - 0.847] (12.50 - 6.25)

(0.575 - 0.847)

= 11.1%.

205

ICp ' Cj%[m1 (1&p/100)&Mj]

(Cj%1&Cj)

(Mj%1&Mj

)

13.13.3.8.8 Using the equation from Section 4.2 of Appendix L, the estimate of the IC50 is calculated as follows:

IC50 = 6.25 + [0.847(1 - 50/100) - 0.847] (12.50 - 6.25)

(0.575 - 0.847)

= 17.5%.

13.13.3.8.9 When the ICPIN program was used to analyze this set of data, requesting 80 resamples, the estimate of

the IC25 was 11.1136%. The empirical 95% confidence interval for the true mean was 5.7119% to 19.2112%. The

computer program output for the IC25 for this data set is shown in Figure 13.

13.13.3.8.10 When the ICPIN program was used to analyze this set of data for the IC50, requesting 80 resamples,

the estimate of the IC50 was 17.4896%. The empirical 95% confidence interval for the true mean was 6.4891% to

22.4754% effluent. The computer program output is shown in Figure 14.

13.14 PRECISION AND ACCURACY

13.14.1 PRECISION – Data on single-laboratory and multilaboratory precision are described below (Subsections

13.14.1.1 and 13.14.1.2). Single-laboratory precision is a measure of the reproducibility of test results when tests are

conducted using a specific method under reasonably constant conditions in the same laboratory. Single-laboratory

precision is synonymous with the terms within-laboratory precision and intralaboratory precision. Multilaboratory

precision is a measure of the reproducibility of test results from different laboratories using the same test method and

analyzing the same test material. Multilaboratory precision is synonymous with the term interlaboratory precision. 

Interlaboratory precision, as used in this document, includes both within-laboratory and between-laboratory

components of variability. In recent multilaboratory studies, these two components of interlaboratory precision have

been displayed separately (termed within-laboratory and between-laboratory variability) and combined (termed total

interlaboratory variability). The total interlaboratory variability that is reported from these studies is synonymous

with interlaboratory variability reported from other studies where individual variability components are not

separated.

13.14.1.1 Single-Laboratory Precision

13.14.1.1.1 Data on the single-laboratory precision of the inland silverside larval survival and growth test using

copper (CU) sulfate and sodium dodecyl sulfate (SDS) as reference toxicants, in natural seawater and GP2 are

provided in Tables 20-22. In Tables 20-21, the coefficient of variation for copper based on the IC25 is 43.2% and

for SDS is 43.2% indicating acceptable precision. In the five tests with each reference toxicant, the NOEC's varied

by only one concentration interval, indicating good precision. The coefficient of variation for all reference toxicants

based on the IC50 in two types of seawater (GP2 and natural) ranges from l.8% to 50.7% indicating acceptable

precision. Data in Table 22 show no detectable differences between tests conducted in natural and artificial

seawaters.

13.14.1.1.2 EPA evaluated within-laboratory precision of the Inland Silverside, Menidia beryllina, Larval Survival

and Growth Test using a database of routine reference toxicant test results from 16 laboratories (USEPA, 2000b). 

The database consisted of 193 reference toxicant tests conducted in 16 laboratories using a variety of reference

toxicants including: chromium, copper, potassium chloride, and sodium dodecyl sulfate. Among the 16 laboratories,

the median within-laboratory CV calculated for routine reference toxicant tests was 27% for the IC25 growth

endpoint. In 25% of laboratories, the within-laboratory CV was less than 18%; and in 75% of laboratories, the

within-laboratory CV was less than 43%.
206

13.14.1.2 Multilaboratory Precision

13.14.1.2.1 In 2000, EPA conducted an interlaboratory variability study of the Inland Silverside, Menidia beryllina,

Larval Survival and Growth Test (USEPA, 2001a; USEPA, 2001b). In this study, each of 10 participant laboratories

tested 4 blind test samples that included some combination of blank, effluent, reference toxicant, and receiving water

sample types. The blank sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater, the effluent

sample was an industrial wastewater spiked with CuSO4, the receiving water sample was a natural seawater spiked

with CuSO4, and the reference toxicant sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater

spiked with CuSO4. Of the 40 Menidia beryllina Larval Survival and Growth tests conducted in this study, 100%

were successfully completed and met the required test acceptability criteria. Of seven tests that were conducted on

blank samples, none showed false positive results for survival endpoints or for the growth endpoint. Results from the

reference toxicant, effluent, and receiving water sample types were used to calculate the precision of the method. 

Table 23 shows the precision of the IC25 for each of these sample types. Averaged across sample types, the total

interlaboratory variability (expressed as a CV%) was 43.8% for IC25 results. Table 24 shows the frequency

distribution of survival and growth NOEC endpoints for each sample type. For the survival endpoint, NOEC values

spanned five concentrations for the effluent, four concentrations for the reference toxicant sample type, and three

concentrations for the receiving water sample type. The percentage of values within one concentration of the median

was 90.9%, 84.6%, and 85.7% for the reference toxicant, effluent, and receiving water sample types, respectively. 

For the growth endpoint, NOEC values spanned four concentrations for the reference toxicant and effluent sample

types and three concentrations for the receiving water sample type. The percentage of values within one

concentration of the median was 90.9%, 91.7%, and 85.7% for the reference toxicant, effluent, and receiving water

sample types, respectively. 

13.14.2 ACCURACY 

13.14.2.1 The accuracy of toxicity tests cannot be determined. 
207

-------------------------------------------------------------------------------------------------------------------------------------------

Conc. ID 1 2 3 4 5 6

-------------------------------------------------------------------------------------------------------------------------------------------

Conc. Tested 0 6.25 12.5 25 50 100

-------------------------------------------------------------------------------------------------------------------------------------------

Response 1 .751 .727 .722 .196 0 0

Response 2 .849 .922 .285 .312 0 0

Response 3 .907 .927 .718 .079 0 0

-------------------------------------------------------------------------------------------------------------------------------------------

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: Effluent

Test Start Date: Test Ending Date: 

Test Species: Menidia beryllina

Test Duration: 7-d

DATA FILE: silver.icp

OUTPUT FILE: silver.i25

-------------------------------------------------------------------------------------------------------------------------------------------

Conc. Number Concentration Response Std. Pooled

 ID Replicates % Means Dev. Response Means

-------------------------------------------------------------------------------------------------------------------------------------------

1 3 0.000 0.836 0.079 0.847

2 3 6.250 0.859 0.114 0.847

3 3 12.500 0.575 0.251 0.575

4 3 25.000 0.196 0.117 0.196

5 3 50.000 0.000 0.000 0.000

6 3 100.000 0.000 0.000 0.000

-------------------------------------------------------------------------------------------------------------------------------------------

The Linear Interpolation Estimate: 11.1136 Entered P Value: 25

-------------------------------------------------------------------------------------------------------------------------------------------

Number of Resamplings: 80

The Bootstrap Estimates Mean: 11.5341 Standard Deviation: 2.1155

Original Confidence Limits: Lower: 8.5413 Upper: 14.9696

Expanded Confidence Limits: Lower: 5.7119 Upper: 19.2112

Resampling time in Seconds: 1.43 Random Seed: -1912403737

Figure 13. ICPIN program output for the IC25.
208

-------------------------------------------------------------------------------------------------------------------------------------------

Conc. ID 1 2 3 4 5 6

-------------------------------------------------------------------------------------------------------------------------------------------

Conc. Tested 0 6.25 12.5 25 50 100

-------------------------------------------------------------------------------------------------------------------------------------------

Response 1 .751 .727 .722 .196 0 0

Response 2 .849 .922 .285 .312 0 0

Response 3 .907 .927 .718 .079 0 0

-------------------------------------------------------------------------------------------------------------------------------------------

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: Effluent

Test Start Date: Test Ending Date: 

Test Species: Menidia beryllina

Test Duration: 7-d

DATA FILE: silver.icp

OUTPUT FILE: silver.i50

-------------------------------------------------------------------------------------------------------------------------------------------

Conc. Number Concentration Response Std. Pooled

 ID Replicates % Means Dev. Response Means

-------------------------------------------------------------------------------------------------------------------------------------------

1 3 0.000 0.836 0.079 0.847

2 3 6.250 0.859 0.114 0.847

3 3 12.500 0.575 0.251 0.575

4 3 25.000 0.196 0.117 0.196

5 3 50.000 0.000 0.000 0.000

6 3 100.000 0.000 0.000 0.000

-------------------------------------------------------------------------------------------------------------------------------------------

The Linear Interpolation Estimate: 17.4896 Entered P Value: 50

-------------------------------------------------------------------------------------------------------------------------------------------

Number of Resamplings: 80

The Bootstrap Estimates Mean: 16.9032 Standard Deviation: 2.49.73

Original Confidence Limits: Lower: 12.2513 Upper: 19.8638

Expanded Confidence Limits: Lower: 6.4891 Upper: 22.4754

Resampling time in Seconds: 1.43 Random Seed: -1440337465

Figure 14. ICPIN program output for the IC50.
209

TABLE 20. SINGLE-LABORATORY PRECISION OF THE INLAND SILVERSIDE, MENIDIA BERYLLINA,

SURVIVAL AND GROWTH TEST PERFORMED IN NATURAL SEAWATER, USING LARVAE

FROM FISH MAINTAINED AND SPAWNED IN NATURAL SEAWATER, AND COPPER (CU)

AS A REFERENCE TOXICANT1,2,3,4,5,6,7

 Most

Test NOEC IC25 IC50 Sensitive

Number (μg/L) (μg/L) (μg/L) Endpoint6

1 63 96.2 148.6 S

2 125 207.2 NC8 S

3 63 218.9 493.4 G

4 125 177.5 241.4 S

5 31 350.1 479.8 G

n: 5 5 4

Mean: NA 209.9 340.8

CV(%): NA 43.7 50.7

1 Data from USEPA (1988a) and USEPA (1991a) 2 Tests performed by George Morrison and Elise Torello, ERL-N, USEPA, Narragansett, RI. 3 Three replicate exposure chambers with 10-15 larvae were used for the control and each copper concentration. 

Copper concentrations were: 31, 63, 125, 250, and 500 μg/L. 4 Adults collected in the field. 5 S = Survival effects. G = Growth data at these toxicant concentrations were disregarded because there was a 

significant reduction in survival. 6 NOEC Range: 31 - 125 μg/L (this represents a difference of two exposure concentrations). 7 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 8 NC = No linear interpolation estimate could be calculated from the ata, since none of the group response means were less

than 50 percent of the control response mean. 
210

TABLE 21. SINGLE-LABORATORY PRECISION OF THE INLAND SILVERSIDE, MENIDIA BERYLLINA,

SURVIVAL AND GROWTH TEST PERFORMED IN NATURAL SEAWATER, USING LARVAE

FROM FISH MAINTAINED AND SPAWNED IN NATURAL SEAWATER, AND SODIUM

DODECYL SULFATE (SDS) AS A REFERENCE TOXICANT1,2,3,4,5,6,7

Most

Test NOEC IC25 IC50 Sensitive

Number (mg/L) (mg/L) (mg/L) Endpoint

1 1.3 0.3 1.7 S

2 1.3 1.6 1.9 S

3 1.3 1.5 1.9 S

4 1.3 1.5 1.9 S

5 1.3 1.6 2.2 S

n: 5 5 5

Mean: NA 1.3 1.9

CV(%): NA 43.2 9.4

 1 Data from USEPA (1988a) and USEPA (1991a) 2 Tests performed by George Morrison and Elise Torello, ERL-N, USEPA, Narragansett, RI. 3 Three replicate exposure chambers with 10-15 larvae were used for the control and each SDS concentration. 

SDS concentrations were: 0.3, 0.6, 1.3, 2.5, and 5.0 mg/L. 4 Adults collected in the field. 5 S = Survival Effects. Growth data at these toxicant concentrations were disregarded because there was a

significant reduction in survival. 6 NOEC Range: 1.3 mg/L. 7 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 
211

TABLE 22. COMPARISON OF THE SINGLE-LABORATORY PRECISION OF THE INLAND 

SILVERSIDE, MENIDIA BERYLLINA, LARVAL SURVIVAL (LC50) AND GROWTH (IC50) 

VALUES EXPOSED TO SODIUM DODECYL SULFATE (SDS) OR COPPER (CU) SULFATE

IN GP2 ARTIFICIAL SEAWATER MEDIUM OR NATURAL SEAWATER (NSW)1,2,3,4

 Survival Growth 

SDS (mg/L) GP2 NSW GP2 NSW

3.59 3.69 3.60 3.55

4.87 4.29 5.54 5.27

5.95 8.05 6.70 8.53

 

Mean 4.81 5.34 5.28 5.79

CV (%) 24.6 44.2 29.6 43.8

 

Copper (μg/L) GP2 NSW GP2 NSW

247 256 260 277

215 211 236 223

268 240 NC5 238

 

Mean 243 236 248 246

CV (%) 10.9 9.8 6.9 11.2

 

1 Tests performed by George Morrison and Glen Modica, ERL-N, USEPA, Narragansett, RI. 2 Three replicate exposure chambers with 10-15 larvae per treatment. 3 Adults collected in the field. 4 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 5 NC= No linear interpolation estimate could be calculated from the data, since none of the group response 

means were less than 50 percent of the control response mean.
212

TABLE 23. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPES1

Test Endpoint Sample Type

CV (%)2

Within-lab3 Between-lab4 Total5

IC25 Reference toxicant 22.0 29.1 36.4

Effluent 7.24 55.5 56.0

Receiving water - - 39.1

Average 14.6 42.3 43.8

1 From EPA’s WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b). 2 CVs were calculated based on the within-laboratory component of variability, the between-laboratory

component of variability, and the total interlaboratory variability (including both within-laboratory and betweenlaboratory components). For the receiving water sample type, within-laboratory and between-laboratory

components of variability could not be calculated since the study design did not provide within-laboratory

replication for this sample type. 3 The within-laboratory (intralaboratory) component of variability for duplicate samples tested at the same time in

the same laboratory. 4 The between-laboratory component of variability for duplicate samples tested at different laboratories. 5 The total interlaboratory variability, including within-laboratory and between-laboratory components of

variability. The total interlaboratory variability is synonymous with interlaboratory variability reported from

other studies where individual variability components are not separated. 
213

TABLE 24. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR VARIOUS

SAMPLE TYPES1

Test Endpoint Sample Type

Median

NOEC

Value

% of Results at

the Median

% of Results

±12

% of Results 

$23

Survival NOEC Reference toxicant 12.5% 72.7 18.2 9.09

Effluent 25% 38.5 46.1 15.4

Receiving water 25% 57.1 28.6 14.3

Growth 

NOEC Reference toxicant 12.5% 72.7 18.2 9.09

Effluent 25% 41.7 50.0 8.33

Receiving water 25% 57.1 28.6 14.3

1 From EPA’s WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b). 2 Percent of values at one concentration interval above or below the median. Adding this percentage to the

percent of values at the median yields the percent of values within one concentration interval of the median. 3 Percent of values two or more concentration intervals above or below the median.
214

SECTION 14

TEST METHOD

MYSID, MYSIDOPSIS BAHIA, SURVIVAL,

GROWTH, AND FECUNDITY TEST

METHOD 1007.0

14.1 SCOPE AND APPLICATION 

14.1.1 This method, adapted in part from USEPA (1987d), estimates the chronic toxicity of effluents and receiving

waters to the mysid, Mysidopsis bahia, during a seven-day, static renewal exposure. The effects include the

synergistic, antagonistic, and additive effects of all the chemical, physical, and additive components which

adversely affect the physiological and biochemical functions of the test organisms.

14.1.2 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).

14.1.3 Detection limits of the toxicity of an effluent or pure substance are organism dependent. 

14.1.4 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

volatile and highly degradable toxicants present in the source may not be detected in the test. 

14.1.5 This test 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. 

14.2 SUMMARY OF METHOD 

14.2.1 Mysidopsis bahia 7-day old juveniles are exposed to different concentrations of effluent, or to receiving

water in a static system, during the period of egg development. The test endpoints are survival, growth (measured as

dry weight), and fecundity (measured as the percentage of females with eggs in the oviduct and/or brood pouch). 

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 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). 

14.3.3 The test results can be confounded by (1) the presence of pathogenic and/or predatory organisms in the

dilution water, effluent, and receiving water, (2) the condition of the brood stock from which the test animals were

taken, (3) the amount and type of natural food in the effluent, receiving water, or dilution water, (4) nutritional

value of the brine shrimp, Artemia nauplii, fed during the test, and (5) the quantity of brine shrimp, Artemia nauplii,

or other food added during the test, which may sequester metals and other toxic substances, and lower the DO. 

14.3.4 pH drift during the test may contribute to artifactual toxicity when ammonia or other pH-dependent

toxicants (such as metals) are present. As pH increases, the toxicity of ammonia also increases (see Subsection

8.8.6), so upward pH drift may increase sample toxicity. For metals, toxicity may increase or decrease with

increasing pH. Lead and copper were found to be more acutely toxic at pH 6.5 than at pH 8.0 or 8.5, while nickel

and zinc were more toxic at pH 8.5 than at pH 6.5 (USEPA, 1992). In situations where sample toxicity is confirmed
215

to be artifactual and due to pH drift (as determined by parallel testing as described in Subsection 14.3.4.1), the

regulatory authority may allow for control of sample pH during testing using procedures outlined in Subsection

14.3.4.2. It should be noted that artifactual toxicity due to pH drift is not likely to occur unless pH drift is large

(more than 1 pH unit) and/or the concentration of some pH-dependent toxicant in the sample is near the threshold

for toxicity. 

14.3.4.1 To confirm that toxicity is artifactual and due to pH drift, parallel tests must be conducted, one with

controlled pH and one with uncontrolled pH. In the uncontrolled-pH treatment, the pH is allowed to drift during the

test. In the controlled-pH treatment, the pH is maintained using the procedures described in Subsection 14.3.4.2. 

The pH to be maintained in the controlled-pH treatment (or target pH) will depend on the objective of the test. If

the objective of the WET test is to determine the toxicity of the effluent in the receiving water, the pH should be

maintained at the pH of the receiving water (measured at the edge of the regulatory mixing zone). If the objective

of the WET test is to determine the absolute toxicity of the effluent, the pH should be maintained at the pH of the

sample after adjusting the sample salinity for use in marine testing. 

14.3.4.1.1 During parallel testing, the pH must be measured in each treatment at the beginning (i.e., initial pH) and

end (i.e., final pH) of each 24-h exposure period. For each treatment, the mean initial pH (e.g., averaging the initial

pH measured each day for a given treatment) and the mean final pH (e.g., averaging the final pH measured each day

for a given treatment) must be reported. pH measurements taken during the test must confirm that pH was

effectively maintained at the target pH in the controlled-pH treatment. For each treatment, the mean initial pH and

the mean final pH should be within ± 0.3 pH units of the target pH. Test procedures for conducting toxicity

identification evaluations (TIEs) also recommend maintaining pH within ± 0.3 pH units in pH-controlled tests

(USEPA, 1996).

14.3.4.1.2 Total ammonia also should be measured in each treatment at the outset of parallel testing. Total

ammonia concentrations greater than 5 mg/L in the 100% effluent are an indicator that toxicity observed in the test

may be due to ammonia (USEPA, 1992). 

14.3.4.1.3 Results from both of the parallel tests (pH-controlled and uncontrolled treatments) must be reported to

the regulatory authority. If the uncontrolled test meets test acceptability criteria and shows no toxicity at the

permitted instream waste concentration, then the results from this test should be used for determining compliance. 

If the uncontrolled test shows toxicity at the permitted instream waste concentration, then the results from the pHcontrolled test should be used for determining compliance, provided that this test meets test acceptability criteria

and pH was properly controlled (see Subsection 14.3.4.1.1). 

14.3.4.1.4 To confirm that toxicity observed in the uncontrolled test was artifactual and due to pH drift, the results

of the controlled and uncontrolled-pH tests are compared. If toxicity is removed or reduced in the pH-controlled

treatment, artifactual toxicity due to pH drift is confirmed for the sample. To demonstrate that a sample result of

artifactual toxicity is representative of a given effluent, the regulatory authority may require additional information

or additional parallel testing before pH control (as described in Subsection 14.3.4.2) is applied routinely to

subsequent testing of the effluent. 

14.3.4.2 The pH can be controlled with the addition of acids and bases and/or the use of a CO2-controlled

atmosphere over the test chambers. pH is adjusted with acids and bases by dropwise adding 1N NaOH or 1N HCl

(see Subsection 8.8.9). The addition of acids and bases should be minimized to reduce the amount of additional

ions (Na or Cl) added to the sample. pH is then controlled using the CO2-controlled atmosphere technique. This

may be accomplished by placing test solutions and test organisms in closed headspace test chambers, and then

injecting a predetermined volume of CO2 into the headspace of each test chamber (USEPA, 1991b; USEPA, 1992);

or by placing test chambers in an atmosphere flushed with a predetermined mixture of CO2 and air (USEPA, 1996). 

Prior experimentation will be needed to determine the appropriate CO2/air ratio or the appropriate volume of CO2 to

inject. This volume will depend upon the sample pH, sample volume, container volume, and sample constituents. 

If more than 5% CO2 is needed, adjust the solutions with acids (1N HCl) and then flush the headspace with no more

than 5% CO2 (USEPA, 1992). If the objective of the WET test is to determine the toxicity of the effluent in the
216

receiving water, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH of the receiving

water (measured at the edge of the regulatory mixing zone). If the objective of the WET test is to determine the

absolute toxicity of the effluent, atmospheric CO2 in the test chambers is adjusted to maintain the test pH at the pH

of the sample after adjusting the sample salinity for use in marine testing. USEPA (1996) and Mount and Mount

(1992) provide techniques and guidance for controlling test pH using a CO2-controlled atmosphere. In pHcontrolled testing, control treatments must be subjected to all manipulations that sample treatments are subjected to. 

These manipulations must be shown to cause no lethal or sublethal effects on control organisms. In pH-controlled

testing, the pH also must be measured in each treatment at the beginning and end of each 24-h exposure period to

confirm that pH was effectively controlled at the target pH level. 

14.4 SAFETY 

14.4.1 See Section 3, Health and Safety. 

14.5 APPARATUS AND EQUIPMENT 

14.5.1 Facilities for holding and acclimating test organisms. 

14.5.2 Brine shrimp, Artemia, culture unit -- see Subsection 14.6.12 below and Section 4, Quality Assurance. 

14.5.3 Mysid, Mysidopsis bahia, culture unit -- see Subsection 14.13 below. This test requires a minimum of 240

7-day old (juvenile) mysids. It is preferable to obtain the test organisms from an in-house culture unit. If it is not

feasible to culture mysids in-house, juveniles can be obtained from other sources, if shipped in well oxygenated

saline water in insulated containers. 

14.5.4 Samplers -- automatic sampler, preferably with sample cooling capability, that can collect a 24-h composite

sample of 5 L. 

14.5.5 Environmental chamber or equivalent facility with temperature control (26 ± 1°C). 

14.5.6 Water purification system -- Millipore Milli-Q®, deionized water or equivalent. 

14.5.7 Balance -- Analytical, capable of accurately weighing to 0.00001 g. 

14.5.8 Reference weights, Class S -- for checking performance of balance. Weights should bracket the expected

weights of the weighing pans and weighing pans plus organisms. 

14.5.9 Drying oven -- 50-105°C range, for drying organisms. 

14.5.10 Desiccator -- for holding dried organisms. 

14.5.11 Air pump -- for oil-free air supply. 

14.5.12 Air lines, and air stones -- for aerating cultures, brood chambers, and holding tanks, and supplying air to

test solutions with low DO. 

14.5.13 Meters, pH and DO -- for routine physical and chemical measurements. 

14.5.14 Tray -- for test vessels; approximately 90 X 48 cm to hold 56 vessels. 

14.5.15 Standard or micro-Winkler apparatus -- for determining DO and checking DO meters. 
217

14.5.16 Dissecting microscope (350-400X magnification) -- for examining organisms in the test vessels to

determine their sex and to check for the presence of eggs in the oviducts of the females. 

14.5.17 Light box -- for illuminating organisms during examination. 

14.5.18 Refractometer or other method -- for determining salinity. 

14.5.19 Thermometers, glass or electronic, laboratory grade -- for measuring water temperatures. 

14.5.20 Thermometers, bulb-thermograph or electronic-chart type -- for continuously recording temperature. 

14.5.21 Thermometer, National Bureau of Standards Certified (see USEPA Method 170.1, USEPA, 1979b) -- to

calibrate laboratory thermometers. 

14.5.22 Test chambers -- 200 mL borosilicate glass beakers or non-toxic 8 oz disposable plastic cups or other

similar containers. Forty-eight (48) test vessels are required for each test (eight replicates at each of five effluent

concentrations and a control). 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).

14.5.23 Beakers or flasks -- six, borosilicate glass or non-toxic plasticware, 2000 mL for making test solutions. 

14.5.24 Wash bottles -- for deionized water, for washing organisms from containers and for rinsing small

glassware and instrument electrodes and probes. 

14.5.25 Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-toxic plastic labware,

50-2000 mL for making test solutions. 

14.5.26 Separatory funnels, 2-L -- Two-four for culturing Artemia. 

14.5.27 Pipets, volumetric -- Class A, 1-100 mL. 

14.5.28 Pipets, automatic -- adjustable, 1-100 mL. 

14.5.29 Pipets, serological -- 1-10 mL, graduated. 

14.5.30 Pipet bulbs and fillers -- PROPIPET®, or equivalent. 

14.5.31 Droppers, and glass tubing with fire polished edges, 4 mm ID -- for transferring organisms. 

14.5.32 Forceps -- for transferring organisms to weighing pans. 

14.5.33 NITEX® or stainless steel mesh sieves (#150 μm, 500-1000 μm, 3-5 mm) -- for concentrating organisms. 

14.5.34 Depression glass slides or depression spot plates -- two, for observing organisms. 

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). 

14.6.2 Data sheets (one set per test) -- for data recording (Figures 2, 7, and 8). 

14.6.3 Tape, colored -- for labeling test chambers. 
218

14.6.4 Markers, waterproof -- for marking containers, etc.

14.6.5 Weighing pans, aluminum -- to determine the dry weight of organisms.

14.6.6 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument manufacturer) -- for standards and

calibration check (see USEPA Method 150.1, USEPA, 1979b). 

14.6.7 Membranes and filling solutions -- for dissolved oxygen probe (see USEPA Method 360.1, USEPA,

1979b), or reagents for modified Winkler analysis. 

14.6.8 Laboratory quality assurance samples and standards -- for the above methods. 

14.6.9 Reference toxicant solutions -- see Section 4, Quality Assurance. 

14.6.10 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). 

14.6.11 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. Dilution water

containing organisms that might prey upon or otherwise interfere with the test organisms should be filtered through

a fine mesh net (with 150 μm or smaller openings). 

14.6.11.1 Saline test and dilution water -- The salinity of the test water must be in the range of 20‰ to 30‰. The

salinity should vary by no more than ± 2‰ among the chambers on a given day. If effluent and receiving water

tests are conducted concurrently, the salinities of these tests should be similar. 

14.6.11.2 The overwhelming majority of industrial and sewage treatment effluents entering marine and estuarine

systems contain little or no measurable salts. Exposure of mysids to these effluents will require adjustments in the

salinity of the test solutions. It is important to maintain a constant salinity across all treatments. In addition, it may

be desirable to match the test salinity with that of the receiving water. Two methods are available to adjust salinities

– a hypersaline brine (HSB) derived from natural seawater or artificial sea salts. 

14.6.11.3 HSB has several advantages that make it desirable for use in toxicity testing. It can be made from any

high quality, filtered seawater by evaporation, and can be added to the effluent or to deionized water to increase the

salinity. Brine derived from natural seawater contains the necessary trace metals, biogenic colloids, and some of the

microbial components necessary for adequate growth, survival, and/or reproduction of marine and estuarine

organisms, and may be stored for prolonged periods without any apparent degradation. However, if 100‰ HSB is

used as a diluent, the maximum concentration of effluent that can be tested is 80% effluent at 30‰ salinity and 70%

effluent at 30‰ salinity. 

14.6.11.3.1 The ideal container for making brine from natural seawater is one that (1) has a high surface to volume

ratio, (2) is made of a non-corrosive material, and (3) is easily cleaned (fiberglass containers are ideal). Special care

should be used to prevent any toxic materials from coming in contact with the seawater being used to generate the

brine. If a heater is immersed directly into the seawater, ensure that the heater materials do not corrode or leach any

substances that would contaminate the brine. One successful method used is a thermostatically controlled heat

exchanger made from fiberglass. If aeration is used, only oil-free air compressors should be used to prevent

contamination. 

14.6.11.3.2 Before adding seawater to the brine generator, thoroughly clean the generator, aeration supply tube,

heater, and any other materials that will be in direct contact with the brine. A good quality biodegradable detergent

should be used, followed by several (at least three) thorough deionized water rinses. 
219

14.6.11.3.3 High quality (and preferably high salinity) seawater should be filtered to at least 10 μm before placing

into the brine generator. Water should be collected on an incoming tide to minimize the possibility of

contamination. 

14.6.11.3.4 The temperature of the seawater is increased slowly to 40°C. The water should be aerated to prevent

temperature stratification and to increase water evaporation. The brine should be checked daily (depending on the

volume being enerated) to ensure that the salinity does not exceed 100‰ and that the temperature does not exceed

40°C. Additional seawater may be added to the brine to obtain the volume of brine required. 

14.6.11.3.5 After the required salinity is attained, the HSB should be filtered a second time through a 1 mm filter

and poured directly into portable containers (20-L cubitainers or polycarbonate water cooler jugs are suitable). The

containers should be capped and labeled with the date the brine was generated and its salinity. Containers of HSB

should be stored in the dark and maintained under room temperature until used. 

14.6.11.3.6 If a source of HSB is available, test solutions can be made by following the directions below. 

Thoroughly mix together the deionized water and HSB before mixing in the effluent. 

14.6.11.3.7 Divide the salinity of the HSB by the expected test salinity to determine the proportion of deionized

water to brine. For example, if the salinity of the brine is 100‰ and the test is to be conducted at 20‰, 100‰

divided by 20‰ = 5.0. The proportion of brine is 1 part in 5 (one part brine to four parts deionized water). To

make 1 L of seawater at 20‰ salinity from a HSB of 100‰, 200 mL of brine and 800 mL of deionized water are

required. 

14.6.11.3.8 Table 2 illustrates the composition of 1800 mL test solutions at 20‰ if they are made by combining

effluent (0‰), deionized water and HSB of 100‰ (only). The volume (mL) of brine required is determined by

using the amount calculated above. In this case, 200 mL of brine is required for 1 L; therefore, 360 mL would be

required for 1.8 L of solution. The volumes of HSB required are constant. The volumes of deionized water are

determined by subtracting the volumes of effluent and brine from the total volume of solution: 1800 mL - mL

effluent - mL brine = mL deionized water. 

14.6.11.4 Artificial sea salts: FORTY FATHOMS® brand sea salts have been used successfully to culture and

perform life cycle tests with mysids (Horne, et al., 1983; ASTM, 1993) (see Section 7, Dilution Water). HW

MARINEMIX® sea salts have been used successfully to culture mysids and perform the mysid toxicity test (USEPA

Region 6 Houston Laboratory; EMSL-Cincinnati). In addition, a slightly modified version of the GP2 medium

(Spotte et al., 1984) has been successfully used to perform the mysid survival, growth, and fecundity test (Table 1). 

14.6.11.4.1 Synthetic sea salts are packaged in plastic bags and mixed with deionized water or equivalent. The

instructions on the package of sea salts should be followed carefully, and the salts should be mixed in a separate

container -- not in the culture tank. The deionized water used in hydration should be in the temperature range of 21-

26°C. Seawater made from artificial sea salts is conditioned (Spotte, 1973; Spotte, et al., 1984; Bower, 1983)

before it is used for culturing or testing. After adding the water, place an airstone in the container, cover, and aerate

the solution mildly for 24 h before use.

14.6.11.4.2 The GP2 reagent grade chemicals (Table 1) should be mixed with deionized (DI) water or its

equivalent in a container other than the culture or testing tanks. The deionized water used for hydration should be

between 21-26°C. The artificial seawater must be conditioned (aerated) for 24 h before use as the testing medium. 

If the solution is to be autoclaved, sodium bicarbonate is added after the solution has cooled. A stock solution of

sodium bicarbonate is made up by dissolving 33.6 g NaHCO3 in 500 mL of deionized water. Add 2.5 mL of this

stock solution for each liter of the GP2 artificial seawater.
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TABLE 1. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2 ARTIFICIAL

SEAWATER FOR THE MYSID, MYSIDOPSIS BAHIA, TOXICITY TEST1,2,3

Compound Concentration Amount (g)

 (g/L) Required for

 20L

NaCl 21.03 420.6

Na2SO4 3.52 70.4

Kcl 0.61 12.2

KBr 0.088 1.76

Na2B4O7@10 H2O 0.034 0.68

MgCl2@6 H2O 9.50 190.0

CaCl2@2 H2O 1.32 26.4

SrCl2@6 H2O 0.02 0.400

NaHCO3 0.17 3.40

1 Modified GP2 from Spotte et al. (1984). 2 The constituent salts and concentrations were taken from USEPA (1990b). The salinity is 30.89 g/L. 3 GP2 can be diluted with deionized (DI) water to the desired test salinity.

14.6.12 BRINE SHRIMP, ARTEMIA, NAUPLII -- for feeding cultures and test organisms.

14.6.12.1 Newly hatched Artemia nauplii are used for food for the stock cultures and test organisms. Although

there are many commercial sources of brine shrimp cysts, the Brazilian or Colombian strains are preferred because

the supplies examined have had low concentrations of chemical residues and produce nauplii of suitably small size. 
221

TABLE 2. QUANTITIES OF EFFLUENT, DEIONIZED WATER, AND HYPERSALINE BRINE (100‰)

NEEDED TO PREPARE 1800 ML VOLUMES OF TEST SOLUTION WITH A SALINITY OF

20‰

 Effluent Volume of Volume of Volume of

Concentration Effluent Deionized Hypersaline Total Volume

 (%) (0‰) Water Brine (mL)

 (mL) (mL) (mL)

80 1440 0 360 1800

40 720 720 360 1800

20 360 1080 360 1800

10 180 1260 360 1800

5 90 1350 360 1800

Control 0 1440 360 1800

Total 2790 5850 2160 10800

14.6.12.2 Each new batch of Artemia cysts must be evaluated for size (Vanhaecke and Sorgeloos, 1980, and

Vanhaecke et al., 1980) and nutritional suitability (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. 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 organic chlorine exceeds 0.15 μg/g wet weight or the total

concentration of organochlorine pesticides plus PCBs exceeds 0.30 μg/g wet weight (For analytical methods see

USEPA, 1982).

14.6.12.2.1 Artemia nauplii are obtained as follows: 

1 Add 1 L of seawater, or an aqueous uniodized salt (NaCl) solution prepared with 35 g salt or

artificial sea salts to 1 L of deionized water, to a 2-L separatory funnel, or equivalent. 

2. Add 10 mL Artemia cysts to the separatory funnel and aerate for 24 h at 27°C. Hatching time

varies with incubation temperature and the geographic strain of Artemia used (see USEPA, 1985a;

USEPA, 2002a; 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 minutes. 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 μm NITEX® or stainless steel screen,

and rinse with seawater or equivalent before use.

14.6.12.3 Testing Artemia nauplii as food for toxicity test organisms. 

14.6.12.3.1 The primary criteria for acceptability of each new supply of brine shrimp, cysts is adequate survival,

growth, and reproduction of the mysids. The mysids used to evaluate the acceptability of the brine shrimp nauplii

must be of the same geographical origin and stage of development (7 days old) as those used routinely in the

toxicity tests. Sufficient data to detect differences in survival and growth should be obtained by using eight

replicate test chambers, each containing 5 mysids, for each type of food. 
222

 14.6.12.3.2 The feeding rate and frequency, test vessels, volume of control water, duration of the test, and age of

the Artemia nauplii at the start of the test, should be the same as used for the routine toxicity tests. 

14.6.12.3.3 Results of the brine shrimp, Artemia, nauplii 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, growth, and reproduction of the mysids fed the two sources of nauplii. 

14.6.13 TEST ORGANISMS, Mysidopsis bahia (see Rodgers et al., 1986 and USEPA, 2002a for information on

mysid ecology). The genus name of this organism was formally changed to Americamysis (Price et al., 1994);

however, the method manual will continue to refer to Mysidopsis bahia to maintain consistency with previous

versions of the method.

14.6.13.1 Brood Stock

14.6.13.1.1 To provide an adequate supply of juveniles for a test, mysid, Mysidopsis bahia, cultures should be

started at least four weeks before the test animals are needed. At least 200 mysids, Mysidopsis bahia, should be

placed in each culture tank to ensure that 1500 to 2000 animals will be available by the time preparations for a test

are initiated. 

14.6.13.1.2 Mysids, Mysidopsis bahia, may be shipped or otherwise transported in polyethylene bottles or

CUBITAINERS®. Place 50 animals in 700 mL of seawater in a 1-L shipping container. To control bacterial growth

and prevent DO depletion during shipment, do not add food. Before closing the shipping container, oxygenate the

water for 10 min. The mysids, Mysidopsis bahia, will starve if not fed within 36 h, therefore, they should be

shipped so that they are not in transit more than 24 h. 

14.6.13.1.3 The identification of the Mysidopsis bahia stock culture should be verified using the key from Heard

(1982), Price (1978), Price, (1982), Stuck et al. (1979a), and Stuck et al. (1979b). Records of the verification

should be retained along with a few of the preserved specimens. 

14.6.13.1.4 Glass aquaria (120- to 200-L) are recommended for cultures. Other types of culture chambers may

also be convenient. Three or more separate cultures should be maintained to protect against loss of the entire

culture stock in case of accident, low DO, or high nitrite levels, and to provide sufficient numbers of juvenile

mysids, Mysidopsis bahia, for toxicity tests. Fill the aquaria about three-fourths full of seawater. A flow-through

system is recommended if sufficient natural seawater is available, but a closed, recirculating or static renewal

system may be used if proper water conditioning is provided and care is exercised to keep the pH above 7.8 and

nitrite levels below 0.05 mg/L. 

14.6.13.1.5 Standard aquarium undergravel filters should be used with either the flow-through or recirculating

system to provide aeration and a current conducive to feeding (Gentile et al., 1983). The undergravel filter is

covered with a prewashed, coarse (2-5 mm) dolomite substrate, 2.5 cm deep for flow-through cultures or 10 cm

deep for recirculating cultures. 

14.6.13.1.6 The recirculating culture system is conditioned as follows: 

1. After the dolomite has been added, the filter is attached to the air supply and operated for 24 h. 

2. Approximately 4 L of seed water obtained from a successfully operating culture is added to the

culture chamber. 

3. The nitrite level is checked daily with an aquarium test kit or with EPA Method 354.1 

(USEPA, 1979b). 

4. Add about 30 mL of concentrated Artemia nauplii every other day until the nitrite level reaches at

least 2.0 mg/L. The nitrite will continue to rise for several days without adding more Artemia

nauplii and will then slowly decrease to less than 0.05 mg/L. 
223

5. After the nitrite level falls below 0.05 mg/L, add another 30 mL of Artemia nauplii concentrate

and check the nitrite concentration every day. 

6. Continue this cycle until the addition of Artemia nauplii does not cause a rise in the nitrite

concentration. The culture chamber is then conditioned and is ready to receive mysids. 

7. Add only a few (5-20) mysids at first, to determine if conditions are favorable. If these mysids are

still doing well after a week, several hundred more can be added. 

14.6.13.1.7 It is important to add enough food to keep the adult animals from cannibalizing the young, but not so

much that the DO is depleted or that there is a buildup of toxic concentrations of ammonia and nitrite. Just enough

newly-hatched Artemia nauplii are fed twice a day so that each feeding is consumed before the next feeding. 

14.6.13.1.8 Natural seawater is recommended as the culture medium, but HSB may be used to make up the culture

water if natural seawater is not available. EMSL-Cincinnati has successfully used FORTY FATHOMS® artificial

sea salts for culturing and toxicity tests of mysids, and USEPA, Region 6 has used HW MARINEMIX® artificial sea

salts. 

14.6.13.1.9 Mysids, Mysidopsis bahia, should be cultured at a temperature of 26 ± 1°C. No water temperature

control equipment is needed if the ambient laboratory temperature remains in the recommended range, and if there

are no frequent, rapid, large temperature excursions in the culture room. 

14.6.13.1.10 The salinity should be maintained at 30 ± 2‰, or at a lower salinity (but not less than 20‰) if most of

the tests will be conducted at a lower salinity. 

14.6.13.1.11 Day/night cycles prevailing in most laboratories will provide adequate illumination for normal growth

and reproduction. A 16-h/8-h day/night cycle in which the light is gradually increased and decreased to simulate

dawn and dusk conditions, is recommended. 

14.6.13.1.12 Mysid, Mysidopsis bahia, culture may suffer if DOs fall below 5 mg/L for extended periods. The

undergravel filter will usually provide sufficient DO. If the DO drops below 5 mg/L at 25°C and 30‰, additional

aeration should be provided. Measure the DO in the cultures daily the first week and then at least weekly thereafter. 

14.6.13.1.13 Suspend a clear glass or plastic panel over the cultures, or use some other means of excluding dust

and dirt, but leave enough space between the covers and culture tanks to allow circulation of air over the cultures. 

14.6.13.1.14 If hydroids or worms appear in the cultures, remove the mysids and clean the chambers thoroughly,

using soap and hot water. Rinse once with acid (10% HCl) and three times with distilled or deionized water. 

Mysids with attached hydroids should be discarded. Those without hydroids should be transferred by hand

pipetting into three changes of clean seawater before returning them to the cleaned culture chamber. To guard

against predators, natural seawater should be filtered through a net with 30 μm mesh openings before entering the

culture vessels. 

14.6.13.1.15 Mysids, Mysidopsis bahia, are very sensitive to low pH and sudden changes in temperature. Care

should be taken to maintain the pH at 8.0 ± 0.3, and to limit rapid changes in water temperature to less than 3°C. 

14.6.13.1.16 Mysids, Mysidopsis bahia, should be handled carefully and as little as possible so that they are not

unnecessarily stressed or injured. They should be transferred between culture chambers with long handled cups

with netted bottoms. Animals should be transferred to the test vessels with a large bore pipette (4-mm), taking care

to release the animals under the surface of the water. Discard any mysids that are injured during handling. 
224

14.6.13.1.17 Culture Maintenance (Also See USEPA, 2002a)

14.6.13.1.17.1 Cultures in closed, recirculating systems are fed twice a day. If no nauplii are present in the culture

chamber after four hours, the amount of food should be increased slightly. In flow-through systems, excess food

can be a problem by promoting bacterial growth and low dissolved oxygen. 

14.6.13.1.17.2 Careful culture maintenance is essential. The organisms should not be allowed to become too

crowded. The cultures should be cropped as often as necessary to maintain a density of about 20 mysids per liter. 

At this density, at least 70% of the females should have eggs in their brood pouch. If they do not, the cultures are

probably under stress, and the cause should be found and corrected. If the cause cannot be found, it may be

necessary to restart the cultures with a clean culture chamber, a new batch of culture water, and clean gravel. 

14.6.13.1.17.3 In closed, recirculating systems, about one third of the culture water should be replaced with newly

prepared seawater every week. Before siphoning the old media from the culture, it is recommended that the sides of

the vessel be scraped and the gravel carefully turned over to prevent excessive buildup of algal growth. Twice a

year the mysids should be removed from the recirculating cultures, the gravel rinsed in clean seawater, the sides of

the chamber washed with clean seawater, and the gravel and animals returned to the culture vessel with newly

conditioned seawater. No detergent should be used, and care should be taken not to rinse all the bacteria from the

gravel. 

14.6.13.2 Test Organisms 

14.6.13.2.1 The test is begun with 7-day-old juveniles. To have the test animals available and acclimated to test

conditions at the start of the test, gravid females must be obtained from the stock culture eight days in advance of

the test. Whenever possible, brood stock should be obtained from cultures having similar salinity, temperature, light

regime, etc., as are to be used in the toxicity test. 

14.6.13.2.2 Eight days before the test is to start, sufficient gravid females are placed in brood chambers. Assuming

that 240 juveniles are needed for each test, approximately half this number (120) of gravid females should be

transferred to brood chambers. The mysids are removed from the culture tank with a net or netted cup and placed in

20-cm diameter finger bowls. The gravid females are transferred from the finger bowls to the brood chambers with

a large-bore pipette or, alternatively, are transferred by pouring the contents of the finger bowls into the water in the

brood chambers. 

14.6.13.2.3 The mysid juveniles may be collected for the toxicity tests by transferring gravid females from the

stock cultures to netted (1000 μm) flow-through containers (Figure 1) held within 4-L glass, wide-mouth separatory

funnels. Newly released juveniles can pass through the netting, whereas the females are retained. The gravid

females are fed newly hatched Artemia nauplii, and are held overnight to permit the release of young. The juvenile

mysids are collected by opening the stopcock on the funnel and collecting them in another container from which

they are transferred to holding tanks using a wide bore (4 mm ID) pipette. The brood chambers usually require

aeration to maintain sufficient DO and to keep the food in suspension.
225

Figure 1. Apparatus (brood chamber) for collection of juvenile mysids,

Mysidopsis bahia. From USEPA (1987d). 

14.6.13.2.4 The temperature in the brood chamber should be maintained at the upper acceptable culture limit (26 -

27°C), or 1°C higher than the cultures, to encourage faster brood release. At this temperature, sufficient juveniles

should be produced for the test. 

14.6.13.2.5 The newly released juveniles (age = 0 days) are transferred to 20-L glass aquaria (holding vessels)

which are gently aerated. Smaller holding vessels may be used, but the density of organisms should not exceed 10

mysids per liter. The test animals are held in the holding vessel for six days prior to initiation of the test. The

holding medium is renewed every other day. 
226

14.6.13.2.6 During the holding period, the mysids are acclimated to the salinity at which the test will be conducted,

unless already at that salinity. The salinity should be changed no more than 2‰ per 24 h to minimize stress on the

juveniles. 

14.6.13.2.7 The temperature during the holding period is critical to mysid development, and must be maintained at

26 ± 1°C. If the temperature cannot be maintained in this range, it is advisable to hold the juveniles an additional

day before beginning the test.

14.6.13.2.8 During the holding period, just enough newly-hatched Artemia nauplii are fed twice a day (a total of at

least 150 nauplii per mysid per day) so that some food is constantly present.

14.6.13.2.9 If the test is to be performed in the field, the juvenile mysids, Mysidopsis bahia, should be gently

siphoned into 1-L polyethylene wide-mouth jars with screw-cap lids filled two-thirds full with clean seawater from

the holding tank. The water in these jars is aerated for 10 min, and the jars are capped and packed in insulated

boxes for shipment to the test site. Food should not be added to the jars to prevent the development of excessive

bacterial growth and a reduction in DO. 

14.6.13.2.10 Upon arrival at the test site (in less than 24 h) the mysids, Mysidopsis bahia, are gently poured from

the jars into 20-cm diameter glass culture dishes. The jars are rinsed with salt water to dislodge any mysids that may

adhere to the sides. If the water appears milky, siphon off half of it with a netted funnel (to avoid siphoning the

mysids) and replace with clean salt water of the same salinity and temperature. If no Artemia nauplii are present in

the dishes, feed about 150 Artemia nauplii per mysid.

14.6.13.2.11 The pre-test holding conditions of test organisms (as well as the test conditions) have been shown to

significantly influence the success of achieving the test acceptability criteria for the fecundity endpoint (egg

production by 50% or more of control females). Temperature, feeding, and organism density are important factors

in the rate of mysid development. Laboratories should optimize these factors (within the limits of the test

procedure) during both the pre-test holding period and the testing period to encourage achieving the test

acceptability criteria for the fecundity endpoint. If test organisms are purchased, the testing laboratory should also

confer with the supplier to ensure that pre-test holding conditions are optimized to successfully achieve the

fecundity endpoint. Lussier et al. (1999) found that by increasing holding temperature and test temperature from

26°C ± 1°C to 26°C - 27°C and maintaining holding densities to #10 organisms / L, the percentage of tests meeting

the test acceptability criteria for fecundity increased from 60% to 97%. While the fecundity endpoint is an optional

endpoint, it is often the most sensitive measure of toxicity, and the 7-d mysid test estimates the chronic toxicity of

effluents most effectively when all three endpoints (survival, growth, and fecundity) are measured (Lussier et al.

1999). 

14.7 EFFLUENT AND RECEIVING WATER 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. 

14.9.2 The reference toxicant recommended for use with the mysid 7-day test is copper sulfate or sodium dodecyl

sulfate. 
227

14.10 TEST PROCEDURES 

14.10.1 TEST DESIGN 

14.10.1.1 The test consists of at least five effluent concentrations plus a site water control and a reference water

treatment (natural seawater or seawater made up from hypersaline brine, or equivalent).

14.10.1.2 Effluent concentrations are expressed as percent effluent. 

14.10.1.3 Eight replicate test vessels, each containing 5 to 7 day old animals, are used per effluent concentration

and control. 

14.10.2 TEST SOLUTIONS 

14.10.2.1 Receiving waters 

14.10.2.1.1 The sampling point(s) is determined by the objectives of the test. At estuarine and marine sites,

samples are usually collected at mid-depth. Receiving water toxicity is determined with samples used directly as

collected or with samples passed through a 60 μm NITEX® filter and compared without dilution, against a control. 

Using eight replicate chambers per test, each containing 150 mL, and 400 mL for chemical analysis, would require

approximately 1.6 L or more of sample per test per day.

14.10.2.2 Effluents 

14.10.2.2.1 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 ± 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 dilution factors are increased beyond 0.5 and declines

rapidly if smaller dilution factors are used. Therefore, USEPA recommends the use of the $ 0.5 dilution factor.

If 100‰ HSB is used as a diluent, the maximum concentration of effluent that can be tested will be 80% at 20‰

and 70% at 30‰ salinity. 

14.10.2.2.2 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 high mortality is observed during the first 1-to-2 h of

the test, additional dilutions at the lower range of effluent concentrations should be added.

14.10.2.2.3 The volume of effluent required for daily renewal of eight replicates per concentration for five

concentrations of effluent and a control, each containing 150 mL of test solution, is approximately 1200 mL. 

Prepare enough test solution (approximately 1600 mL) at each effluent concentration to provide 400 mL additional

volume for chemical analyses. 

14.10.2.2.4 Just prior to test initiation (approximately 1 h), the temperature of a sufficient quantity of the sample to

make the test solutions should be adjusted to the test temperature (26 ± 1°C) and maintained at that temperature

during the addition of dilution water. 

14.10.2.2.5 Higher effluent concentrations (i.e., 25%, 50%, and 100%) may require aeration to maintain adequate

dissolved oxygen concentrations. However, if one solution is aerated, all concentrations must be aerated. Aerate

effluent as it warms and continue to gently aerate test solutions in the test chambers for the duration of the test.

14.10.2.2.6 Effluent dilutions should be prepared for all replicates in each treatment in one flask to minimize

variability among the replicates. The test chambers (cups) are labeled with the test concentration and replicate

number. Dispense 150 mL of the appropriate effluent dilution to each test chamber. 
228

14.10.2.3 Dilution Water

14.10.2.3.1 Dilution water may be uncontaminated natural seawater (receiving water), HSB prepared from natural

seawater, or artificial seawater prepared from FORTY FATHOMS® or GP2 sea salts (see Table 1 and Section 7,

Dilution Water). Other artificial sea salts may be used for culturing mysid and for the survival, growth, and

fecundity test if the control criteria for acceptability of test data are satisfied.

14.10.3 START OF THE TEST 

14.10.3.1 The test should begin as soon as possible, preferably within 24 h after 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 test be started more than 72 h

after sample collection (see Section 8, Effluent and Receiving Water Sampling, Sample Handling, and Sample

Preparation for Toxicity Tests). 

14.10.3.2 Begin the test by randomly placing five animals (one at a time) in each test cup of each treatment using a

large bore (4 mm ID) pipette (see Appendix A for an example of randomization). It is easier to capture the animals

if the volume of water in the dish is reduced and the dish is placed on a light table. It is recommended that the

transfer pipette be rinsed frequently because mysids tend to adhere to the inside surface. 

14.10.4 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE 

14.10.4.1 The light quality and intensity under ambient laboratory conditions are generally adequate. Light

intensity of 10-20 μE/m2

/s, or 50 to 100 foot candles (ft-c), with a photoperiod of 16 h light and 8 h darkness. It is

critical that the test water temperature be maintained at 26 ± 1°C. It is recommended that the test water temperature

be continuously recorded. The salinity should vary no more than ± 2‰ among chambers on a given day. If effluent

and receiving water tests are conducted concurrently, the salinities of these tests should be similar. 

14.10.4.1.1 If a water bath is used to maintain the test temperature, the water depth surrounding the test cups

should be at least 2.5 cm deep. 

14.10.4.1.2 Rooms or incubators with high volume ventilation should be used with caution because the

volatilization of the test solutions and evaporation of dilution water may cause wide fluctuations in salinity. 

Covering the test cups with clear polyethylene plastic may help prevent volatilization and evaporation of the test

solutions. 

14.10.5 DISSOLVED OXYGEN (DO) CONCENTRATION

14.10.5.1 Aeration may affect the toxicity of effluents and should be used only as a last resort to maintain a

satisfactory DO. The DO should be measured on new solutions at the start of the test (Day 0) and before daily

renewal of test solutions on subsequent days. The DO 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 treatments and the control should be aerated. The aeration rate should not exceed 100 bubbles/minute,

using a pipet with a 1-2 mm orifice, such as a 1-mL KIMAX® serological pipet, or equivalent. Care should be taken

to ensure that turbulence resulting from aeration does not cause undue stress on the mysid.

14.10.6 FEEDING

14.10.6.1 Artemia nauplii are prepared as described above.

14.10.6.2 During the test, the mysids in each test chamber should be fed Artemia nauplii, (less than 24-h old), at

the rate of 150 nauplii per mysid per day. Adding the entire daily ration at a single feeding immediately after test

solution renewal may result in a significant DO depression. Therefore, it is preferable to feed half of the daily
229

ration immediately after test solution renewal, and the second half 8 - 12 h later. Increase the feeding if the nauplii

are consumed in less than 4 h. It is important that the nauplii be washed before introduction to the test chamber. 

14.10.7 DAILY CLEANING OF TEST CHAMBERS

14.10.7.1 Before the daily renewal of test solutions, uneaten and dead Artemia, dead mysids and other debris are

removed from the bottom of the test chambers with a pipette. As much of the uneaten Artemia as possible should be

removed from each chamber to ensure that the mysids principally eat new hatched nauplii. By placing the test

chambers on a light box, inadvertent removal of live mysids can be greatly reduced because they can be more easily

seen. Any incidence of removal of live mysids from the test chambers during cleaning, and subsequent return to the

chambers should be noted in the test records.

14.10.8 OBSERVATIONS DURING THE TEST

14.10.8.1 Routine Chemical and Physical Determinations

14.10.8.1.1 DO is measured at the beginning and end of each 24-h exposure period in one test chamber at each test

concentration and in the control.

14.10.8.1.2 Temperature, pH, and salinity are measured at the end of each 24-h exposure period in one test

chamber at each concentration and in the control. Temperature should also be monitored continuously 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 chambers at least at the end of the test to determine temperature variation in

environmental chamber.

14.10.8.1.3 The pH is measured in the effluent sample each day before new test solutions are made.

14.10.8.2 Routine Biological Observations

14.10.8.2.1 The number of live mysids are counted and recorded each day when the test solutions are renewed

(Figure 7). Dead animals and excess food should be removed with a pipette before test solutions are renewed.

14.10.8.2.2 Protect the mysids from unnecessary disturbance during the test by carrying out the daily test

observations, solution renewals, and removal of the dead mysids, carefully. Make sure the mysids remain immersed

during the performance of the above operations.

14.10.9 TEST SOLUTION RENEWAL 

14.10.9.1 Before the daily renewal of test solutions, slowly pour off all but 10 mL of the old test medium into a 20

cm diameter culture dish on a light table. Be sure to check for animals that may have adhered to the sides of the test

chamber. Rinse them back into the test cups. Add 150 mL of new test solution slowly to each cup. Check the

culture dish for animals that may have been poured out with the old media, and return them to the test chamber. 

14.10.10 TERMINATION OF THE TEST 

14.10.10.1 After measuring the DO, pH, temperature, and salinity and recording survival, terminate the test by

pouring off the test solution in all the cups to a one cm depth and refilling the cups with clean seawater. This will

keep the animals alive, but not exposed to the toxicant, while waiting to be examined for sex and the presence of

eggs. 

14.10.10.2 The live animals must be examined for eggs and the sexes determined within 12 h of the termination of

the test. If the test was conducted in the field, and the animals cannot be examined on site, the live animals should

be shipped back to the laboratory for processing. Pour each replicate into a labeled 100 mL plastic screw capped
230

jar, and send to the laboratory immediately. 

14.10.10.3 If the test was conducted in the laboratory, or when the test animals arrive in the laboratory from the

field test site, the test organisms must be processed immediately while still alive as follows: 

14.10.10.3.1 Examine each replicate under a stereomicroscope (240X) to determine the number of immature

animals, the sex of the mature animals, and the presence or absence of eggs in the oviducts or brood sacs of the

females (see Figures 3-6). This must be done while the mysids are alive because they turn opaque upon dying. 

This step should not be attempted by a person who has not had specialized training in the determination of sex and

presence of eggs in the oviduct. NOTE: Adult females without eggs in the oviduct or brood sac look like

immature mysids (see Figure 6).
231

TEST: 

START DATE:

SALINITY:

TRTMT TEMP SALINITY D.O. pH TRTMT TEMP SALINITY D.O. pH

DAY 1 REP

REP

DAY 2 REP

REP

DAY 3 REP

REP

DAY 4 REP

REP

DAY 5 REP

REP

DAY 6 REP

REP

DAY 7 REP

REP

TRTMT TEMP SALINITY D.O. pH TRTMT TEMP SALINITY D.O pH

DAY 1 REP

REP

DAY 2 REP

REP

DAY 3 REP

REP

DAY 4 REP

REP

DAY 5 REP

REP

DAY 6 REP

REP

DAY 7 REP

REP

Figure 2. Data form for the mysid, Mysidopsis bahia, water quality measurements. From USEPA (1987d).
232

Figure 3. Mature female mysid, Mysidopsis bahia, with eggs in oviducts. From USEPA (1987d).
233

Figure 4. Mature female mysid, Mysidopsis bahia, with eggs in oviducts and developing embryos in the

brood sac. Above: lateral view. Below: dorsal view. From USEPA (1987d). 
234

14.10.10.3.2 Record the number of immatures, males, females with eggs and females without eggs on data sheets

(Figure 7).

14.10.10.3.3 Rinse the mysids by pipetting them into a small netted cup and dipping the cup into a dish containing

deionized water. Using forceps, place the mysids from each replicate cup on tared weighing boats and dry at 60°C

for 24 h or at 105°C for at least 6 h. 

14.10.10.3.4 Immediately upon removal from the drying oven, the weighing pans were placed in a dessicator until

weighed, to prevent absorption of moisture from the air. Weigh to the nearest mg. Record weighing pans and

subtract the tare weight to determine the dry weight of the mysid in each replicate. Record the weights (Figure 8). 

For each test chamber, divide the first dry weight by the number of original mysids per replicate to determine the

average individual dry weight and record data. For the controls also calculate the mean weight per surviving mysid

in the test chamber to evaluate if weights met test acceptability criteria (see Subsection 14.12).

14.10.9.3.5 Pieces of aluminum foil (1-cm square) or small aluminum weighing pans can be used for dry weight

analyses. The weighing pans should not exceed 10 mg in weight.

14.10.9.3.6 Number each pan with a waterproof pen with the treatment concentration and replicate number. 

Forty-eight (48) weigh pans are required per test if all the organisms survive.
235

Figure 5. Mature male mysid, Mysidopsis bahia. From USEPA (1987d). 

14.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

14.11.1 A summary of test conditions and test acceptability criteria is listed in Table 3. 

14.12 ACCEPTABILITY OF TEST RESULTS 

14.12.1 The minimum requirements for an acceptable test are 80% survival and an average weight of at least 0.20

mg/surviving mysid in the controls. If fecundity in the controls is adequate (egg production by 50% of females),

fecundity should be used as a criterion of effect in addition to survival and growth. 
236

Figure 6. Immature mysid, Mysidopsis bahia, (A) lateral view, (B) dorsal view. From USEPA (1987d). 

14.13 DATA ANALYSIS

14.13.1 GENERAL

14.13.1.1 Tabulate and summarize the data. Table 4 presents a sample set of survival, growth, and fecundity data.

14.13.1.2 The endpoints of the mysid 7-day chronic test are based on the adverse effects on survival, growth, and

egg development. 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, growth, and

fecundity 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

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, growth, and fecundity, 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. 

14.13.1.3 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. 
237

TEST: 

START DATE: 

SALINITY: 

TREATMENT/

REPLICATE

DAY 1

# ALIVE

DAY 2

# ALIVE

DAY 3

# ALIVE

DAY 4

# ALIVE

DAY 5

# ALIVE

DAY 6

# ALIVE

DAY 7

# ALIVE

FEMALES

W/EGGS

FEMALES

NO EGGS MALES IMMATURES

 

 

 

C 

 

 

 

 

 

 

 

 1 

 

 

 

 

 

 

 

 2 

 

 

 

 

Figure 7. Data form for the mysid, Mysidopsis bahia, survival and fecundity data. From USEPA (1987d).
238

TEST: 

START DATE: 

SALINITY: 

TREATMENT/

REPLICATE

DAY 1

# ALIVE

DAY 2

# ALIVE

DAY 3

# ALIVE

DAY 4

# ALIVE

DAY 5

 # ALIVE

DAY 6

# ALIVE

DAY 7

# ALIVE

FEMALES

W/EGGS

FEMALES

NO EGGS MALES IMMATURES

 1

 2

 3

 3 4

 5

 6

 7

 8

 1

 2

 3

 4 4

 5

 6

 7

 8

 1

 2

 3

 5 4

 5

 6

 7

 8

Figure 7. Data form for the mysid, Mysidopsis bahia, survival and fecundity data (CONTINUED). From

USEPA (1987d).
239

TEST: 

START DATE: 

SALINITY: 

TREATMENT/REPLICATE PAN # TARE WT. TOTAL WT. ANIMAL WT. # OF ANIMALS WT./ANIMAL

1

2

3

C 4

5

6

7

8

1

2

3

1 4

5

6

7

8

1

2

3

2 4

5

6

7

8

Figure 8. Data form for the mysid, Mysidopsis bahia, dry weight measurements. From USEPA (1987d).
240

TEST: 

START DATE: 

SALINITY: 

TREATMENT/REPLICATE PAN # TARE WT. TOTAL WT. ANIMAL WT. # OF ANIMALS WT./ANIMAL

1

2

3

3 4

5

6

7

8

1

2

3

4 4

5

6

7

8

1

2

3

5 4

5

6

7

8

Figure 8. Data form for the mysid, Mysidopsis bahia, dry weight measurements (CONTINUED). From

USEPA (1987d).
241

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE

MYSID, MYSIDOPSIS BAHIA, SEVEN DAY SURVIVAL, GROWTH, AND FECUNDITY

TEST WITH EFFLUENTS AND RECEIVING WATERS (TEST METHOD 1007.0)1

1. Test type: Static renewal (required) 

2. Salinity: 20‰ to 30‰ (± 2‰ of the selected test salinity) 

(recommended) 

3. Temperature: 26 ± 1°C (recommended) 

Test temperatures must not deviate (i.e., maximum minus

minimum temperature) by more than 3°C during the test

(required) 

4. Light quality: Ambient laboratory illumination (recommended) 

5. Light intensity: 10-20 μE/m2

/s (50-100 ft-c.) 

(ambient laboratory levels) (recommended) 

6. Photoperiod: 16 h light, 8 h darkness, with phase in/out period 

(recommended)

7. Test chamber: 8 oz plastic disposable cups, or 400 mL glass beakers 

(recommended) 

8. Test solution volume: 150 mL per replicate (recommended minimum) 

9. Renewal of test solutions: Daily (required) 

10. Age of test organisms: 7 days (required) 

11. No. organisms per test chamber: 5 (required minimum)

12. No. replicate chambers per concentration: 8 (required minimum)

13. No. larvae per concentration: 40 (required minimum)

14. Source of food: Newly hatched Artemia nauplii (less than 24 h old)(required) 

15. Feeding regime: Feed 150 24 h old nauplii per mysid daily, half after test 

solution renewal and half after 8-12 h (recommended)

16. Cleaning: Pipette excess food from cups daily immediately before test 

solution renewal and feeding (recommended) 

1

 For the purposes of reviewing WET test data submitted under NPDES permits, each test condition listed above

is identified as required or recommended (see Subsection 10.2 for more information on test review). Additional

requirements may be provided in individual permits, such as specifying a given test condition where several

options are given in the method. 
242

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE

MYSID, MYSIDOPSIS BAHIA, SEVEN DAY SURVIVAL, GROWTH, AND FECUNDITY

TEST WITH EFFLUENTS AND RECEIVING WATERS (TEST METHOD 1007.0)

(CONTINUED)

17. Aeration: None unless DO falls below 4.0 mg/L, then gently aerate in all cups

(recommended)

18. Dilution water: Uncontaminated source of natural seawater, deionized water mixed

with hypersaline brine or artificial sea salts (HW MARINEMIX®,

FORTY FATHOMS®, GP2 or equivalent) (available options) 

19. Test concentrations: Effluents: 5 and a control (required) 

Receiving waters: 100% receiving water (or minimum of 5) and a

control (recommended) 

20. Dilution factor: Effluents: $ 0.5 series (required) 

Receiving waters: None, or $ 0.5 (recommended)

21. Test duration: 7 days (required) 

22. Endpoints: Survival and growth (required); and egg development 

(recommended)

23. Test acceptability criteria: 80% or greater survival, average dry weight 0.20 mg or greater in

controls (required); fecundity may be used if 50% or more of females

in controls produce eggs (required if fecundity endpoint used)

24. Sampling requirements: 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 (e.g., 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) (required) 

25. Sample volume required: 3 L per day (recommended) 
243

TABLE 4. DATA FOR MYSIDOPSIS BAHIA 7-DAY SURVIVAL, GROWTH, AND FECUNDITY TEST1

Treatment Replicate Total No. Total Females Mean

Chamber Mysids Alive Females w/Eggs Weight

Control 1 5 4 1 1 0.146

2 5 4 2 2 0.118

3 5 5 3 2 0.216

4 5 5 1 1 0.199

5 5 5 2 2 0.176

6 5 5 5 4 0.243

7 5 5 2 2 0.213

8 5 4 3 3 0.144

50 ppb 1 5 4 2 1 0.154

2 5 5 3 1 0.193

3 5 4 3 2 0.190

4 5 4 0 0 0.190

5 5 5 5 2 0.256

6 5 5 2 1 0.191

7 5 4 4 1 0.122

8 5 5 3 1 0.177

100 ppb 1 5 3 3 1 0.114

2 5 5 2 1 0.172

3 5 5 1 0 0.160

4 5 5 2 1 0.199

5 5 5 3 2 0.165

6 5 3 1 0 0.145

7 5 4 4 1 0.207

8 5 4 0 0 0.186

210 ppb 1 5 5 1 0 0.153

2 5 4 2 0 0.094

3 5 1 1 0 0.017

4 5 4 3 0 0.122

5 5 3 1 0 0.052

6 5 4 2 0 0.154

7 5 4 1 0 0.110

8 5 4 3 0 0.103

450 ppb 1 5 0 0 0 - -

2 5 1 0 0 0.012

3 5 0 0 0 - -

4 5 1 0 0 0.002

5 5 0 0 0 - -

6 5 0 0 0 - -

7 5 0 0 0 - -

8 5 2 1 0 0.081

1 Data provided by Lussier, Kuhn and Sewall, Environmental Research Laboratory, U.S. Environmental

Protection Agency, Narragansett, RI.
244

14.13.2 EXAMPLE OF ANALYSIS OF MYSID, MYSIDOPSIS BAHIA, SURVIVAL DATA 

14.13.2.1 Formal statistical analysis of the survival data is outlined in Figures 9 and 10. 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 endpoint. 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 LC, EC, and IC endpoints. 

14.13.2.2 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. 

14.13.2.3 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.

14.13.2.4 Probit Analysis (Finney, 1971; see Appendix H) 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 model, the Spearman-Karber method, the Trimmed

Spearman-Karber method, or the Graphical method may be used (see Appendices I-K). 

14.13.2.5 The proportion of survival in each replicate must first be transformed by the arc sine transformation

procedure described in Appendix B. The raw and transformed data, means and variances of the transformed

observations at each concentration including the control are listed in Table 5. A plot of the survival data is provided in

Figure 11.
245

Figure 9. Flowchart for statistical analysis of mysid, Mysidopsis bahia, survival data by hypothesis testing.
246

Figure 10. Flowchart for statistical analysis of mysid, Mysidopsis bahia, survival data by point estimation. 
247

Figure 11. Plot of survival proportions of mysids, Mysidopsis bahia, at each treatment level.
248

D ' j

n

i'1

(Xi & X ̄)

2

TABLE 5. MYSID, MYSIDOPSIS BAHIA, SURVIVAL DATA

 Concentration (ppb) 

Replicate Control 50.0 100.0 210.0 450.0 

1 0.80 0.80 0.60 1.00 0.00 

2 0.80 1.00 1.00 0.80 0.20 

3 1.00 0.80 1.00 0.20 0.00 

RAW 4 1.00 0.80 1.00 0.80 0.20 

5 1.00 1.00 1.00 0.60 0.00 

6 1.00 1.00 0.60 0.80 0.00

7 1.00 0.80 0.80 0.80 0.00 

8 0.80 1.00 0.80 0.80 0.40 

1 1.107 1.107 0.886 1.345 0.225 

ARC SINE 2 1.107 1.345 1.345 1.107 0.464

TRANS- 3 1.345 1.107 1.345 0.464 0.225 

FORMED 4 1.345 1.107 1.345 1.107 0.464 

5 1.345 1.345 1.345 0.886 0.225 

6 1.345 1.345 0.886 1.107 0.225 

7 1.345 1.107 1.107 1.107 0.225 

8 1.107 1.345 1.107 1.107 0.685 

Mean (Yi

) 1.256 1.226 1.171 1.029 0.342 

S 0.015 0.016 0.042 0.067 0.031 2

i

i 1 2 3 4 5 

14.13.2.6 Test for Normality

14.13.2.6.1 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 listed

in Table 6.

14.13.2.6.2 Calculate the denominator, D, of the test statistic: 

Where: Xi

 = the ith centered observation 

X ̄ = the overall mean of the centered observations 

n = the total number of centered observations. 
249

W ' 1

D [j

k

i'1

ai(X (n&i%1) & X (i))]2

TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE 

 Concentration (ppb) 

Replicate Control 50.0 100.0 210.0 450.0 

(Site Water)

1 -0.149 -0.119 -0.285 0.316 -0.117

2 -0.149 0.119 0.174 0.078 0.121

3 0.089 -0.119 0.174 -0.565 -0.117

4 0.089 -0.119 0.174 0.078 0.121

5 0.089 0.119 0.174 -0.142 -0.117

6 0.089 0.119 -0.285 0.078 -0.117

7 0.089 -0.119 -0.064 0.078 -0.117

8 -0.149 0.119 -0.064 0.078 0.342

14.13.2.6.3 For this set of data, n = 40 

X' = 1 (-0.006) = 0.0 

40

D = 1.197 

14.13.2.6.4 Order the centered observations from smallest to largest: 

X(1) # X(2) # ... # X(n)

Where X(i) is the ith ordered observation. These ordered observations are listed in Table 7.

14.13.2.6.5 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-1)/2 if n is odd. For the data in this example, n = 40 and k = 20. The ai

 values are listed in Table 8.

14.13.2.6.6 Compute the test statistic, W, as follows:

The differences X(n-i+1) - X(i) are listed in Table 8. For this data in this example:

W = 1 (1.0475)2

 = 0.9167 

1.197
250

TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

i X(i) i X(i)

1 -0.565 21 0.078

2 -0.285 22 0.078

3 -0.285 23 0.078

4 -0.149 24 0.089

5 -0.149 25 0.089

6 -0.149 26 0.089

7 -0.143 27 0.089

8 -0.119 28 0.089

9 -0.119 29 0.119

10 -0.119 30 0.119

11 -0.119 31 0.119

12 -0.117 32 0.119

13 -0.117 33 0.121

14 -0.117 34 0.121

15 -0.117 35 0.174

16 -0.117 36 0.174

17 -0.064 37 0.174

18 -0.064 38 0.174

19 0.078 39 0.316

20 0.078 40 0.342

14.13.2.6.7 The decision rule for this test is to compare W as calculated in Subsection 14.13.2.6.6 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 set of data, the critical value at a significance level of 0.01 and n = 40 observations is 0.919. Since

W = 0.9167 is less than the critical value, conclude that the data are not normally distributed. 

14.13.2.6.8 Since the data do not meet the assumption of normality, Steel's Many-one Rank Test will be used to analyze

the survival data. 

14.13.2.7 Steel's Many-one Rank Test 

14.13.2.7.1 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, ... , 16) 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. 

14.13.2.7.2 An example of assigning ranks to the combined data for the control and 50.0 ppb concentration is given in

Table 9. This ranking procedure is repeated for each control/concentration combination. The complete set of rankings is

summarized in Table 10. The ranks are then summed for each concentration level, as shown in Table 11. 
251

TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE 

i ai X(n-i+1) - X(i)

1 0.3964 0.907 X(40) - X(1)

2 0.2737 0.601 X(39) - X(2)

3 0.2368 0.459 X(38) - X(3)

4 0.2098 0.323 X(37) - X(4)

5 0.1878 0.323 X(36) - X(5)

6 0.1691 0.323 X(35) - X(6)

7 0.1526 0.264 X(34) - X(7)

8 0.1376 0.240 X(33) - X(8)

9 0.1237 0.238 X(32) - X(9)

10 0.1108 0.238 X(31) - X(10)

11 0.0986 0.238 X(30) - X(11)

12 0.0870 0.236 X(29) - X(12)

13 0.0759 0.206 X(28) - X(13)

14 0.0651 0.206 X(27) - X(14)

15 0.0546 0.206 X(26) - X(15)

16 0.0444 0.206 X(25) - X(16)

17 0.0343 0.153 X(24) - X(17)

18 0.0244 0.142 X(23) - X(18)

19 0.0146 0.0 X(22) - X(19)

20 0.0049 0.0 X(21) - X(20)

14.13.2.7.3 For this example, determine if the survival in any of the concentrations is significantly lower than the

survival in the control. 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 survival at each of the various concentration levels with some

"minimum" or critical rank sum, at or below which the survival would be considered significantly lower than the

control. At a significance level of 0.05, the minimum rank sum in a test with four concentrations (excluding the

control) and eight replicates is 47 (See Table 5, Appendix E).

14.13.2.7.4 Since the rank sum for the 450 ppb concentration level is less than the critical value, the proportion

surviving in that concentration is considered significantly less than that in the control. Since no other rank sums are

less than or equal to the critical value, no other concentrations have a significantly lower proportion surviving than the

control. Hence, the NOEC and the LOEC are assumed to be 210.0 ppb and 450.0 ppb, respectively.

14.13.2.8 Calculation of the LC50

14.13.2.8.1 The data used for the Probit Analysis is summarized in Table 12. For the Probit Analysis, run the USEPA

Probit Analysis Program. An example of the program output is provided in Figure 12.

14.13.2.8.2 For this example, the chi-square test for heterogeneity was not significant. Thus Probit Analysis appears

to be appropriate for this set of data.
252

TABLE 9. ASSIGNING RANKS TO THE CONTROL AND 50 PPB CONCENTRATION LEVEL FOR

STEEL'S MANY-ONE RANK TEST 

Rank Transformed Proportion Concentration

 of Total Mortality

4 1.107 Control 

4 1.107 Control 

4 1.107 Control 

4 1.107 50 ppb 

4 1.107 50 ppb 

4 1.107 50 ppb 

4 1.107 50 ppb 

12 1.571 Control 

12 1.571 Control 

12 1.571 Control 

12 1.571 Control 

12 1.571 Control 

12 1.571 50 ppb 

12 1.571 50 ppb 

12 1.571 50 ppb 

12 1.571 50 ppb 

14.13.3 EXAMPLE OF ANALYSIS OF MYSID, MYSIDOPSIS BAHIA, GROWTH DATA

14.13.3.1 Formal statistical analysis of the growth data is outlined in Figure 13. The response used in the statistical

analysis is mean weight per original of males and females combined per replicate. Because this measurement is based

on the number of original organisms exposed (rather than the number surviving), the measured response is a combined

survival and growth endpoint that can be termed biomass. The IC25 and IC50 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 an NOEC and LOEC for growth. Concentrations above the NOEC for survival are

excluded from the hypothesis test for growth effects.

TABLE 10. TABLE OF RANKS1

 Concentration (ppb) 

Replicate Control 50 100 210 450

1 1.107(4,5,6.5,10) 1.107(4) 0.886(1.5) 1.345(13.5) 0.225(3)

2 1.107(4,5,6.5,10) 1.345(12) 1.345(12) 1.107(6.5) 0.464(6.5)

3 1.345(12,12,13.5,14) 1.107(4) 1.345(12) 0.464(1) 0.225(3)

4 1.345(12,12,13.5,14) 1.107(4) 1.345(12) 1.107(6.5) 0.464(6.5)

5 1.345(12,12,13.5,14) 1.345(12) 1.345(12) 0.886(2) 0.225(3)

6 1.345(12,12,13.5,14) 1.345(12) 0.886(1.5) 1.107(6.5) 0.225(3)

7 1.345(12,12,13.5,14) 1.107(4) 1.107(5) 1.107(6.5) 0.225(3)

8 1.107(4,5,6.5,10) 1.345(12) 1.107(5) 1.107(6.5) 0.685(8)

1

Control ranks are given in the order of the concentration with which they were ranked. 
253

TABLE 11. RANK SUMS

Concentration Rank Sum

50 64

100 61

210 49

450 36

14.13.3.2 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.

14.13.3.3 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. 

The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the nonparametric alternative. For detailed

information on the Bonferroni adjustment, see Appendix D.
254

Probit Analysis of Mysidopsis bahia Survival Data

Proportion 

Observed Responding 

Number Number Proportion Adjusted for

Conc. Exposed Resp. Responding Controls

Control 40 3 0.0750 0.0000

50.0000 40 4 0.1000 -0.0080

100.0000 40 6 0.1500 0.0480

210.0000 40 11 0.2750 0.1880

450.0000 40 36 0.9000 0.8880

Chi - Square for Heterogeneity (calculated) = 0.725

Chi - Square for Heterogeneity (tabular value) = 5.991

Probit Analysis of Mysidopsis bahia Survival Data

Estimated LC/EC Values and Confidence Limits

Exposure Lower Upper

Point Conc. 95% Confidence Limits

LC/EC 1.00 123.112 65.283 165.552

LC/EC 50.00 288.873 239.559 335.983

Figure 12. Output for USEPA Probit Analysis Program, Version 1.5.
255

TABLE 12. DATA FOR PROBIT ANALYSIS

 Concentration (ppb) 

Control 50.0 100.0 210.0 450.0

No Dead 3 4 6 11 36

No Exposed 40 40 40 40 40

14.13.3.4 The data, mean and variance of the observations at each concentration including the control for this

example are listed in Table 13. A plot of the data is provided in Figure 14. Since there is significant mortality in the

450 ppb concentration, its effect on growth is not considered. 

TABLE 13. MYSID, MYSIDOPSIS BAHIA, GROWTH DATA

 Concentration (ppb) 

 

Replicate Control 50.0 100.0 210.0 450.0

1 0.146 0.157 0.114 0.153 - 

2 0.118 0.193 0.172 0.071 0.012 

3 0.216 0.190 0.160 0.017 - 

4 0.199 0.190 0.199 0.112 0.002 

5 0.176 0.256 0.165 0.052 - 

6 0.243 0.191 0.145 0.154 - 

7 0.213 0.122 0.207 0.110 - 

8 0.144 0.177 0.186 0.103 0.081 

Mean (Yi

) 0.182 0.184 0.168 0.101 -

S 0.00186 0.00145 0.00091 0.00222 - 2

i

i 1 23 4 5

14.13.3.5 Test for Normality

14.13.3.5.1 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 listed

in Table 14. 
256

Figure 13. Flowchart for statistical analysis of mysid, Mysidopsis bahia, growth data.
257

Figure 14. Plot of mean growth data for mysid, Mysidopsis bahia, test.
258

D ' j

n

i'1

(Xi&X ̄)

2

TABLE 14. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE 

Concentration (ppb)

Replicate Control 50.0 100.0 210.0

1 -0.036 -0.030 -0.054 0.052 

2 -0.064 0.009 0.004 -0.007 

3 0.034 0.006 -0.008 -0.084 

4 0.017 0.006 0.031 0.021 

5 -0.006 0.072 -0.003 -0.049 

6 0.061 0.007 -0.023 0.053 

7 0.031 -0.062 0.039 0.009 

8 -0.038 -0.007 0.018 0.002 

14.13.3.5.2 Calculate the denominator, D, of the statistic: 

Where: Xi

 = the ith centered observation

X ̄ = the overall mean of the centered observations 

 n = the total number of centered observations 

14.13.3.5.3 For this set of data, n = 32

 = 1 X ̄ (0.007) = 0.000

 32 

D = 0.0451 

14.13.3.5.4 Order the centered observations from smallest to largest 

X(1) # X(2) # ... # X(n)

Where X(i) denotes the ith ordered observation. The ordered observations for this example are listed in Table 15. 
259

W ' 1

D[j

k

i'1

ai(x (n&i%1)&x (i)]

2

TABLE 15. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE 

i X(i) i X(i)

1 -0.084 17 0.006 

2 -0.064 18 0.006 

3 -0.062 19 0.007 

4 -0.054 20 0.009 

5 -0.049 21 0.009 

6 -0.038 22 0.017 

7 -0.036 23 0.018 

8 -0.030 24 0.021 

9 -0.023 25 0.031 

10 -0.008 26 0.031 

11 -0.007 27 0.034 

12 -0.007 28 0.039 

13 -0.006 29 0.052 

14 -0.003 30 0.053 

15 0.002 31 0.061 

16 0.004 32 0.072 

14.13.3.5.5 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-1)/2 if n is odd. For the data in this example, n = 32 and k = 16. The ai

 values are listed in

Table 16. 

14.13.3.5.6 Compute the test statistic, W, as follows: 

The differences X(n-i+1) - X(i) are listed in Table 16. For this set of data: 

W = 1 (0.2097)2

 = 0.9752 

 0.045

14.13.3.5.7 The decision rule for this test is to compare W as calculated in Subsection 14.13.3.5.6 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 this set of data, the critical value at a significance level of 0.01 and n = 32 observations is 0.904. Since

W = 0.9752 is greater than the critical value, conclude that the data are normally distributed. 
260

B '

[(j

p

i'1

Vi) lnS ̄2 & j

p

i'1

Vi ln S 2

i ]

C

TABLE 16. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE 

i a(i) X(n-i+1)

1 0.4188 0.156 X(32) - X(1)

2 0.2898 0.125 X(31) - X(2)

3 0.2462 0.115 X(30) - X(3)

4 0.2141 0.106 X(29) - X(4)

5 0.1878 0.088 X(28) - X(5)

6 0.1651 0.072 X(27) - X(6)

7 0.1449 0.067 X(26) - X(7)

8 0.1265 0.061 X(25) - X(8)

9 0.1093 0.044 X(24) - X(9)

10 0.0931 0.026 X(23) - X(10)

11 0.0777 0.024 X(22) - X(11)

12 0.0629 0.016 X(21) - X(12)

13 0.0485 0.015 X(20) - X(13)

14 0.0344 0.010 X(19) - X(14)

15 0.0206 0.004 X(18) - X(15)

16 0.0068 0.002 X(17) - X(16)

14.13.3.6 Test for Homogeneity of Variance 

14.13.3.6.1 The test used to examine whether the variation in mean weight of the mysids is the same across all

concentration levels including the control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as

follows:

Where: Vi = degrees of freedom for each copper concentration and control, Vi

 = (ni - 1) 

p = number of concentration levels including the control 

ln = loge

i = 1, 2, ..., p where p is the number of concentrations including the control 

ni

 = the number of replicates for concentration i. 
261

S ̄2 '

(j

P

i'1

ViS 2

i )

j

P

i'1

Vi

C ' 1 % [3(p&1)]&1[j

p

i'1

1/Vi&(j

p

i'1

Vi)

&1

B ' [(28)ln(0.00162)&7j

p

i'1

ln(S 2

1 )]/1.06

14.13.3.6.2 For the data in this example (see Table 13), all concentrations including the control have the same number

of replicates (ni

 = 8 for all i). Thus, Vi = 7 for all i.

14.13.3.6.3 Bartlett's statistic is therefore: 

= [28(-6.427) - 7(-25.9329)]/1.06 

= [-179.973 - (-181.530)]/1.06 

= 1.469 

14.13.3.6.4 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 three degrees of

freedom, is 11.34. Since B = 1.469 is less than the critical value of 11.34, conclude that the variances are not different. 

14.13.3.7 Dunnett's Procedure 

14.13.3.7.1 To obtain an estimate of the pooled variance for the Dunnett's Procedure, construct an ANOVA table as

described in Table 17.
262

SSB ' j

p

i'1

T 2

i /ni

&G 2 /N

SST ' j

p

i'1

j

n

j'1

&j Y 2

ij &G 2 /N

SSW ' SST&SSB

TABLE 17. ANOVA TABLE 

Source df Sum of Squares Mean Square (MS)

(SS) (SS/df)

Between p - 1 SSB = SSB/(p-1) S 2

B

Within N - p SSW = SSW/(N-p) S 2

W

Total N - 1 SST

Where: p = number of concentration levels including the control

N = total number of observations n1 + n2 ... + np

ni

 = number of observations in concentration i

Between Sum of Squares

Total Sum of Squares

Within Sum of Squares 

G = the grand total of all sample observations, G ' j

p

i'1

Ti

Ti = the total of the replicate measurements for concentration i

Yij = the jth observation for concentration i (represents the mean dry weight of the mysids for 

concentration i in test chamber j) 

14.13.3.7.2 For the data in this example: 

n1 = n2 = n3 = n4 = 8 

N = 32

T1 = Y11 + Y12 + ... + Y18 = 1.455 

T2 = Y21 + Y22 + ... + Y28 = 1.473 

T3 = Y31 + Y32 + ... + Y38 = 1.348 

T4 = Y41 + Y42 + ... + Y48 = 0.805 
263

SSB ' j

p

i'1

T 2

i /ni

&G 2 /N

SST ' j

p

i'1

j

nj

j'1

Y 2

ij &G 2 /N

SSW ' SST&SSB

t

i ' (Y ̄

1&Y ̄

i

)

Sw (1/n1)%(1/ni

)

G = T1 + T2 + T3 + T4 = 5.081 

= 1 (6.752) - (5.081)2

 = 0.0372 

 8 32

= 0.889 - (5.081)2

 = 0.0822

32

 = 0.0822 - 0.0372 = 0.0450 

S = SSB / (p -1) = 0.0372 / (4 -1) = 0.0124 2

B

S = SSW / (N-p) = 0.0450 / (32-4) = 0.0016 2

W

14.13.3.7.3 Summarize these calculations in the ANOVA table (Table 18).

TABLE 18. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE 

Source df Sum of Squares Mean Square(MS) 

(SS) (SS/df) 

Between 3 0.0372 0.0127 

Within 28 0.0450 0.0016 

Total 31 0.0822 

14.13.3.7.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control

combination as follows: 

Where: = mean dry weight for concentration i Y ̄

i

 

Y ̄ = mean dry weight for the control 1

SW = square root of the within mean square 
264

t

2 ' (0.182&0.184)

[0.040 (1/8) % (1/8)]

MSD ' d Sw (1/n1) % (1/n)

n1 = number of replicates for the control 

ni

 = number of replicates for concentration i

14.13.3.7.5 Table 19 includes the calculated t values for each concentration and control combination. In this example,

comparing the 50.0 ppb concentration with the control the calculation is as follows: 

= -0.100

TABLE 19. CALCULATED T VALUES

Concentration (ppb) i ti

50.0 2 -0.150

100.0 3 0.700

210.0 4 4.050

14.13.3.7.6 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, 28 degrees of freedom for error and three concentrations (excluding the control) the approximate critical value is

2.15. The mean weight for concentration "i" is considered significantly less than the mean weight for the control if ti

 is

greater than the critical value. Therefore, the 210.0 ppb concentration has significantly lower mean weight than the

control. Hence the NOEC and the LOEC for growth are 100.0 ppb and 210.0 ppb, respectively.

14.13.3.7.7 To quantify the sensitivity of the test, the minimum significant difference (MSD) that can be detected

statistically may be calculated. 

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) 

n1 = the number of replicates in the control. 
265

MSD ' 2.15(0.04) (1/8) % (1/8)

14.13.3.7.8 In this example:

= 2.15 (0.04)(0.5) 

= 0.043 

14.13.3.7.9 Therefore, for this set of data, the minimum difference that can be detected as statistically significant is

0.043 mg.

14.13.3.7.10 This represents a 23.6% reduction in mean weight from the control.

14.13.3.8 Calculation of the ICp

14.13.3.8.1 The growth data from Table 13 are utilized in this example. As seen in, the observed means are not

monotonically non-increasing with respect to concentration. Therefore, it is necessary to smooth the means prior to

calculating the ICp. In the following discussion, the observed means are represented by and the smoothed means by Y ̄

i

Mi.

14.13.3.8.2 Starting with the control mean, = 0.182 and = 0.184, we see that < . Calculate the smoothed Y ̄

1 Y ̄2 Y ̄1 Y ̄2

means:

M1 = M2 = ( )/2 = 0.183 Y ̄

1 % Y ̄2

14.13.3.8.3 Since = 0.025 < = 0.101 < = 0.168 < M2, set M3 = 0.168 and M4 = 0.101, and M5 Y ̄ = 0.025. 5 Y ̄

4 Y ̄3

Table 20 contains the smoothed means and Figure 15 gives a plot of the smoothed response curve.

TABLE 20. MYSID, MYSIDOPSIS BAHIA, MEAN GROWTH RESPONSE AFTER SMOOTHING

Toxicant Response Smoothed

Conc. Means Mean

(ppb) i Yi

 (mg) Mi (mg)

Control 1 0.182 0.183

50.0 2 0.184 0.183

100.0 3 0.168 0.168

210.0 4 0.101 0.101

450.0 5 0.012 0.012

14.13.3.8.4 An IC25 and IC50 can be estimated using the Linear Interpolation Method. A 25% reduction in weight,

compared to the controls, would result in a mean weight of 0.136 mg, where M1(1-p/100) = 0.183(1-25/100). A 50%

reduction in mean dry weight, compared to the controls, would result in a mean weight of 0.091 mg. Examining the

smoothed means and their associated concentrations (Table 20), the response, 0.136 mg, is bracketed by C3 = 100 ppb and

C4 = 210 ppb. The response, 0.091 mg, is bracketed by C4 = 210 ppb and C5 = 450 ppb.
266

Figure 15. Plot of raw data, observed means, and smoothed means for the mysid, Mysidopsis bahia, growth data from Tables 13 and 20
267

ICp ' Cj

%[M1 (1&p/100)&Mj

]

(C(j%1)&Cj)

M(j%1)&Mj

ICp ' Cj%[M1 (1&p/100)&Mj]

(C(j%1)&Cj)

M(j'1)&Mj

14.13.3.8.5 Using the equation in Section 4.2 from Appendix L, the estimate of the IC25 is calculated as follows:

IC25 = 100 + [0.183(1 - 25/100) - 0.168] (210 - 100) 

(0.101 - 0.168) 

= 151 ppb. 

14.13.3.8.6 Using Equation 1 from Appendix L, the estimate of the IC50 is calculated as follows:

IC50 = 210 + [0.183(1 - 50/100) - 0.101] (450 - 210) 

(0.012 - 0.101)

= 236 ppb.

14.13.3.8.7 When the ICPIN program was used to analyze this set of data, requesting 80 resamples, the estimate of the

IC25 was 150.6446 ppb. The empirical 95.0% confidence interval for the true mean was 97.0905 ppb and 186.6383 ppb.

The computer program output for the IC25 for this data set is shown in Figure 16.

14.13.3.8.8 When the ICPIN program was used to analyze this set of data for the IC50, requesting 80 resamples, the

estimate of the IC50 was 234.6761 ppb. The empirical 95.0% confidence interval for the true mean was (183.8187 ppb

to 277.9211 ppb). The computer program output for the IC50 for this data set is shown in Figure 17.

14.13.4 EXAMPLE OF ANALYSIS OF MYSID, MYSIDOPSIS BAHIA, FECUNDITY DATA

14.13.4.1 Formal statistical analysis of the fecundity data is outlined in Figure 18. The response used in the statistical

analysis is the proportion of females with eggs in each test or control chamber. If no females were present in a replicate,

a response of zero should not be used. Instead there are no data available for that replicate and the number of replicates for

that level of concentration or the control should be reduced by one. Separate analyses are performed for the estimation of

the NOEC and LOEC endpoints, and for the estimation of the EC, LC, and IC endpoints. The data for a concentration are

excluded from the statistical analysis of the NOEC and LOEC endpoints if no eggs were produced in all of the replicates

in which females existed. However, all data are included in the estimation of the IC25 and IC50.
268

Conc. ID 1 2 3 4. 5

Conc. Tested 0 50 100 210 450

Response 1 .146 .154 .114 .153 0

Response 2 .118 .19 .172 .094 .012

Response 3 .216 .193 .160 .017 0

Response 4 .199 .190 .199 .122 .002

Response 5 .176 .190 .165 .052 0

Response 6 .243 .191 .145 .154 0

Response 7 .213 .122 .207 .110 0

Response 8 .144 .177 .186 .103 .081

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: Effluent

Test Start Date: Test Ending Date: 

Test Species: MYSID SHRIMP, Mysidopsis bahia

Test Duration: growth test

DATA FILE: mysidwt.icp

OUTPUT FILE: mysid.i25

Conc. Number Concentration Response Standard. Pooled

 ID Replicates Fg/l Means Dev. Response Means

1 8 0.000 0.182 0.043 0.183

2 8 50.000 0.184 0.038 0.183

3 8 100.000 0.168 0.030 0.168

4 8 210.000 0.101 0.047 0.101

5 8 450.000 0.102 0.028 0.012

The Linear Interpolation Estimate: 150.6446 Entered P Value: 25

Number of Resamplings: 80

The Bootstrap Estimates Mean: 147.1702 Standard Deviation: 23.7984

Original Confidence Limits: Lower: 97.0905 Upper: 186.6383

Resampling time in Seconds: 0.11 Random Seed: -1623038650

Figure 16. ICPIN program output for the IC25.
269

Conc. ID 1 2 3 4. 5

Conc. Tested 0 50 100 210 450

Response 1 .146 .154 .114 .153 0

Response 2 .118 .193 .172 .094 .012

Response 3 .216 .190 .160 .017 0

Response 4 .199 .190 .199 .122 .002

Response 5 .176 .256 .165 .052 0

Response 6 .243 .191 .145 .154 0

Response 7 .213 .122 .207 .110 0

Response 8 .144 .177 .186 .103 .081

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: 

Test Start Date: Test Ending Date: 

Test Species: MYSID SHRIMP, Mysidopsis bahia

Test Duration: growth test

DATA FILE: mysidwt.icp

OUTPUT FILE: mysidwt.i50

Conc. Number Concentration Response Standard. Pooled

 ID Replicates Fg/L Means Dev. Response Means

 1 8 0.000 0.182 0.043 0.183

 2 8 50.000 0.184 0.038 0.183

 3 8 100.000 0.168 0.030 0.168

 4 8 210.000 0.101 0.047 0.101

 5 8 450.000 0.012 0.028 0.01

The Linear Interpolation Estimate: 234.6761 Entered P Value: 50

Number of Resamplings: 80

The Bootstrap Estimates Mean: 230.7551 Standard Deviation: 30.6781

Original Confidence Limits: Lower: 183.8197 Upper: 277.9211

Resampling time in Seconds: 0.16 Random Seed: -628896314

 Figure 17. ICPIN program output for the IC50.
270

Figure 18. Flowchart for statistical analysis of mysid, Mysidopsis bahia, fecundity data.
271

D ' j

n

i'1

(Xi&X ̄)

2

14.13.4.2 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. 

14.13.4.3 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 (Appendix D). 

The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the nonparametric alternative.

14.13.4.4 The proportion of female mysids, Mysidopsis bahia, with eggs in each replicate must first be transformed by

the arc sine square root transformation procedure described in Appendix B. Since the denominator of the proportion of

females with eggs varies with the number of females occurring in that replicate, the adjustment of the arc sine square

root transformation for 0% and 100% is not used for this data. The raw and transformed data, means and variances of

the transformed observations at each test concentration including the control are listed in Table 21. Since there is

significant mortality in the 450 ppb concentration, its effect on reproduction is not considered. Additionally, since no

eggs were produced by females in any of the replicates for the 210 ppb concentration, it is not included in this statistical

analysis and is considered a qualitative reproductive effect. A plot of the mean proportion of female mysids with eggs

is illustrated in Figure 19.

14.13.4.5 Test for Normality 

14.13.4.5.1 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 listed in

Table 22.

14.13.4.5.2 Calculate the denominator, D, of the statistic: 

Where: Xi

 = the ith centered observation 

X ̄ = the overall mean of the centered observations 

n = the total number of centered observations 
272

Figure 19. Proportion of female mysids, Mysidopsis bahia, with eggs.
273

TABLE 21. MYSID, MYSIDOPSIS BAHIA, FECUNDITY DATA: PERCENT FEMALES WITH EGGS 

Test Concentration (ppb)

Replicate Control 50.0 100.0 210.0 

1 1.00 0.50 0.33 0.0 

2 1.00 0.33 0.50 0.0 

3 0.67 0.67 0.00 0.0 

RAW 4 1.00 - 0.50 0.0 

5 1.00 0.40 0.67 0.0 

6 0.80 0.50 0.00 0.0 

7 1.00 0.25 0.25 0.0 

8 1.00 0.33 - 0.0 

 1 1.57 0.78 0.61 - 

ARC SINE 2 1.57 0.61 0.78 - 

TRANS- 3 0.96 0.96 0.00 - 

FORMED1 4 1.57 - 0.78 - 

5 1.57 0.68 0.96 - 

6 1.12 0.78 0.00 - 

7 1.57 0.52 0.52 - 

8 1.57 0.61 - - 

 

Mean(Yi

) 1.44 0.71 0.52 - 

S2

i 0.064 0.021 0.147 - 

i 1 2 3 4 

1

 Since the denominator of the proportion of females with eggs varies with the number of females occurring in that

replicate, the adjustment of the arc sine square root transformation for 0% and 100% is not used for this data.

TABLE 22. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE 

 

 Test Concentration (ppb) 

 Replicate Control 50.0 100.0

1 0.13 0.07 0.09 

2 0.13 -0.10 0.26 

3 -0.48 0.25 -0.52 

4 0.13 - 0.26 

5 0.13 -0.03 0.44 

6 -0.32 0.07 -0.52 

7 0.13 -0.19 0.00 

8 0.13 -0.10 -
274

W ' 1

D [j

k

i'1

ai(X (n&i%1)&X (i))]2

14.13.4.5.3 For this set of data, n = 22 

X = 1 (0.000) = 0.000 

22

D = 1.4412

14.13.4.5.4 Order the centered observations from smallest to largest: 

X(1) # X(2) # ... # X(n)

Where X(i) denotes the ith ordered observation. The ordered observations for this example are listed in Table 23. 

14.13.4.5.5. 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-1)/2 if n is odd. For the data in this example, n = 22 and k = 11. The ai

 values are listed in

Table 24. 

14.13.4.5.6 Compute the test statistic, W, as follows:

The differences X(n-i+1) - X(I) are listed in Table 24. For the data in this example: 

W = 1 (1.1389)2

 = 0.900 

1.4412 

TABLE 23. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE 

i X(i) i X(i)

1 -0.52 12 0.09 

2 -0.52 13 0.13 

3 -0.48 14 0.13 

4 -0.32 15 0.13 

5 -0.19 16 0.13 

6 -0.10 17 0.13 

7 -0.10 18 0.13 

8 0.03 19 0.25 

9 0.00 20 0.26 

10 0.07 21 0.26 

11 0.07 22 0.44 
275

B '

[(j

p

i'1

Vi) lnS ̄2 & j

p

i'1

Vi ln S 2

i ]

C

S ̄2 '

(j

p

i'1

Vi S 2

i )

j

p

i'1

Vi

TABLE 24. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE 

i ai X(n-i+1) - X(i)

 1 0.4590 0.96 X(22) - X(1)

2 0.3156 0.78 X(21) - X(2)

3 0.2571 0.74 X(20) - X(3)

4 0.2131 0.57 X(19) - X(4)

5 0.1764 0.32 X(18) - X(5)

6 0.1443 0.23 X(17) - X(6)

7 0.1150 0.23 X(16) - X(7)

8 0.0878 0.16 X(15) - X(8)

9 0.0618 0.13 X(14) - X(9)

10 0.0368 0.06 X(13) - X(10)

11 0.0122 0.02 X(12) - X(11)

14.13.4.5.7 The decision rule for this test is to compare W as calculated in Subsection 14.13.4.5.6 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 this set of data, the critical value at a significance level of 0.01 and n = 22 observations is 0.878. Since

W = 0.900 is greater than the critical value, conclude that the data are normally distributed.

14.13.4.6 Test for Homogeneity of Variance 

14.13.4.6.1 The test used to examine whether the variation in proportion of female mysids with eggs is the same

across all concentration levels including the control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic

is as follows: 

Where: Vi

 = degrees of freedom for each copper concentration and control, Vi = (ni - 1) 

p = number of concentration levels including the control

ln = loge

i = 1, 2, ..., p where p is the number of concentrations including the control 

ni

 = the number of replicates for concentration i.
276

C ' 1%[3(p&1)&1 [j

p

i'1

1/Vi& (j

p

i'1

Vi)

&1

]

14.13.4.6.2 For the data in this example (see Table 21), n1 = 8, n2 = 7 and n3 = 7. Thus, the respective degrees of

freedom are 7, 6 and 6. 

14.13.4.6.3 Bartlett's statistic is therefore:

 B = [(19)ln(0.077) - (7 ln(0.064) + 6 ln(0.021) + 6 ln(0.147))]/1.07 

= [19(-2.564) - (-53.925)]/1.07

= [-48.716 - (-53.925)]/1.07 

= 4.868 

14.13.4.6.4 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 two degrees of

freedom, is 9.210. Since B = 4.868 is less than the critical value of 9.210, conclude that the variances are not different. 

14.13.4.7 T test with the Bonferroni Adjustment 

14.13.4.7.1 A t test with the Bonferroni adjustment is used as an alternative to Dunnett's Procedure when, as in this set

of data, the number of replicates is not the same for all concentrations. Like Dunnett's Procedure, it uses a pooled

estimate of the variance, which is equal to the error value calculated in an analysis of variance. To obtain an estimate of

the pooled variance, construct an ANOVA table as described in Table 25. 

TABLE 25. ANOVA TABLE 

Source df Sum of Squares Mean Square (MS)

(SS) (SS/df)

Between p - 1 SSB = SSB/(p-1) S 2

B

Within N - p SSW = SSW/(N-p) S 2

W

Total N - 1 SST

Where: p = number of concentration levels including the control

N = total number of observations n1 + n2 ... + np

ni

 = number of observations in concentration i
277

SSB ' j

p

i'1

T 2

i /ni

&G 2 /N

SST ' j

p

i'1

j

nj

j&1

Y 2

ij &G 2 /N

SSW 'SST&SSB

SSB ' j

p

i'1

T 2

i /ni

&G 2 /N

SST ' j

p

i'1

j

nj

j'1

Y 2

ij &G 2 /N

Between Sum of Squares

Total Sum of Squares

Within Sum of Squares 

G = the grand total of all sample observations, G ' j

p

i'1

Ti

Ti = the total of the replicate measurements for concentration i

Yij = the jth observation for concentration i (represents the mean dry weight of the mysids for

concentration i in test chamber j) 

14.13.4.7.2 For the data in this example:

n1 = 8 n2 = 7 n3 = 7 

N = 22 

T1 = Y11 + Y12 + ... + Y18 = 11.5 

T2 = Y21 + Y22 + ... + Y27 = 4.94 

T3 = Y31 + Y32 + ... + Y37 = 3.65 

G = T1 + T2 + T3 = 20.09 

= 132.25 + 24.40 + 13.32 - 403.61 = 3.57

8 7 7 22

= 23.396 - 403.61 = 5.05

22 

SSW = SST - SSB = 5.05 - 3.57 = 1.48 

S = SSB/(p-1) = 3.57/(3-1) = 1.785 2

B

S = SSW/(N-p) = 1.48/(22-3) = 0.078 2

W
278

t

i ' (Y ̄

1&Y ̄

i

)

Sw (1/n1) % (1/ni

)

t

2 ' (1.44 &0.52)

[0.279 (1/8)&(1/7)]

14.13.4.7.3 Summarize these calculations in the ANOVA table (Table 26).

TABLE 26. ANOVA TABLE FOR THE T TEST WITH BONFERRONI’S ADJUSTMENT EXAMPLE 

Source df Sum of Squares Mean Square (MS) 

(SS) (SS/df) 

Between 2 3.57 1.785 

Within 19 1.48 0.078 

Total 21 5.05 

14.13.4.7.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control

combination as follows: 

Where: = mean proportion of females with eggs for concentration i Y ̄

i

Y ̄ = mean proportion of females with eggs for the control 1

SW = square root of the within mean square 

n1 = number of replicates for the control 

ni = number of replicates for concentration i 

14.13.4.7.5 Table 27 includes the calculated t values for each concentration and control combination. In this example,

comparing the 50.0 ppb concentration with the control the calculation is as follows: 

= 5.05 
279

MSD ' t Sw (1/n1) % (1/n)

MSD ' 2.094(0.279) (1/8)%(1/7)

TABLE 27. CALCULATED T VALUES 

Test Concentration (ppb) i ti

50.0 2 5.05 

100.0 3 6.37

14.13.4.7.6 Since the purpose of this test is to detect a significant reduction in mean proportion of females with eggs, a

one-sided test is appropriate. The critical value for this one-sided test is found in Table 5, Appendix D, Critical Values

for the t test with Bonferroni's adjustment. For an overall alpha level of 0.05, 19 degrees of freedom for error and two

concentrations (excluding the control) the approximate critical value is 2.094. The mean proportion for concentration

"i" is considered significantly less than the mean proportion for the control if ti

 is greater than the critical value. 

Therefore, the 50.0 ppb and the 100.0 ppb concentrations have significantly lower mean proportion of females with

eggs than the control. Hence the LOEC for fecundity is 50.0 ppb.

14.13.4.7.7 To quantify the sensitivity of the test, the minimum significant difference (MSD) that can be detected

statistically may be calculated. 

Where: t = the critical value for the t test with Bonferroni's adjustment 

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) 

n1 = the number of replicates in the control

14.13.4.7.8 In this example: 

= 2.094 (0.279)(0.518)

= 0.303 

14.13.4.7.9 Therefore, for this set of data, the minimum difference that can be detected as statistically significant is

0.30.

14.13.4.7.10 The MSD (0.30) is in transformed units. To determine the MSD in terms of percent of females with

eggs, carry out the following conversion.
280

1. Subtract the MSD from the transformed control mean. 

1.44 - 0.30 = 1.14 

2. Obtain the untransformed values for the control mean and the difference calculated in 4.10.1. 

[ Sine (1.44) ]2

 = 0.983 

[ Sine (1.14) ]2

 = 0.823 

3. The untransformed MSD (MSDu) is determined by subtracting the untransformed values from 

 14.13.4.7.10.2.

MSDu = 0.983 - 0.823 = 0.16 

14.13.4.7.11 Therefore, for this set of data, the minimum difference in mean proportion of females with eggs between

the control and any copper concentration that can be detected as statistically significant is 0.16. 

14.13.4.7.12 This represents a 17% decrease in proportion of females with eggs from the control. 

14.13.4.8 Calculation of the ICp

14.13.4.8.1 The fecundity data in Table 4 are utilized in this example. Table 28 contains the mean proportion of

females with eggs for each toxicant concentration. As can be seen, the observed means are monotonically

nonincreasing with respect to concentration. Therefore, it is not necessary to smooth the means prior to calculating the

IC. Figure 20 gives a plot of the response curve.

TABLE 28. MYSID, MYSIDOPSIS BAHIA, MEAN MEAN PROPORTION OF FEMALES WITH EGGS

Toxicant Response Smoothed

Conc. Means Mean

(ppb) i Yi

 (mg) Mi (mg)

Control 1 0.934 0.934

50.0 2 0.426 0.426

100.0 3 0.317 0.317

210.0 4 0.000 0.000

450.0 5 0.010 0.000

14.13.4.8.2 An IC25 and IC50 can be estimated using the Linear Interpolation Method. A 25% reduction in mean

proportion of females with eggs, compared to the controls, would result in a mean proportion of 0.701, where M1(1-

p/100) = 0.934(1-25/100). A 50% reduction in mean proportion of females with eggs, compared to the could would

result in a mean proportion of 0.467. Examining the means and their associated concentrations (Table 28), the

response, 0.701, is bracketed by C1 = 0 ppb and C2 = 50 ppb. The response, 0.467, is bracketed by C1 = 0 ppb and C2 =

50 ppb.

14.13.4.8.3 Using the equation in Section 4.2 from Appendix L, the estimate of the IC25 is calculated as follows:

ICp ' Cj

%[M1 (1&p/100)&Mj

] (C(j%1)&Cj

)

(M(j%1)&Mj

)
281

ICp ' Cj

%[M1 (1&p/100)&Mj

]

C(j%1)&Cj)

(M(j%1)&Mj

)

IC25 = 0 + [0.934(1 - 25/100) - 0.934] (50 - 0) 

(0.426 - 0.934)

= 23 ppb.

14.13.4.8.4 Using the equation in Section 4.2 from Appendix L, the estimate of the IC50 is calculated as follows:

IC50 = 0 + [0.934(1 - 50/100) - 0.934] (50 - 0) 

 (0.426 - 0.934)

= 46 ppb.

14.13.4.8.5 When the ICPIN program was used to analyze this set of data, requesting 80 resamples, the estimate of the

IC25 was 29.9745 ppb. The empirical 95.0% confidence interval for the true mean was 20.0499 ppb to 30.5675 ppb. 

The computer program output for the IC25 for this data set is shown in Figure 21. This value is extrapolated below the

lowest test concentration and data should be used cautiously.

14.13.4.8.6 When the ICPIN program was used to analyze this set of data for the IC50, requesting 80 resamples, the

estimate of the IC50 was 45.9490 ppb. The empirical 95.0% confidence interval for the true mean was 40.1467 ppb to

63.0931 ppb. The computer program output for the IC50 for this data set is shown in Figure 22.
282

Figure 20. Plot of the mean proportion of female mysids, Mysidopsis bahia, with eggs
283

Conc. ID 1 2 3 4

Conc. Tested 0 50 100 210

Response 1 1 .5 .3 0

Response 2 1 .33 .5 0

Response 3 .67 .67 0 0

Response 4 1 .4 .5 0

Response 5 1 .5 .67 0

Response 6 .8 .25 0 0

Response 7 1 .33 .25 0

Response 8 1 0

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: Effluent

Test Start Date: Test Ending Date: 

Test Species: MYSID SHRIMP, Mysidopsis bahia

Test Duration: fecundity

DATA FILE: mysidfe.icp

OUTPUT FILE: mysidfe.i25

Conc. Number Concentration Response Standard. Pooled

 ID Replicates Fg/l Means Dev. Response Means

 1 8 0.000 0.934 0.127 0.934

 2 7 50.000 0.426 0.142 0.426

 3 7 100.000 0.317 0.257 0.317

 4 8 210.000 0.000 0.000 0.000

The Linear Interpolation Estimate: 29.9745 Entered P Value: 25

Number of Resamplings: 80

The Bootstrap Estimates Mean: 23.8871 Standard Deviation: 3.0663

Original Confidence Limits: Lower: 20.0499 Upper: 30.5765

Resampling time in Seconds: 1.37 Random Seed: 1918482350

Figure 21. ICPIN program output for the IC25.
284

Conc. ID 1 2 3 4

Conc. Tested 0 50 100 210

Response 1 1 .5 .3 0

Response 2 1 .33 .5 0

Response 3 .67 .67 0 0

Response 4 1 .4 .5 0

Response 5 1 .5 .67 0

Response 6 .8 .25 0 0

Response 7 1 .33 .25 0

Response 8 1 0

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: Effluent

Test Start Date: Test Ending Date: 

Test Species: MYSID SHRIMP

Test Duration: fecundity

DATA FILE: mysidfe.icp

OUTPUT FILE: mysidfe.i50

-Conc. Number Concentration Response Std. Pooled

 ID Replicates Fg/l Means Dev. Response Means

 1 8 0.000 0.934 0.127 0.934

 2 7 50.000 0.426 0.142 0.426

 3 7 100.000 0.317 0.257 0.317

 4 8 210.000 0.000 0.000 0.000

The Linear Interpolation Estimate: 45.9490 Entered P Value: 50

Number of Resamplings: 80

The Bootstrap Estimates Mean: 47.8720 Standard Deviation: 8.2908

Original Confidence Limits: Lower: 40.1467 Upper: 63.0931

Resampling time in Seconds: 1.32 Random Seed: -391064242

Figure 22. ICPIN program output for the IC50.
285

14.14 PRECISION AND ACCURACY 

14.14.1 PRECISION – Data on single-laboratory and multilaboratory precision are described below (Subsections

14.14.1.1 and 14.14.1.2). Single-laboratory precision is a measure of the reproducibility of test results when tests are

conducted using a specific method under reasonably constant conditions in the same laboratory. Single-laboratory

precision is synonymous with the terms within-laboratory precision and intralaboratory precision. Multilaboratory

precision is a measure of the reproducibility of test results from different laboratories using the same test method and

analyzing the same test material. Multilaboratory precision is synonymous with the term interlaboratory precision. 

Interlaboratory precision, as used in this document, includes both within-laboratory and between-laboratory

components of variability. In recent multilaboratory studies, these two components of interlaboratory precision have

been displayed separately (termed within-laboratory and between-laboratory variability) and combined (termed total

interlaboratory variability). The total interlaboratory variability that is reported from these studies is synonymous with

interlaboratory variability reported from other studies where individual variability components are not separated.

14.14.1.1 Single-Laboratory Precision

14.14.1.1.1 Data on the single-laboratory precision of the mysid survival, growth, and fecundity using copper (Cu)

sulfate and sodium dodecyl sulfate (SDS) in natural seawater and in artificial seawater (GP2) are shown in Tables 29-

33. In Tables 29-30 the coefficient of variation for the IC25, ranges from 18.0 to 35.0 and the IC50, ranges from 5.8 to

47.8, indicating acceptable test precision. Data in Tables 31-33 show no detectable differences between tests conducted

in natural or artificial seawaters.

14.14.1.1.2 EPA evaluated within-laboratory precision of the Mysid, Mysidopsis bahia, Survival, Growth, and

Fecundity Test using a database of routine reference toxicant test results from 10 laboratories (USEPA, 2000b). The

database consisted of 130 reference toxicant tests conducted in 10 laboratories using a variety of reference toxicants

including: chromium, copper, and potassium chloride. Among the 10 laboratories, the median within-laboratory CV

calculated for routine reference toxicant tests was 28% for the IC25 growth endpoint. In 25% of laboratories, the

within-laboratory CV was less than 24%; and in 75% of laboratories, the within-laboratory CV was less than 32%. 

14.14.1.2 Multilaboratory Precision

14.14.1.2.1 In 2000, EPA conducted an interlaboratory variability study of the Mysid, Mysidopsis bahia, Survival,

Growth, and Fecundity Test (USEPA, 2001a; USEPA, 2001b). In this study, each of 11 participant laboratories tested

4 blind test samples that included some combination of blank, effluent, reference toxicant, and receiving water sample

types. The blank sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater, the effluent sample was

a municipal wastewater spiked with KCl, the receiving water sample was a natural seawater spiked with KCl, and the

reference toxicant sample consisted of bioassay-grade FORTY FATHOMS® synthetic seawater spiked with KCl. Of

the 44 Mysidopsis bahia Survival, Growth, and Fecundity tests conducted in this study, 97.7% were successfully

completed and met the required test acceptability criteria. Of seven tests that were conducted on blank samples, none

showed false positive results for survival, growth, or fecundity endpoints. Results from the reference toxicant, effluent,

and receiving water sample types were used to calculate the precision of the method. Table 34 shows the precision of

the IC25 for each of these sample types. Averaged across sample types, the total interlaboratory variability (expressed

as a CV%) was 41.3% for growth IC25 results. Table 35 shows the frequency distribution of survival and growth

NOEC endpoints for each sample type. For the survival endpoint, NOEC values spanned three concentrations for the

reference toxicant, effluent, and receiving water sample types. The percentage of values within one concentration of

the median was 100% for each of the sample types. For the growth endpoint, NOEC values spanned four

concentrations for the reference toxicant sample type and three concentrations for the effluent and receiving water

sample types. The percentage of values within one concentration of the median was 92.3%, 100%, and 100% for the

reference toxicant, effluent, and receiving water sample types, respectively. For the fecundity endpoint, NOEC values

spanned three concentrations for the reference toxicant, the effluent, and the receiving water sample types. The

percentage of values within one concentration of the median was 75.0%, 87.5%, and 66.7% for the reference toxicant,

effluent, and receiving water sample types, respectively. 
286

14.14.2 ACCURACY 

14.14.2.1 The accuracy of toxicity tests cannot be determined.

TABLE 29. SINGLE-LABORATORY PRECISION OF THE MYSID, MYSIDOPSIS BAHIA, SURVIVAL,

GROWTH, AND FECUNDITY TEST PERFORMED IN NATURAL SEAWATER, USING

JUVENILES FROM MYSIDS CULTURED AND SPAWNED IN NATURAL SEAWATER, AND

COPPER (Cu) SULFATE AS A REFERENCE TOXICANT1,2,3,4,5,6

Most

Test NOEC IC25 IC50 Sensitive

Number (μg/L) (μg/L) (μg/L) Endpoint7

1 63 96.1 NC8 S

2 125 138.3 175.5 S

3 125 156.3 187.5 S

4 125 143.0 179.9 S

5 125 157.7 200.3 S

n: 5 5 4

Mean: NA 138.3 185.8

CV(%): NA 18.0 5.8

1

 Data from USEPA (1988a) and USEPA (1991a).

2 Tests performed by Randy Cameleo, ERL-N, USEPA, Narragansett, RI. 3 Eight replicate exposure chambers, each with five juveniles, were used for the control and each toxicant 

 concentration. The temperature of the test solutions was maintained at 26 ± 1°C.

4 Copper concentrations in Tests 1-2 were: 8, 16, 31, 63, and 125 mg/L. Copper concentrations in Tests 3-6 were, 

 16, 31, 63, 125, and 250 μg/L.

5 NOEC Range: 63 - 125 μg/L (this represents a difference of two exposure concentrations). 6 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 7 Endpoints: G=Growth; S=Survival. 8

 NC = No linear interpolation estimate could be calculated from the data, since none of the group response means 

 were less than 50 percent of the control concentrations.
287

TABLE 30. SINGLE-LABORATORY PRECISION OF THE MYSID, MYSIDOPSIS BAHIA, SURVIVAL,

GROWTH, AND FECUNDITY TEST PERFORMED IN NATURAL SEAWATER, USING

JUVENILES FROM MYSIDS CULTURED AND SPAWNED IN NATURAL SEAWATER, AND

SODIUM DODECYL SULFATE (SDS) AS A REFERENCE TOXICANT1,2,3,4,5,6

Most

Test NOEC IC25 IC50 Sensitive

Number (mg/L) (mg/L) (mg/L) Endpoint7

1 2.5 4.5 NC9 S

2 < 0.3 NC8 NC9 S

3 < 0.6 NC8 NC9 S

4 5.0 7.8 NC9 S

5 2.5 3.6 4.6 S

6 5.0 7.0 9.3 S

n: 4 4 2

Mean: NA 5.7 6.9

CV(%): NA 35.0 47.8

 1 Data from USEPA (1988a) and USEPA (1991a). 2 Tests performed by Randy Cameleo, ERL-N, USEPA, Narragansett, RI. 3 Eight replicate exposure chambers, each with five juveniles, were used for the control and each toxicant 

 concentration. The temperature of the test solutions was maintained at 26 ± 1°C. 

4 SDS concentrations in Tests 1-2 were: 0.3, 0.6, 1.3, 2.5, and 5.0 mg/L. SDS concentrations in Tests 3-4 were: 0.6, 

 1.3, 2.5, 5.0 and 10.0 mg/L. SDS concentrations in Tests 5-6 were: 1.3, 2.5, 5.0, 10.0, and 20.0 mg/L. 5 NOEC Range: < 0.3 - 5.0 mg/L (this represents a difference of four exposure concentrations). 6 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 7 Endpoints: G=Growth; S=Survival. 8 NC = No linear interpolation estimate could be calculated from the data, since none of the group response means 

 were less than 75 percent of the control response mean. 9 NC = No linear interpolation estimate could be calculated from the data, since none of the group response means 

 were less than 50 percent of the control response mean.
288

TABLE 31. COMPARISON OF SURVIVAL (LC50)1, GROWTH AND FECUNDITY (IC50)1 RESULTS

FROM 7-DAY TESTS WITH THE MYSID, MYSIDOPSIS BAHIA, USING NATURAL

SEAWATER (NSW) AND ARTIFICIAL SEAWATER (GP2) AS DILUTION WATER AND

SODIUM DODECYL SULFATE (SDS) AS A REFERENCE TOXICANT

 Survival LC50 Growth IC50 Fecundity IC50 

Test NSW GP2 NSW GP2 NSW GP2

1 16.2 16.3 16.8 16.3 12.0 10.9

2 20.5 19.2 24.2 23.3 20.1 18.5

3 --2 21.9 --2 24.4 --2 21.7

1 All LC50/IC50 values in mg/L. 2 No test performed.
289

TABLE 32. COMPARISON OF SURVIVAL (LC50)1, GROWTH AND FECUNDITY (IC50)1 RESULTS 

FROM 7-DAY TESTS WITH THE MYSID, MYSIDOPSIS BAHIA, USING NATURAL

SEAWATER (NSW) AND ARTIFICIAL SEAWATER (GP2) AS DILUTION WATER AND

COPPER (Cu) SULFATE AS A REFERENCE TOXICANT

 Survival LC50 Growth IC50 Fecundity IC50 

Test NSW GP2 NSW GP2 NSW GP2

1 177 182 208 186 177 125

2 --2 173 --2 210 --2 142

3 190 174 195 179 168 186

1 All LC50/IC50 values in μg/L. 2 No test performed.
290

TABLE 33. CONTROL RESULTS FROM 7-DAY SURVIVAL, GROWTH, AND FECUNDITY TESTS

WITH THE MYSID, MYSIDOPSIS BAHIA, USING NATURAL SEAWATER AND ARTIFICIAL

SEAWATER (GP2) AS A DILUTION WATER

Control 1

 Survival (%) Growth (mg) Fecundity (%) 

Test NSW GP2 NSW GP2 NSW GP2

1 98 93 0.32 0.32 73 77

2 80 90 0.40 0.43 100 95

3 --2 95 --2 0.40 --2 100

4 94 84 0.34 0.37 89 83

5 --2 94 --2 0.36 --2 83

6 80 75 0.40 0.41 79 93

1 Survival as percent of mysids alive after 7 days; growth as mean individual dry weight; fecundity as percent 

 females with eggs.

2 No test performed.
291

TABLE 34. PRECISION OF POINT ESTIMATES FOR VARIOUS SAMPLE TYPES1

Test Endpoint Sample Type

CV (%)2

Within-lab3 Between-lab4 Total5

IC25 for

Growth Reference toxicant 8.69 40.0 40.9

Effluent 5.26 36.6 37.0

Receiving water - - 45.9

Average 6.98 38.3 41.3

1 From EPA’s WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b). 2 CVs were calculated based on the within-laboratory component of variability, the between-laboratory component of

variability, and the total interlaboratory variability (including both within-laboratory and between-laboratory

components). For the receiving water sample type, within-laboratory and between-laboratory components of

variability could not be calculated since the study design did not provide within-laboratory replication for this

sample type. 3 The within-laboratory (intralaboratory) component of variability for duplicate samples tested at the same time in the

same laboratory. 4 The between-laboratory component of variability for duplicate samples tested at different laboratories. 5 The total interlaboratory variability, including within-laboratory and between-laboratory components of variability. 

The total interlaboratory variability is synonymous with interlaboratory variability reported from other studies

where individual variability components are not separated. 
292

TABLE 35. FREQUENCY DISTRIBUTION OF HYPOTHESIS TESTING RESULTS FOR VARIOUS

SAMPLE TYPES1

Test Endpoint Sample Type

Median

NOEC

Value

% of Results

at the Median

% of Results

±12

% of Results

$23

Survival

NOEC Reference toxicant 25% 53.8 46.2 0.00

Effluent 12.5% 46.7 53.3 0.00

Receiving water 12.5% 37.5 62.5 0.00

Growth 

NOEC Reference toxicant 25% 53.8 38.5 7.69

Effluent 12.5% 46.7 53.3 0.00

Receiving water 12.5% 50.0 50.0 0.00

Fecundity

NOEC Reference toxicant 18.8% -4 75.0 25.0

Effluent 25% 62.5 25.0 12.5

Receiving water 9.38% -4 66.7 33.3

1 From EPA’s WET Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2001b). 2 Percent of values at one concentration interval above or below the median. Adding this percentage to the percent of

values at the median yields the percent of values within one concentration interval of the median. 3 Percent of values two or more concentration intervals above or below the median. 4 The median NOEC fell between test concentrations, so no test results fell precisely on the median.
293

SECTION 15

TEST METHOD

SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST

METHOD 1008.0

15.1 SCOPE AND APPLICATION 

15.1.1 This method, adapted in part from USEPA (1987e), measures the toxicity of effluents and receiving water

to the gametes of the sea urchin, Arbacia punctulata, during a 1 h and 20 min exposure. The purpose of the sperm

cell toxicity test is to determine the concentration of a test substance that reduces fertilization of exposed gametes

relative to that of the control.

15.1.2 Detection limits of the toxicity of an effluent or chemical substance are organism dependent. 

15.1.3 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

volatile and highly degradable toxicants in the source may not be detected in the test. 

15.1.4 This test 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. 

15.2 SUMMARY OF METHOD 

15.2.1 The method consists of exposing dilute sperm suspensions to effluents or receiving waters for 1 h. Eggs are

then added to the sperm suspensions. Twenty minutes after the eggs are added, the test is terminated by the addition

of preservative. The percent fertilization is determined by microscopic examination of an aliquot from each

treatment. The test results are reported as the concentration of the test substance which causes a statistically

significant reduction in fertilization. 

15.3 INTERFERENCES 

15.3.1 Toxic substances may be introduced by contaminants in dilution water, glassware, sample hardware, and

testing equipment (see Section 5, Facilities, Equipment, and Supplies). 

15.3.2 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). 

15.4 SAFETY 

15.4.1 See Section 3, Health and Safety. 

15.5. APPARATUS AND EQUIPMENT 

15.5.1 Facilities for holding and acclimating test organisms. 

15.5.2 Laboratory sea urchins, Arbacia punctulata, culture unit -- See Subsection 15.6.19, culturing methods

below and Section 4, Quality Assurance. To test effluent or receiving water toxicity, sufficient eggs and sperm

must be available. 
294

15.5.3 Samplers -- automatic sampler, preferably with sample cooling capability, that can collect a 24-h composite

sample of 5 L. 

15.5.4 Environmental chamber or equivalent facility with temperature control (20 ± 1°C). 

15.5.5 Water purification system -- Millipore Milli-Q®, deionized water (DI) or equivalent. 

15.5.6 Balance -- Analytical, capable of accurately weighing to 0.00001 g.

15.5.7 Reference weights, Class S -- for checking performance of balance. Weights should bracket the expected

weights of materials to be weighed. 

15.5.8 Air pump -- for oil-free air supply. 

15.5.9 Air lines, and air stones -- for aerating water containing adults, or for supplying air to test solutions with

low DO. 

15.5.10 Vacuum suction device -- for washing eggs. 

15.5.11 Meters, pH and DO -- for routine physical and chemical measurements. 

15.5.12 Standard or micro-Winkler apparatus -- for determining DO (optional). 

15.5.13 Transformer, 10-12 Volt, with steel electrodes -- for stimulating release of eggs and sperm. 

15.5.14 Centrifuge, bench-top, slant-head, variable speed -- for washing eggs. 

15.5.15 Fume hood -- to protect the analyst from formaldehyde fumes. 

15.5.16 Dissecting microscope -- for counting diluted egg stock.

15.5.17 Compound microscope -- for examining and counting sperm cells and fertilized eggs. 

15.5.18 Sedgwick-Rafter counting chamber -- for counting egg stock and examining fertilized eggs. 

15.5.19 Hemacytometer, Neubauer -- for counting sperm. 

15.5.20 Count register, 2-place -- for recording sperm and egg counts. 

15.5.21 Refractometer -- for determining salinity. 

15.5.22 Thermometers, glass or electronic, laboratory grade -- for measuring water temperatures. 

15.5.23 Thermometers, bulb-thermograph or electronic-chart type -- for continuously recording temperature. 

15.5.24 Thermometer, National Bureau of Standards Certified (see USEPA Method 170.1, USEPA, 1979b) -- to

calibrate laboratory thermometers. 

15.5.25 Ice bucket, covered -- for maintaining live sperm. 

15.5.26 Centrifuge tubes, conical -- for washing eggs. 

15.5.27 Cylindrical glass vessel, 8-cm diameter -- for maintaining dispersed egg suspension. 
295

15.5.28 Beakers -- six Class A, borosilicate glass or non-toxic plasticware, 1000 mL for making test solutions. 

15.5.29 Glass dishes, flat bottomed, 20-cm diameter -- for holding urchins during gamete collection. 

15.5.3 Wash bottles -- for deionized water, for rinsing small glassware and instrument electrodes and probes. 

15.5.31 Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-toxic plastic labware,

10-1000 mL for making test solutions. 

15.5.32 Syringes, 1-mL, and 10-mL, with 18 gauge, blunt-tipped needles (tips cut off) -- for collecting sperm and

eggs. 

15.5.33 Pipets, volumetric -- Class A, 1-100 mL. 

15.5.34 Pipets, automatic -- adjustable 1-100 mL. 

15.5.35 Pipets, serological -- 1-10 mL, graduated. 

15.6.36 Pipet bulbs and fillers -- PROPIPET®, or equivalent. 

15.6 REAGENTS AND CONSUMABLE MATERIALS 

15.6.1 Sea Urchins, Arbacia punctulata minimum 12 of each sex. 

15.6.2 Food -- kelp, Laminaria sp., or romaine lettuce for the sea urchin, Arbacia punctulata. 

15.6.3 Standard salt water aquarium or Instant Ocean Aquarium (capable of maintaining seawater at 15°C) -- with

appropriate filtration and aeration system. 

15.6.4 Sample containers -- for sample shipment and storage (see Section 8, Effluent and Receiving Water

Sampling, Sample Handling, and Sample Preparation for Toxicity Tests). 

15.6.5 Scintillation vials, 20 mL, disposable -- to prepare test concentrations. 

15.6.6 Tape, colored -- for labeling tubes. 

15.6.7 Markers, waterproof -- for marking containers, etc. 

15.6.8 Parafilm -- to cover tubes and vessels containing test materials. 

15.6.9 Gloves, disposable; labcoat and protective eyewear – for personal protection from contamination. 

15.6.10 Data sheets (one set per test) -- for data recording (see Figures 1, 2, and 3). 

15.6.11 Acetic acid, 10%, reagent grade, in seawater -- for preparing killed sperm dilutions. 

15.6.12 Formalin, 1%, in 2 mL of seawater -- for preserving eggs (see Subsection 15.10.9 Termination of the

Test). 

15.6.13 Buffers, pH 4, pH 7, and pH 10 (or as per instructions of instrument manufacturer) -- for standards and

calibration check (see USEPA Method 150.1, USEPA, 1979b). 

15.6.14 Membranes and filling solutions for dissolved oxygen probe (see USEPA Method 360.1, USEPA, 1979b),

or reagents -- for modified Winkler analysis. 
296

15.6.15 Laboratory quality assurance samples and standards -- for the above methods. 

15.6.16 Reference toxicant solutions -- see Section 4, Quality Assurance. 

15.6.17 Reagent water -- defined as distilled or deionized water that does not contain substances which are toxic to

the test organisms. 

15.6.18 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.
297

TEST DATE: 

SAMPLE: 

COMPLEX EFFLUENT SAMPLE: 

COLLECTION DATE: 

SALINITY/ADJUSTMENT: 

PH/ADJUSTMENT REQUIRED: 

PHYSICAL CHARACTERISTICS: 

STORAGE: 

COMMENTS:

 

 

 

SINGLE COMPOUND: 

SOLVENT (CONC): 

TEST CONCENTRATIONS: 

DILUTION WATER: 

CONTROL WATER: 

TEST TEMPERATURE: 

TEST SALINITY: 

COMMENTS: 

 

Figure 1. Data form (1) for fertilization test using sea urchin, Arbacia punctulata. 
298

TEST DATE: 

SAMPLE: 

 

SPERM DILUTIONS: 

HEMACYTOMETER COUNT, E: __________ x 104

 = SPM SOLUTION E = __________ 

SPERM CONCENTRATIONS: SOLUTION E x 40 = SOLUTION A = __________ SPM 

SOLUTION E x 20 = SOLUTION B = __________ SPM 

SOLUTION E x 5 = SOLUTION D = __________ SPM 

SOLUTION SELECTED FOR TEST ( = 5 x 107

 SPM): 

DILUTION: SPM/(5 x 107

) = __________ DF 

[(DF) x 10) - 10 = __________ + SW, mL 

FINAL SPERM COUNTS = 

EGG DILUTIONS: 

INITIAL EGG COUNT = __________

ORIGINAL EGG STOCK CONCENTRATION = 10X (INITIAL EGG COUNT) = __________

VOLUME OF SW TO ADD TO DILUTE EGG STOCK TO 2000/mL: 

EGG COUNT) - 200 = __________

CONTROL WATER TO ADD EGG STOCK, mL = __________

FINAL EGG COUNT = __________

TEST TIMES: 

SPERM COLLECTED: 

EGGS COLLECTED: 

 SPERM ADDED: 

EGGS ADDED: 

FIXATIVE ADDED: 

SAMPLES READ: 

Figure 2. Data form (2) for fertilization test using sea urchin, Arbacia punctulata. 
299

DATE TESTED: 

SAMPLE:

TOTAL AND UNFERTILIZED EGG COUNT AT END OF TEST: 

EFFLUENT REPLICATE VIAL 

CONC (%) 1 2 3 4 

TOTAL-UNFERT TOTAL-UNFERT TOTAL-UNFERT TOTAL-UNFERT 

STATISTICAL ANALYSIS: 

ANALYSIS OF VARIANCE: 

CONTROL: 

DIFFERENT FROM CONTROL (P): 

COMMENTS: 

Figure 3. Data form (3) for fertilization test using sea urchin, Arbacia punctulata. 
300

15.6.18.1 Saline test and dilution water -- the salinity of the test water must be 30‰. The salinity should vary by no

more than ± 2‰ among the replicates. If effluent and receiving water tests are conducted concurrently, the salinities of

these tests should be similar.

15.6.18.2 The overwhelming majority of industrial and sewage treatment effluents entering marine and estuarine

systems contain little or no measurable salts. Exposure of sea urchin eggs and sperm to these effluents will require

adjustments in the salinity of the test solutions. It is important to maintain a constant salinity across all treatments. In

addition it may be desirable to match the test salinity with that of the receiving water. Two methods are available to

adjust salinities – hypersaline brine (HSB) derived from natural seawater or artificial sea salts. 

15.6.18.3 Hypersaline brine (HSB): HSB has several advantages that make it desirable for use in toxicity testing. It

can be made from any high quality, filtered seawater by evaporation, and can be added to the effluent or to deionized

water to increase the salinity. HSB derived from natural seawater contains the necessary trace metals, biogenic

colloids, and some of the microbial components necessary for adequate growth, survival, and/or reproduction of marine

and estuarine organisms, and may be stored for prolonged periods without any apparent degradation. However, if

100‰ HSB is used as a diluent, the maximum concentration of effluent that can be tested will be 80% at 20‰ salinity

and 70% at 30‰ salinity. 

15.6.18.3.1 The ideal container for making HSB from natural seawater is one that (1) has a high surface to volume

ratio, (2) is made of a noncorrosive material, and (3) is easily cleaned (fiberglass containers are ideal). Special care

should be used to prevent any toxic materials from coming in contact with the seawater being used to generate the

brine. If a heater is immersed directly into the seawater, ensure that the heater materials do not corrode or leach any

substances that would contaminate the brine. One successful method used is a thermostatically controlled heat

exchanger made from fiberglass. If aeration is utilized, use only oil-free air compressors to prevent contamination. 

15.6.18.3.2 Before adding seawater to the brine generator, thoroughly clean the generator, aeration supply tube,

heater, and any other materials that will be in direct contact with the brine. A good quality biodegradable detergent

should be used, followed by several (at least three) thorough deionized water rinses. 

15.6.18.3.3 High quality (and preferably high salinity) seawater should be filtered to at least 10 μm before placing into

the brine generator. Water should be collected on an incoming tide to minimize the possibility of contamination. 

15.6.18.3.4 The temperature of the seawater is increased slowly to 40EC. The water should be aerated to prevent

temperature stratification and to increase water evaporation. The brine should be checked daily (depending on the

volume being generated) to ensure that the salinity does not exceed 100‰ and that the temperature does not exceed

40EC. Additional seawater may be added to the brine to obtain the volume of brine required. 

15.6.18.3.5 After the required salinity is attained, the HSB should be filtered a second time through a 1 mm filter and

poured directly into portable containers, (20 L cubitainers or polycarbonate water cooler jugs are suitable). The

containers should be capped and labeled with the date the brine was generated and its salinity. Containers of HSB

should be stored in the dark and maintained under room temperature until used. 

15.6.18.3.6 If a source of HSB is available, test solutions can be made by following the directions below. 

Thoroughly mix together the deionized water and brine before mixing in the effluent. 

15.6.18.3.7 Divide the salinity of the HSB by the expected test salinity to determine the proportion of deionized water

to brine. For example, if the salinity of the brine is 100‰ and the test is to be conducted at 30‰, 100‰ divided by

30‰ = 3.3. The proportion of brine is 1 part in 3.3 (one part brine to 2.3 parts deionized water). To make 1 L of

seawater at 30‰ salinity from a HSB of 100‰, 300 mL of brine and 700 mL of deionized water are required. 

15.6.18.3.8 Table 1 illustrates the preparation of test solutions at 30‰ if they are made by combining effluent (0‰),

deionized water and HSB (100‰), or FORTY FATHOMS® sea salts. 
301

15.6.18.4 Artificial sea salts: FORTY FATHOMS® brand sea salts have been used successfully at the EMSLCincinnati, for long-term (6-12 months) maintenance of stock cultures of sexually mature sea urchins and to perform

the sea urchin fertilization test. GP2 seawater formulation (Table 2) has also been used successfully at ERLNarragansett, RI. 

15.6.18.4.1 Synthetic sea salts are packaged in plastic bags and mixed with deionized water or equivalent. The

instructions on the package of sea salts should be followed carefully, and the salts should be mixed in a separate

container -- not in the culture tank. The deionized water used in hydration should be in the temperature range of 21-

26°C. Seawater made from artificial sea salts is conditioned (Spotte, 1973; Spotte, et al., 1984; Bower, 1983).

15.6.18.4.2 The GP2 reagent grade chemicals (Table 2) should be mixed with deionized (DI) water or its equivalent in

a container other than the culture or testing tanks. The deionized water used for hydration should be between 21-26°C. 

The artificial seawater must be conditioned (aerated) for 24 h before use as the testing medium. If the solution is to be

autoclaved, sodium bicarbonate is added after the solution has cooled. A stock solution of sodium bicarbonate is made

up by dissolving 33.6 g NaHCO3 in 500 mL of deionized water. Add 2.5 mL of this stock solution for each liter of the

GP2 artificial seawater.

TABLE 1. PREPARATION OF TEST SOLUTIONS AT A SALINITY OF 30‰ USING NATURAL

SEAWATER, HYPERSALINE BRINE, OR ARTIFICIAL SEA SALTS 1

 Solutions To Be Combined 

Effluent Volume of Volume of Diluent 

Effluent Concentration Effluent Seawater (30‰) 

Solution (%) Solution

 (mL) (mL) 

 

1 1001 840 —

2 50 420 Solution 1 + 420

3 25 420 Solution 2 + 420

4 12.5 420 Solution 3 + 420

5 6.25 420 Solution 4 + 420

Control 0.0 420

 

Total 2080

 

1 This illustration assumes: (1) the use of 5 mL of test solution in each of four replicates (total of 20 mL) for the 

control and five concentrations of effluent, (2) an effluent dilution factor of 0.5, (3) the effluent lacks appreciable

salinity, and (4) 400 mL of each test concentration is used for chemical analysis. A sufficient initial volume (840

mL) of effluent is prepared by adjusting the salinity to 30‰. In this example, the salinity is adjusted by adding

artificial sea salts to the 100% effluent, and preparing a serial dilution using 30‰ seawater (natural seawater,

hypersaline brine, or artificial seawater). Stir solutions 1 h to ensure that the salts dissolve. The salinity of the

initial 840 mL of 100% effluent is adjusted to 30‰ by adding 25.2 g of dry artificial sea salts (FORTY

FATHOMS®). Test concentrations are then made by mixing appropriate volumes of salinity adjusted effluent and

30‰ salinity dilution water to provide 840 mL of solution for each concentration. If hypersaline brine alone

(100‰) is used to adjust the salinity of the effluent, the highest concentration of effluent that could be tested

would be 70% at 30‰ salinity. 
302

TABLE 2. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2 ARTIFICIAL

SEAWATER FOR THE SEA URCHIN, ARBACIA PUNCTULATA, TOXICITY TEST1,2,3

Compound Concentration Amount (g)

(g/L) Required for

20 L

NaCl 21.03 420.6

Na2SO4 3.52 70.4

KCl 0.61 12.2

KBr 0.088 1.76

Na2B4O7@10 H2O 0.034 0.68

MgCl2@6 H2O 9.50 190.0

CaCl2@2 H2O 1.32 26.4

SrCl2@6 H2O 0.02 0.400

 NaHCO3 0.17 3.40

1 Modified GP2 from Spotte et al. (1984). 2 The constituent salts and concentrations were taken from USEPA (l990b). The salinity is 30.89 g/L. 3 GP2 can be diluted with deionized (DI) water to the desired test salinity.

15.6.19 TEST ORGANISMS, SEA URCHINS, ARBACIA PUNCTULATA

15.6.19.1 Adult sea urchins, Arbacia punctulata, can be obtained from commercial suppliers. After acquisition, the

animals are sexed by briefly stimulating them with current from a 12 V transformer. Electrical stimulation causes the

immediate release of masses of gametes that are readily identifiable by color -- the eggs are red, and the sperm are

white. 

15.6.19.2 The sexes are separated and maintained in 20-L, aerated fiberglass tanks, each holding about 20 adults. The

tanks are supplied continuously (approximately 5 L/min) with filtered natural seawater, or salt water prepared from

commercial sea salts is recirculated. The animals are checked daily and any obviously unhealthy animals are discarded. 

15.6.19.3 The culture unit should be maintained at 15 ± 3°C, with a water temperature control device. 

15.6.19.4 The food consists of kelp, Laminaria sp., gathered from known uncontaminated zones or obtained from

commercial supply houses whose kelp comes from known uncontaminated areas, or romaine lettuce. Fresh food is

introduced into the tanks at approximately one week intervals. Decaying food is removed as necessary. Ample

supplies of food should always be available to the sea urchins. 

15.6.19.5 Natural or artificial seawater with a salinity of 30‰ is used to maintain the adult animals, for all washing

and dilution steps, and as the control water in the tests (see Subsection 15.6.18). 

15.6.19.6 Adult male and female animals used in field studies are transported in separate or partitioned insulated

boxes or coolers packed with wet kelp or paper toweling. Upon arrival at the field site, aquaria (or a single partitioned
303

aquarium) are filled with control water, loosely covered with a styrofoam sheet and allowed to equilibrate to 15°C

before animals are added. Healthy animals will attach to the kelp or aquarium within hours. 

15.6.19.7 To successfully maintain about 25 adult animals for 7 days at a field site, a screen-partitioned, 40-L glass

aquarium using aerated, recirculating, clean saline water (30‰) and a gravel bed filtration system, is housed within a

water bath, such as FORTY FATHOMS® or equivalent (15°C). The inner aquarium is used to avoid contact of animals

and water bath with cooling coils. 

15.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE 

15.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling, and Sampling Preparation for

Toxicity Tests. 

15.8 CALIBRATION AND STANDARDIZATION 

15.8.1 See Section 4, Quality Assurance.

15.9 QUALITY CONTROL

15.9.1 See Section 4, Quality Assurance.

15.10 TEST PROCEDURES 

15.10.1 TEST SOLUTIONS

15.10.1.1 Receiving Waters 

15.10.1.1.1 The sampling point is determined by the objectives of the test. At estuarine and marine sites, samples are

usually collected at mid-depth. Receiving water toxicity is determined with samples used directly as collected or with

samples passed through a 60 μm NITEX® filter and compared without dilution against a control. Using four replicate

chambers per test, each containing 5 mL, and 400 mL for chemical analysis, would require approximately 420 mL or

more of sample per test. 

15.10.1.2 Effluents 

15.10.1.2.1 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 ± 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 dilution factors are increased beyond 0.5 and declines rapidly if smaller dilution

factors are used. Therefore, USEPA recommends the use of the $ 0.5 dilution factor. If 100‰ HSB is used as a

diluent, the maximum concentration of effluent that can be tested will be 80% at 20‰ and 70% at 30‰ salinity.

15.10.1.2.2 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%). 

15.10.1.2.3 Just prior to test initiation (approximately 1 h), a sufficient quantity of the sample to make the test

solutions should be adjusted to the test temperature (20 ± 1°C) and maintained at that temperature during the addition

of dilution water.

15.10.1.2.4 The test 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 Test).
304

15.10.1.2.5 Effluent dilutions should be prepared for all replicates in each treatment in one beaker to minimize

variability among the replicates. The test chambers are labeled with the test concentration and replicate number. 

Dispense into the appropriate effluent dilution chamber.

15.10.1.3 Dilution Water

15.10.1.3.1 Dilution water may be uncontaminated natural seawater (receiving water), HSB prepared from natural

seawater, or artificial seawater FORTY FATHOMS® or GP2 sea salts (see Table 2 and Section 7, Dilution Water). 

Prepare 3 L of control water at 30‰ using HSB or artificial sea salts (see Table 1). This water is used in all washing

and diluting steps and as control water in the test. Natural seawater and local waters may be used as additional

controls.

15.10.2 COLLECTION OF GAMETES FOR THE TEST 

15.10.2.1 Select four females and place in shallow bowls, barely covering the shell with seawater. Stimulate the

release of eggs by touching the shell with steel electrodes connected to a 10-12 volt transformer (about 30

seconds each time). Collect the eggs from each female using a 10 mL disposable syringe fitted with an 18-gauge,

blunt-tipped needle (tip cut off). Remove the needle from the syringe before adding the eggs to a conical centrifuge

tube. Pool the eggs. The egg stock may be held at room temperature for several hours before use. Note: Eggs should

be collected first to eliminate possibility of pre-fertilization.

15.10.2.2 Select four males and place in shallow bowls, barely covering the animals with seawater. Stimulate the

release of sperm as described above. Collect the sperm (about 0.25 mL) from each male, using a 1-3 mL disposable

syringe fitted with an 18-gauge, blunt-tipped needle. Pool the sperm. Maintain the pooled sperm sample on ice. The

sperm must be used in a toxicity test within 1 h of collection. 

15.10.3 PREPARATION OF SPERM DILUTION FOR USE IN THE TEST 

15.10.3.1 Using control water, dilute the pooled sperm sample to a concentration of about 5 X l07

 sperm/mL (SPM). 

Estimate the sperm concentration as described below: 

1. Make a sperm dilutions of 1:50, 1:100, 1:200, and 1:400, using 30‰ seawater, as follows:

a. Add 400 μL of collected sperm to 20 mL of seawater in Vial A. Mix by gentle pipetting 

 using a 5-mL pipettor, or by inversion;

b. Add 10 mL of sperm suspension from Vial A to 10 mL of seawater in Vial B. Mix by gentle

pipetting using a 5-mL pipettor, or by inversion; 

c. Add 10 mL of sperm suspension from Vial B to 10 mL of seawater in Vial C. Mix by gentle

pipetting using a 5-mL pipettor, or by inversion; 

d. Add 10 mL of sperm suspension from Vial C to 10 mL of seawater in Vial D. Mix by gentle

pipetting using a 5-mL pipettor, or by inversion; 

e. Discard 10 mL from Vial D. (The volume of all suspensions is 10 mL).

2. Make a 1:2000 killed sperm suspension and determine the SPM. 

a. Add 10 mL 10% acetic acid in seawater to Vial C. Cap Vial C and mix by inversion.

b. Add 1 mL of killed sperm from Vial C to 4 mL of seawater in Vial E. Mix by gentle 

 pipetting with a 4-mL pipettor. 

c. Add sperm from Vial E to both sides of the Neubauer hemacytometer. Let the sperm settle 

 15 min. 

d. Count the number of sperm in the central 400 squares on both sides of the hemacytometer 

 using a compound microscope (100X). Average the counts from the two sides. 

e. SPM in Vial E = 104

 x average count. 
305

3. Calculate the SPM in all other suspensions using the SPM in Vial E above: 

SPM in Vial A = 40 x SPM in Vial E 

SPM in Vial B = 20 x SPM in Vial E 

SPM in Vial D = 5 x SPM in Vial E 

SPM in original sperm sample = 2000 x SPM in Vial E 

4. Dilute the sperm suspension with a SPM greater than 5 x 107

 SPM to 5 x 107 SPM. 

Actual SPM/(5 x 107

) = dilution factor (DF) 

[(DF) x 10] - 10 = mL of seawater to add to vial. 

5. Confirm the sperm count by sampling from the test stock. Add 0.1 mL of test stock to 9.9 mL of 10%

acetic acid in seawater, and count with the hemacytometer. The count should average 50 ± 5. 

15.10.4 PREPARATION OF EGG SUSPENSION FOR USE IN THE TEST Note: The egg suspension may be

prepared during the 1-h sperm exposure.

15.10.4.1 Wash the pooled eggs three times using control water with gentle centrifugation (500xg for 3 minutes using

a tabletop centrifuge). If the wash water becomes red, the eggs have lysed and must be discarded.

15.10.4.2 Dilute the egg stock, using control water, to about 2000 eggs/mL. 

1. Transfer the eggs to a glass beaker containing 200 mL of control water ("egg stock"). 

2. Mix the egg stock using an air-bubbling device. Using a wide-mouth pipet tip, transfer 1 mL of eggs

from the egg stock to a vial containing 9 mL of control water. (This vial contains an egg suspension

diluted 1:10 from egg stock). 

3. Mix the contents of the vial by inversion. Using a wide-mouth pipet tip, transfer 1 mL of eggs from the

vial to a Sedgwick-Rafter counting chamber. Count all eggs in the chamber using a dissecting

microscope at 24X "egg count". 

4. Calculate the concentration of eggs in the stock. Eggs/mL = 10X (egg count). Dilute the egg stock to

2000 eggs/mL by the formula below. 

a. If the egg count is equal to or greater than 200: 

(egg count) - 200 = volume (mL) of control water to add to egg stock.

b. If the egg count is less than 200, allow the eggs to settle and remove enough control water to

concentrate the eggs to greater than 200, repeat the count, and dilute the egg stock as in a. above. 

NOTE: It requires 24 mL of a egg stock solution for each test with a control and five exposure

concentrations. 

c. Transfer 1 mL of the diluted egg stock to a vial containing 9 mL of control water. Mix well, then

transfer 1 mL from the vial to a Sedgwick-Rafter counting chamber. Count all eggs using a

dissecting microscope. Confirm that the final egg count = 2000/mL (± 200).

15.10.5 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE

15.10.5.1 The light quality and intensity should be at ambient laboratory levels 10-20 μE/m2

/s (50-100 ft-c) with a

photoperiod of 16 h light and 8 h darkness. The water temperature in the test chambers should be maintained at 20 ±

1°C. The test salinity should be in the range of 28 to 32‰. The salinity should vary by no more that ± 2‰ among the

chambers on a given day. If effluent and receiving water tests are conducted concurrently, the salinities of these tests

should be similar. 
306

15.10.6 DISSOLVED OXYGEN (DO) CONCENTRATION

15.10.6.1 Aeration may affect the toxicity of effluent and should be used only as a last resort to maintain a satisfactory

DO. The DO concentrations should be measured on new solutions at the start of the test (Day 0). The DO 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 treatments and the control should be aerated. The aeration rate

should not exceed 100 bubbles/minute, using a pipet with a 1-2 mm orifice, such as a 1 mL KIMAX® serological pipet,

or equivalent.

15.10.7 OBSERVATIONS DURING THE TEST

15.10.7.1 Routine Chemical and Physical Observations

15.10.7.1.1 DO is measured at the beginning of the exposure period in one test chamber at each test concentration and

in the control.

15.10.7.1.2 Temperature, pH, and salinity are measured at the beginning of the exposure period in one test chamber at

each concentration and in the control. Temperature should also be monitored continuously 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 chambers at least at the end of the test to determine temperature variation in environmental

chamber.

15.10.7.1.3 The pH is measured in the effluent sample each day before new test solutions are made.

15.10.7.1.4 Record all the measurements on the data sheet.

15.10.7.2 Routine Biological Observations

15.10.7.2.1 Fertilization will be determined by the presence of a fertilization membrane surrounding the egg.

15.10.8 START OF THE TEST 

15.10.8.1 Effluent/receiving water samples are adjusted to salinity of 30‰. Four replicates are prepared for each test

concentration, using 5 mL of solution in disposable liquid scintillation vials. A 50% (0.5) concentration series can be

prepared by serially diluting test concentrations with control water. Sufficient test solution is prepared at each effluent

concentration to provide additional volume for chemical analyses, at the high, medium, and low test concentrations. 

15.10.8.2 All test samples are equilibrated at 20°C ± 1°C before addition of sperm.

15.10.8.3 Within 1 h of collection add 100 μL of appropriately diluted sperm to each test vial. Record the time of

sperm addition. 

15.10.8.4 Incubate all test vials at 20 ± 1°C for 1 h. 

15.10.8.5 Mix the diluted egg suspension (2000 eggs/mL), using gentle bubbling. Add 1 mL of diluted egg 

suspension to each test vial using a wide mouth pipet tip. Incubate 20 min at 20 ± 1°C. 

 

15.10.9 TERMINATION OF THE TEST 

15.10.9.1 Terminate the test and preserve the samples by adding 2 mL of 1% formalin in seawater to each vial. 

15.10.9.2 Vials should be evaluated within 48 hours. 
307

15.10.9.3 To determine fertilization, transfer about 1 mL eggs from the bottom of a test vial to a Sedgwick-Rafter

counting chamber. Observe the eggs using a compound microscope (100X). Count between 100 and 200 eggs/sample. 

Record the number counted and the number unfertilized. Fertilization is indicated by the presence of a fertilization

membrane surrounding the egg. NOTE: adjustment of the microscope to obtain proper contrast may be required to

observe the fertilization membrane. Because samples are fixed in formalin, a ventilation hood is set up surrounding the

microscope to protect the analyst from prolonged exposure to formaldehyde fumes. 

15.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA 

15.11.1 A summary of test conditions and test acceptability criteria is listed in Table 3. 

15.12 ACCEPTABILITY OF TEST RESULTS

15.12.1 The sperm:egg ratio routinely employed must result in fertilization of 70%-90% of the eggs in the control

chambers. 
308

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR SEA

URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST WITH EFFLUENT AND

RECEIVING WATERS (TEST METHOD 1008.0)1

1 Test type: Static (required) 

2. Salinity: 30‰ (± 2‰ of the selected test salinity) (recommended) 

3. Temperature: 20 ± 1°C (recommended)

Test temperatures must not deviate (i.e., maximum minus

minimum temperature) by more than 3°C during the test

(required) 

4. Light quality: Ambient laboratory light during test preparation

(recommended) 

5. Light intensity: 10-20 μE/m2

/s, or 50-100 ft-c (Ambient laboratory levels)

(recommended) 

6. Test chamber size: Disposable (glass) liquid scintillation vials (20 mL capacity),

presoaked in control water (recommended) 

7. Test solution volume: 5 mL (recommended) 

8. No. of sea urchins: Pooled sperm from four males and pooled eggs from four

females are used per test (recommended)

9. No. egg and sperm cells per chamber: About 2,000 eggs and 5,000,000 sperm cells per vial

(recommended) 

10. No. replicate chambers per concentration: 4 (required minimum)

11. Dilution water: Uncontaminated source of natural seawater; deionized water

mixed with hypersaline brine or artificial sea salts (HW

MARINEMIX®, FORTY FATHOMS®, GP2, or equivalent)

(available options)

12. Test concentrations: Effluents: 5 and a control (required minimum)

Receiving waters: 100% receiving water (or minimum of 5)

and a control (recommended) 

1

 For the purposes of reviewing WET test data submitted under NPDES permits, each test condition listed above is

identified as required or recommended (see Subsection 10.2 for more information on test review). Additional

requirements may be provided in individual permits, such as specifying a given test condition where several options

are given in the method. 
309

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR SEA

URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST WITH EFFLUENT AND

RECEIVING WATERS (TEST METHOD 1008.0) (CONTINUED)

13. Dilution factor: Effluents: $0.5 (recommended) 

Receiving waters: None or $0.5 (recommended) 

14. Test duration: 1 h and 20 min (required) 

15. Endpoint: Fertilization of sea urchin eggs (required) 

16. Test acceptability criteria: 70% - 90% egg fertilization in controls (required) 

17. Sampling requirements: 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

before first use (see Section 8, Effluent and Receiving Water

Sampling, Sample Handling, and Sample Preparation for

Toxicity Tests, Subsection 8.5.4) (required) 

18. Sample volume required: 1 L per test (recommended) 

15.13 DATA ANALYSIS

15.13.1 GENERAL

15.13.1.1 Tabulate and summarize the data. Calculate the proportion of fertilized eggs for each replicate. A sample

set of test data is listed in Table 4.

15.13.1.2 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.
310

TABLE 4. DATA FROM SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION TEST

Copper 

Concentration No. of Eggs No. of Eggs Proportion

(μg/L) Replicate Counted Fertilized Fertilized

Control A 100 85 0.85

B 100 78 0.78

C 100 87 0.87

2.5 A 100 81 0.81

B 100 65 0.65

C 100 71 0.71

5.0 A 100 63 0.63

B 100 74 0.74

C 100 78 0.78

10.0 A 100 63 0.63

B 100 66 0.66

C 100 51 0.51

20.0 A 100 41 0.41

B 100 41 0.41 

C 100 37 0.37

40.0 A 100 12 0.12

B 100 30 0.30

C 100 26 0.26

1 Tests performed by Dennis M. McMullen, Technology Applications, Inc., EMSL, Cincinnati, OH.

15.13.1.3 The endpoints of toxicity tests using the sea urchin are based on the reduction in proportion of eggs

fertilized. The IC25 and the IC50 are calculated using the Linear Interpolation Method (see Section 9, Chronic

Toxicity Test Endpoints and Data Analysis). LOEC and NOEC values for fecundity 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 IC25 and IC50. See the Appendices for examples of the manual computations, and examples of

data input and program output. 

15.13.2 EXAMPLE OF ANALYSIS OF SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION DATA

15.13.2.1 Formal statistical analysis of the fertilization data is outlined in Figure 4. The response used in the

analysis is the proportion of fertilized eggs 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 IC25 and IC50 endpoints. 

Concentrations at which there are no eggs fertilized in any of the test chambers are excluded from statistical analysis

of the NOEC and LOEC, but included in the estimation of the IC25 and IC50.

15.13.2.2 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
311

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. 

15.13.2.3 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. 

15.13.2.4 Example of Analysis of Fecundity Data

15.13.2.4.1 This example uses toxicity data from a sea urchin, Arbacia punctulata, fertilization test performed with

copper. The response of interest is the proportion of fertilized eggs, thus 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 copper concentration and control are listed in Table 5. The

data are plotted in Figure 5.

15.13.2.5 Test for Normality 

15.13.2.5.1 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 6. 
312

Figure 4. Flowchart for statistical analysis of sea urchin, Arbacia punctulata, by point estimation.
313

Figure 5. Plot of mean percent of fertilized sea urchin, Arbacia punctulata, eggs.
314

D ' j

n

i'1

(Xi&X ̄)

2

TABLE 5. SEA URCHIN, ARBACIA PUNCTULATA, FERTILIZATION DATA

Copper Concentration (μg/L) 

Replicate Control 2.5 5.0 10.0 20.0 40.0

A 0.85 0.81 0.63 0.63 0.41 0.12

RAW B 0.78 0.65 0.74 0.66 0.41 0.30

C 0.87 0.71 0.78 0.51 0.37 0.26

ARC SINE A 1.173 1.120 0.917 0.917 0.695 0.354

TRANSFORMED B 1.083 0.938 1.036 0.948 0.695 0.580

C 1.202 1.002 1.083 0.795 0.654 0.535

Mean ( ) 1.153 1.020 1.012 0.887 0.681 0.490 Y ̄ i

S 0.004 0.009 0.007 0.007 0.001 0.014 2

i

i 1234 5 6

TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE 

Copper Concentration (μg/L) 

 

Replicate Control 2.5 5.0 10.0 20.0 40.0

A 0.020 0.100 -0.095 0.030 0.014 -0.136

B -0.070 -0.082 0.024 0.061 0.014 0.090

C 0.049 -0.018 0.071 -0.092 -0.027 0.045

15.13.2.5.2 Calculate the denominator, D, of the statistic: 

Where: Xi = the ith centered observation 

 

X ̄ = the overall mean of the centered observations 

n = the total number of centered observations 
315

W ' 1

D [j

k

i'1

ai(X (n&i%1)&X (i))]2

W ' 1

0.0822 (0.2782)2 ' 0.942

15.13.2.5.3 For this set of data, n = 18 

X ̄ = 1 (0) = 0 

 18

D = 0.0822 

15.13.2.5.4 Order the centered observations from smallest to largest 

X(1) # X(2) # ... # X(n)

where X(i) denotes the ith ordered observation. The ordered observations for this example are listed in Table 7. 

TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

i X(i) i X(i)

1 -0.136 10 0.020

2 -0.095 11 0.024

3 -0.092 12 0.030 

4 -0.082 13 0.045 

5 -0.070 14 0.049 

6 -0.027 15 0.061 

7 -0.018 16 0.071 

8 0.014 17 0.090 

9 0.014 18 0.100 

15.13.2.5.5 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-1)/2 if n is odd. For the data in this example, n = 18 and k = 9. The ai

 values are listed in Table 8. 

15.13.2.5.6 Compute the test statistic, W, as follows: 

The differences, X(n-i+1) - X(i), are listed in Table 8. For the data in this example: 

15.13.2.5.7 The decision rule for this test is to compare W as calculated in Subsection 15.13.2.5.6 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 = 18 observations is

0.858. Since W = 0.942 is greater than the critical value, conclude that the data are normally distributed. 
316

B '

[(j

p

i'1

Vi) lnS ̄2&j

p

i'1

Vi ln S 2

i ]

C

S ̄2

'

(j

p

i'1

ViS 2

i )

j

p

i'1

Vi

C ' 1%[3(p&1)]&1 [j

p

i'1

1/Vi&(j

p

i'1

Vi)

&1

]

TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE 

i ai X(n-i+1) - X(i)

1 0.4886 0.236 X(18) - X(1)

2 0.3253 0.185 X(17) - X(2)

3 0.2553 0.163 X(16) - X(3)

4 0.2027 0.143 X(15) - X(4)

5 0.1587 0.119 X(14) - X(5)

6 0.1197 0.072 X(13) - X(6)

7 0.0837 0.048 X(12) - X(7)

8 0.0496 0.010 X(11) - X(8)

9 0.0163 0.006 X(10) - X(9)

15.13.2.6 Test for Homogeneity of Variance 

15.13.2.6.1 The test used to examine whether the variation in the proportion of fertilized eggs is the same across all

copper concentrations including the control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as

follows: 

 Where: Vi = degrees of freedom for each copper concentration and control, Vi

 = (ni - 1) 

p = number of levels of copper concentration including the control

ni = the number of replicates for concentration i. 

ln = loge

i = 1,2, ..., p where p is the number of concentrations including the control
317

B ' [(12)ln(0.0007)&2j

p

i'1

ln(S 2

i )]/1.194

15.13.2.6.2 For the data in this example (see Table 5), all copper concentrations including the control have the same

number of replicates (ni

 = 3 for all i). Thus, Vi = 2 for all i. 

15.13.2.6.3 Bartlett's statistic is, therefore: 

= [12(-4.962) - 2(-31.332)]/1.194 

 = 3.122/1.194 

 = 2.615 

15.13.2.6.4 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 5 degrees of

freedom, is 15.09. Since B = 2.615 is less than the critical value of 15.09, conclude that the variances are not different. 

15.13.2.7 Dunnett's Procedure 

15.13.2.7.1 To obtain an estimate of the pooled variance for the Dunnett's Procedure, construct an ANOVA table as

described in Table 9.

TABLE 9. ANOVA TABLE 

Source df Sum of Squares Mean Square (MS)

(SS) (SS/df)

Between p - 1 SSB = SSB/(p-1) S 2

B

Within N - p SSW = SSW/(N-p) S 2

W

Total N - 1 SST

Where: p = number of concentration levels including the control

N = total number of observations n1 + n2 ... + np

ni

 = number of observations in concentration i

SSB ' j Between Sum of Squares

p

i'1

T 2

i /ni

&G 2 /N

SST ' j Total Sum of Squares

p

i'1

j

ni

j'1

Y 2

ij &G 2 /N
318

SSB ' j

p

i'1

T 2

i /ni

&G 2 /N

SST ' j

p

i'1

j

ni

j'1

Y 2

ij &G 2 /N

SSW ' SST&SSB

SSW ' SST&SSB Within Sum of Squares 

G = the grand total of all sample observations, G'j

p

i'1

Ti

Ti = the total of the replicate measurements for concentration i

Yij = the jth observation for concentration i (represents the proportion of fertilized eggs for upper 

concentration i in test chamber j) 

15.13.2.7.2 For the data in this example: 

n1 = n2 = n3 = n4 = n5 = n6 = 3 

N = 18 

T1 = Y11 + Y12 + Y13 = 3.458 

T2 = Y21 + Y22 + Y23 = 3.060 

T3 = Y31 + Y32 + Y33 = 3.036 

T4 = Y41 + Y42 + Y43 = 2.660 

T5 = Y51 + Y52 + Y53 = 2.044 

T6 = Y61 + Y62 + Y63 = 1.469 

G = T1 + T2 + T3 + T4 + T5 + T6 = 15.727 

= (43.950)/3 - (15.727)2

/18 = 0.909 

= 14.732 - (15.727)2

/18 = 0.991 

 = 0.991 - 0.909 = 0.082 

S = SSB/(p-1) = 0.909/(6-1) = 0.182 2

B

S = SSW/(N-p) = 0.082/(18-6) = 0.007 2

W

15.13.2.7.3 Summarize these calculations in the ANOVA table (Table 10). 
319

t

i ' (Y ̄

1&Y ̄

i

)

Sw (1/n1) % (1/ni

)

t

2 ' (1.153&1.020)

[0.084 (1/3)%(1/3)]

'1.939

TABLE 10. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE 

Source df Sum of Squares Mean Square(MS) 

 (SS) (SS/df) 

Between 5 0.909 0.182 

Within 12 0.082 0.007 

Total 17 0.991

15.13.2.7.4 To perform the individual comparisons, calculate the t statistic 

for each concentration, and control combination as follows: 

Where: = mean proportion fertilized Y ̄ eggs for copper concentration i i

Y ̄ = mean proportion fertilized eggs for the control 1

SW = square root of the within mean square 

n1 = number of replicates for the control 

ni = number of replicates for concentration i

Since we are looking for a decreased response from the control in the proportion of fertilized eggs, the concentration

mean is subtracted from the control mean.

15.13.2.7.5 Table 11 includes the calculated t values for each concentration and control combination. In this example,

comparing the 2.5 μg/L concentration with the control the calculation is as follows: 
320

MSD ' d Sw (1/n1) % (1/n)

MSD ' 2.50(0.084) (1/3)%(1/3)

TABLE 11. CALCULATED T VALUES

Copper Concentration (μg/L) i ti

2.5 2 1.939

5.0 3 2.056

10.0 4 3.878

20.0 5 6.882

40.0 6 9.667

15.13.2.7.6 Since the purpose of this test is to detect a significant decrease in the proportion of fertilized eggs, a

one-sided test is appropriate. The critical value for this one-sided test is found in Table 5, Appendix D. 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 proportion of fertilized eggs for concentration i is considered significantly less than the mean

proportion of fertilized eggs for the control if ti

 is greater than the critical value. Therefore, the 10.0 μg/L, 20.0 μg/L

and 40.0 μg/L concentrations have a significantly lower mean proportion of fertilized eggs than the control. Hence the

NOEC is 5.0 μg/L and the LOEC is 10.0 μg/L. 

15.13.2.7.7 To quantify the sensitivity of the test, the minimum significant difference (MSD) that can be statistically

detected may be calculated: 

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) 

n1 = the number of replicates in the control. 

15.13.2.7.8 In this example, 

= 2.50 (0.084)(0.816) 

= 0.171 

15.13.2.7.9 The MSD (0.171) is in transformed units. To determine the MSD in terms of proportion of fertilized

eggs, carry out the following conversion. 

1. Subtract the MSD from the transformed control mean. 

1.153 - 0.171 = 0.982 

2. Obtain the untransformed values for the control mean and the difference calculated in step 1

of 15.13.2.7.9.1 

[ Sine (1.153) ]2

 = 0.835
321

ICp ' Cj

%[M1 (1&p/100)&Mj

] (C(j%1)&Cj

)

(M(j%1)&Mj

)

[ Sine (0.982) ]2

 = 0.692 

3. The untransformed MSD (MSDu) is determined by subtracting the untransformed values from

step 2 in 15.13.2.7.9. 

MSDu = 0.835 - 0.692 = 0.143 

15.13.2.7.10 Therefore, for this set of data, the minimum difference in mean proportion of fertilized eggs between

the control and any copper concentration that can be detected as statistically significant is 0.143. 

15.13.2.7.11 This represents a 17% decrease in the proportion of fertilized eggs from the control. 

15.13.2.8 Calculation of the ICp

15.13.2.8.1 The fertilization data in Table 4 are utilized in this example. Table 12 contains the mean proportion of

fertilized eggs for each toxicant concentration. As can be seen, the observed means are monotonically nonincreasing 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).

15.13.2.8.2 An IC25 and IC50 can be estimated using the Linear Interpolation Method. A 25% reduction in mean

proportion of fertilized eggs, compared to the controls, would result in a mean proportion of 0.625, where M1(1-

p/100) = 0.833(1-25/100). A 50% reduction in mean proportion of fertilized eggs, compared to the controls, would

result in a mean proportion of 0.417. Examining the means and their associated concentrations (Table 12), the

response, 0.625, is bracketed by C3 = 5.0 μg/L copper and C4 = 10.0 μg/L copper. The response, 0.417, is bracketed

by C4 = 10.0 μg/L copper and C5 = 20.0 μg/L copper.

TABLE 12. SEA URCHIN, ARBACIA PUNCTULATA, MEAN PROPORTION OF FERTILIZED EGGS

Copper Response Smoothed

Conc. Means Yi Mean Mi

(μg/L) i (proportion) (proportion)

Control 1 0.833 0.833

2.5 2 0.723 0.723

5.0 3 0.717 0.717

10.0 4 0.600 0.600

20.0 5 0.397 0.397

40.0 6 0.227 0.227

15.13.2.8.3 Using the equation from Section 4.2 in Appendix L, the estimate of the IC25 is calculated as follows:

IC25 = 5.0 + [0.833(1 - 25/100) - 0.717] (10.0 - 5.0) 

(0.600 - 0.717) 

= 8.9 μg/L. 
322

ICp ' Cj%[M1 (1&p/100)&Mj] (C(j%1)&Cj

)

(M(j%1)&Mj

)

15.13.2.8.4 Using the equation from Section 4.2 in Appendix L, the estimate of the IC50 is calculated as follows:

IC50 = 10.0 + [0.833(1 - 50/100) - 0.600] (20.0 - 10.0) 

(0.397 - 0.600) 

= 19.0 μg/L. 

15.13.2.8.5 When the ICPIN program was used to analyze this set of data, requesting 80 resamples, the estimate of

the IC25 was 8.9286 μg/L. The empirical 95.0% confidence interval for the true mean was 3.3036 μg/L to 14.6025

μg/L. The computer program output for the IC25 for this data set is shown in Figure 6.

15.13.2.8.6 When the ICPIN program was used to analyze this set of data, requesting 80 resamples, the estimate of

the IC50 was 19.0164 μg/L. The empirical 95.0% confidence interval for the true mean was 16.1083 μg/L to

23.6429 μg/L. The computer program output for the IC50 for this data set is shown in Figure 7.
323

Conc. ID 1 2 3 4 5 6

Conc. Tested 0 2.5 5.0 10.0 20.0 40.0

Response 1 .85 .81 .63 .63 .41 .12

Response 2 .78 .65 .74 .66 .41 .3

Response 3 .87 .71 .71 .51 .37 .2

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: Copper

Test Start Date: Test Ending Date: 

Test Species: sea urchin, Arbacia punctulata

Test Duration:

DATA FILE: urchin.icp

OUTPUT FILE: urchin.i25

Conc. Number Concentration Response Standard. Pooled

 ID Replicates μg/L Means Dev. Response Means

 1 3 0.000 0.833 0.047 0.833

 2 3 2.500 .723 0.081 0.723

 3 3 5.000 0.717 0.078 0.717

 4 3 10.000 0.600 0.079 0.600

 5 3 20.000 0.397 0.023 0.397

 6 3 40.000 0.227 0.095 0.227

The Linear Interpolation Estimate: 8.9286 Entered P Value: 25

Number of Resamplings: 80

The Bootstrap Estimates Mean: 8.7092 Standard Deviation: 0.8973

Original Confidence Limits: Lower: 6.2500 Upper: 11.6304

Expanded Confidence Limits Lower: 3.3036 Upper: 14.6025

Resampling time in Seconds: 1.59 Random Seed: 1834854321

Figure 6. ICPIN program output for the IC25.
324

Conc. ID 1 2 3 4 5 6

Conc. Tested 0 2.5 5.0 10.0 20.0 40.0

Response 1 .85 .81 .63 .63 .41 .12

Response 2 .78 .65 .74 .66 .41 .3

Response 3 .87 .71 .78 .51 .37 .26

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: Copper

Test Start Date: Test Ending Date: 

Test Species: MYSID SHRIMP

Test Duration: fecundity

DATA FILE: mysidfe.icp

OUTPUT FILE: mysidfe.i50

Conc. Number Concentration Response Standard. Pooled

 ID Replicates μg/l Means Dev. Response Means

 1 8 0.000 0.934 0.127 0.934

 2 7 50.000 0.426 0.142 0.426

 3 7 100.000 0.317 0.257 0.317

 4 8 210.000 0.000 0.000 0.000

-------------------------------------------------------------------------------------------------------------------------------------------

The Linear Interpolation Estimate: 19.0164 Entered P Value: 50

-------------------------------------------------------------------------------------------------------------------------------------------

Number of Resamplings: 80

The Bootstrap Estimates Mean: 19.0013 Standard Deviation: 0.8973

Original Confidence Limits: Lower: 17.6316 Upper: 21.2195

Expanded Confidence Limits: Lower: 16.1083 Upper: 23.6492

Resampling time in Seconds: 1.65 Random Seed: -823775279

Figure 7. ICPIN program output for the IC50.
325

15.14 PRECISION AND ACCURACY

15.14.1 PRECISION

15.14.1.1 Single-Laboratory Precision

15.14.1.1.1 Single-laboratory precision data for the reference toxicants, copper (Cu) and sodium dodecyl sulfate

(SDS), tested in FORTY FATHOMS® artificial seawater, GP2 artificial seawater, and natural seawater are provided

in Tables 13-18. The test results were similar in the three types of seawater. The IC25 and IC50 for the reference

toxicants (copper and sodium dodecyl sulfate) are reported in Tables 13-16. The coefficient of variation, based on

the IC25, is 28.7% to 54.6% for natural and FORTY FATHOMS® seawater, indicating acceptable precision. The

IC50 ranges from 23.3% to 48.2%, showing acceptable precision. 

15.14.1.2 Multilaboratory Precision

15.14.1.2.1 No data are available on the multilaboratory precision of the test. 

15.14.2 ACCURACY

15.14.2.1 The accuracy of toxicity tests cannot be determined.
326

TABLE 13. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA PUNCTULATA,

FERTILIZATION TEST PERFORMED IN FORTY FATHOMS® ARTIFICIAL SEAWATER,

USING GAMETES FROM ADULTS MAINTAINED IN FORTY FATHOMS® ARTIFICIAL

SEAWATER, OR OBTAINED DIRECTLY FROM NATURAL SOURCES, AND COPPER

(CU) AS A REFERENCE TOXICANT1,2,3,4,5

Test LOEC IC25 IC50

Number (μg/L) (μg/L) (μg/L)

1 5.0 8.92 29.07

2 12.5 26.35 38.96

3 <6.2 11.30 23.93

4 6.2 34.28 61.75

5 12.5 36.67 75.14

n: 4 5 5

Mean: NA 23.51 45.77

CV(%): NA 54.60 47.87

1 Data from USEPA (1991a) 2 Tests performed by Dennis McMullen, Technology Applications, Inc., EMSL, Cincinnati, OH. 2 All tests were performed using FORTY FATHOMS® synthetic seawater. 3 Copper test solutions were prepared with copper sulfate. Copper concentrations in Test 1 were: 2.5, 5.0,

10.0, 20.0, and 40.0 μg/L. Copper concentrations in Tests 2-5 were: 6.25, 12.5, 25.0, 50.0, and 100.0 μg/L. 4 NOEC Range: < 5.0 - 12.5 μg/L (this represents a difference of one exposure concentrations). 5 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 
327

TABLE 14. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA PUNCTULATA,

FERTILIZATION TEST PERFORMED IN FORTY FATHOMS® ARTIFICIAL SEAWATER,

USING GAMETES FROM ADULTS MAINTAINED IN FORTY FATHOMS® ARTIFICIAL

SEAWATER, OR OBTAINED DIRECTLY FROM NATURAL SOURCES, AND SODIUM

DODECYL SULFATE (SDS) AS A REFERENCE TOXICANT1,2,3,4,5,6

Test NOEC IC25 IC50

Number (mg/L) (mg/L) (mg/L)

1 <0.9 1.11 1.76

2 0.9 1.27 1.79

3 1.8 2.26 2.87

4 0.9 1.90 2.69

5 1.8 2.11 2.78

n: 4 5 5

Mean: NA 1.73 2.38

CV(%): NA 29.7 23.3

1 Data from USEPA (1991a) 2 Tests performed by Dennis M. McMullen, Technology Applications, Inc., EMSL, Cincinnati, OH. 3 All tests were performed using FORTY FATHOMS® synthetic seawater. 4 NOEC Range: <0.9 - 1.8 mg/L (this represents a difference of two exposure concentration). 5 SDS concentrations for all tests were: 0.9, 1.8, 3.6, 7.2, and 14.4 mg/L. 6 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 
328

TABLE 15. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA PUNCTULATA,

FERTILIZATION TEST PERFORMED IN NATURAL SEAWATER, USING GAMETES FROM

ADULTS MAINTAINED IN NATURAL SEAWATER AND COPPER (CU) SULFATE AS A

REFERENCE TOXICANT 1,2,3,4,5,6

Test NOEC IC25 IC50

Number (μg/L) (μg/L) (μg/L)

1 12.2 14.2 18.4

2 12.2 32.4 50.8

3 24.4 30.3 46.3

4 <6.1 26.2 34.1

5 6.1 11.2 17.2

n: 4 5 5

Mean: NA 22.8 29.9

CV(%): NA 41.9 48.2

1 Data from USEPA (1991a) 2 Tests performed by Ray Walsh and Wendy Greene, ERL-N, USEPA, Narragansett, RI. 3 Copper concentrations were: 6.1, 12.2, 24.4, 48.7, and 97.4 μg/L. 4 NOEC Range: < 6.1 - 24.4 μg/L (this represents a difference of two exposure concentrations). 5 Adults collected in the field. 6 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.
329

TABLE 16. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA PUNCTULATA,

FERTILIZATION TEST PERFORMED IN NATURAL SEAWATER, USING GAMETES FROM

ADULTS MAINTAINED IN NATURAL SEAWATER AND SODIUM DODECYL SULFATE (SDS)

AS A REFERENCE TOXICANT 1,2,3,4,5,6

Test NOEC IC25 IC50

Number (mg/L) (mg/L) (mg/L)

1 1.8 2.3 2.7

2 1.8 3.9 5.1

3 1.8 2.3 2.9

4 0.9 2.1 2.6

5 1.8 2.3 2.7

n: 5 5 5

Mean: NA 2.58 3.2

CV(%): NA 28.7 33.3

1 Data from USEPA (1991a). 2 Tests performed by Ray Walsh and Wendy Greene, ERL-N, USEPA, Narragansett, RI. 3 SDS concentrations were: 0.9, 1.8, 3.6, 7.3, and 14.5 mg/L. 4 NOEC Range: 0.9 - 1.8 mg/L (this represents a difference of one exposure concentration). 5 Adults collected in the field. 6 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.
330

TABLE 17. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA PUNCTULATA,

FERTILIZATION TEST PERFORMED IN GP2, USING GAMETES FROM ADULTS

MAINTAINED IN GP2 ARTIFICIAL SEAWATER AND COPPER (CU) SULFATE AND

SODIUM DODECYL SULFATE (SDS) AS REFERENCE TOXICANTS1,2,3,4,5

 Cu (μg/L) SDS (mg/L) 

Test LC50 CI NOEC LOEC LC50 CI NOEC LOEC

1 29.1 27.3-31.1 6.3 12.5 2.1 2.0-2.1 1.3 2.5 

2 47.6 44.6-50.8 25.0 50.0 1.8 1.8-1.9 1.3 2.5 

3 32.7 29.8-35.8 6.3 12.5 2.2 2.1-2.2 1.3 2.5

4 78.4 73.3-83.9 50.0 100.0 2.3 2.2-2.4 1.3 2.5

5 45.6 41.0-50.7 12.5 25.0 1.8 1.7-2.8 1.3 2.5 

Mean 46.7 2.0

SD 19.5 0.2

CV 41.8 10.0

1 Tests performed by Pamela Comeleo, Science Application International Corp., ERL-N, USEPA, Narragansett, RI. 2 All tests were performed using GP2 artificial seawater. 3 Copper concentrations were: 6.25, 12,5, 25.0, 50.0 and 100 μg/L. 4 SDS concentrations were: 0.6, 1.25, 2.5, 5.0, and l0.0 mg/L. SDS stock (14.645 mg/mL) provided by EMSL,

USEPA, Cincinnati, OH. 5 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 
331

TABLE 18. SINGLE-LABORATORY PRECISION OF THE SEA URCHIN, ARBACIA PUNCTULATA,

FERTILIZATION TEST PERFORMED IN NATURAL SEAWATER, USING GAMETES FROM

ADULTS MAINTAINED IN NATURAL SEAWATER AND COPPER (CU) SULFATE AND

SODIUM DODECYL SULFATE (SDS) AS REFERENCE TOXICANTS1,2,3,4

 Cu (μg/L) SDS (mg/L) 

Test LC50 CI NOEC LOEC LC50 CI NOEC LOEC

1 28.6 26.7-30.6 6.3 12.5 12.5 2.1-2.2 1.3 2.5 

2 13.0 11.9-14.2 6.3 12.5 12.5 1.9-2.0 1.3 2.5 

3 67.8 63.2-72.6 6.3 12.5 12.5 2.1-2.3 1.3 2.5

4 36.7 33.9-398 < 6.3 6.3 6.3 3.3-3.4 < 0.6 0.6

5 356 33.6-37.7 < 6.3 6.3 6.3 2.8-3.1 < 0.6 0.6 

Mean 36.3 2.5

SD 20.0 0.58

CV 55.1 23.2

1 Tests performed by Anne Kuhn-Hines, Catherine Sheehan, Glen Modica, and Pamela Comeleo, Science Application

International Corp., ERL-N, USEPA, Narragansett, RI. 2 Copper concentrations were prepared with copper sulfate. Concentrations were 6.25, 12.5, 25.0, 50.0, and l00 μg/L. 3 SDS concentrations were: 0.6, 1.25, 2.5, 5.0, and 10.0 mg/L. SDS stock (14.64 mg/mL) provided by EMSL, USEPA,

Cincinnati, OH. 4 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance.
332

SECTION 16

TEST METHOD

RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUCTION TEST

METHOD 1009.0

16.1 SCOPE AND APPLICATION

16.1.1 CAUTION: The Red Macroalga, Champia parvula, Reproduction Test Method 1009.0 is not listed at 40

CFR Part 136 for nationwide use. 

16.1.2 This method, adapted in part from USEPA (1987f) measures the effects of toxic substances in effluents and

receiving water on the sexual reproduction of the marine red macroalga, Champia parvula. The method consists of

exposing male and female plants to test substances for two days, followed by a 5-7 day recovery period in control

medium, during which the cystocarps mature. 

16.1.3 Detection limits of the toxicity of an effluent or chemical substance are organism dependent. 

16.1.4 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, highly volatile and highly degradable toxicants present in

the source may not be detected in the test.

16.1.5 This test 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. 

16.2 SUMMARY OF METHOD 

16.2.1 Sexually mature male and female branches of the red macroalga, Champia parvula, are exposed in a static

system for 2 days to different concentrations of effluent, or to receiving water, followed by a 5 to 7 day recovery

period in control medium. The recovery period allows time for the development of cystocarps resulting from

fertilization during the exposure period. The test results are reported as the concentration of the test substance

which causes a statistically significant reduction in the number of cystocarps formed.

16.3 INTERFERENCES 

16.3.1 Toxic substances may be introduced by contaminants in dilution water, glassware, sample hardware, and

testing equipment (see Section 5, Facilities, Equipment, and Supplies). 

16.3.2 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).

16.3.3 Adverse effects of high concentrations of suspended and/or dissolved solids, and extremes of pH, may mask

the presence of toxic substances.

16.3.4 Pathogenic and/or predatory organisms in the dilution water and effluent may affect test organism survival,

and confound test results. 
333

16.4 SAFETY 

16.4.1 See Section 3, Safety and Health. 

16.5 APPARATUS AND EQUIPMENT 

16.5.1 Facilities for holding and acclimating test organisms. 

16.5.2 Laboratory red macroalga, Champia parvula, culture unit -- see culturing methods below. To test effluent

or receiving water toxicity, sufficient numbers of sexually mature male and female plants must be available. 

16.5.3 Samplers -- automatic samplers, preferably with sample cooling capability, that can collect a 24-h

composite sample of 1 L.

16.5.4 Environmental chamber or equivalent facility with temperature control (23 ± 1°C). 

16.5.5 Water purification system -- Millipore Milli-Q®, deionized water (DI) or equivalent. 

16.5.6 Air pump -- for oil-free air supply. 

16.5.7 Air lines, and air stones -- for aerating cultures. 

16.5.8 Balance -- Analytical, capable of accurately weighing to 0.00001 g. 

16.5.9 Reference weights, Class S -- for checking performance of balance.

16.5.10 Meter, pH -- for routine physical and chemical measurements. 

16.5.11 Dissecting (stereoscope) microscope -- for counting cystocarps. 

16.5.12 Compound microscope -- for examining the condition of plants. 

16.5.13 Count register, 2-place -- for recording cystocarp counts. 

16.5.14 Rotary shaker -- for incubating exposure chambers (hand-swirling twice a day can be substituted). 

16.5.15 Drying oven -- to dry glassware. 

16.5.16 Filtering apparatus -- for use with membrane filters (47 mm). 

16.5.17 Refractometer -- for determining salinity. 

16.5.1 Thermometers, glass or electronic, laboratory grade -- for measuring water temperatures. 

16.5.19 Thermometers, bulb-thermograph or electronic-chart type -- for continuously recording temperature. 

16.5.20 Thermometer, National Bureau of Standards Certified (see USEPA Method 170.1, USEPA, l979b) -- to

calibrate laboratory thermometers. 

16.5.21 Beakers -- Class A, borosilicate glass or non-toxic plasticware, 1000 mL for making test solutions. 

16.5.22 Erlenmeyer flasks, 250 mL, or 200 mL disposable polystyrene cups, with covers -- for use as exposure

chambers. 
334

 16.5.23 Bottles -- borosilicate glass or disposable polystyrene cups (200-400 mL) for use as recovery vessels. 

16.5.24 Wash bottles -- for deionized water, for rinsing small glassware and instrument electrodes and probes. 

16.5.25 Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-toxic plastic labware,

10-1000 mL for making test solutions.

16.5.26 Micropipettors, digital, 200 and 1000 μL – to make dilutions.

16.5.27 Pipets, volumetric -- Class A, 1-100 mL. 

16.5.28 Pipettor, automatic -- adjustable, 1-100 mL. 

16.5.29 Pipets, serological -- 1-10 mL, graduated. 

16.5.30 Pipet bulbs and fillers -- PROPIPET®, or equivalent. 

16.5.31 Forceps, fine-point, stainless steel -- for cutting and handling branch tips. 

16.6 REAGENTS AND CONSUMABLE MATERIALS 

16.6.1 Mature red macroalga, Champia parvula, plants -- see Subsection 16.6.14 below. 

16.6.2 Sample containers -- for sample shipment and storage (see Section 8, Effluent and Receiving Water

Sampling, Sample Handling, and Sample Preparation for Toxicity Tests).

16.6.3 Petri dishes, polystyrene -- to hold plants for cystocarp counts and to cut branch tips. Other suitable

containers may be used. 

16.6.4 Disposable tips for micropipettors. 

16.6.5 Aluminum foil, foam stoppers, or other closures -- to cover culture and test flasks. 

16.6.6 Tape, colored -- for labeling test chambers. 

16.6.7 Markers, waterproof -- for marking containers, etc. 

16.6.8 Data sheets (one set per test) -- for data recording. 

16.6.9 Buffers, pH 4, pH 7, and pH l0 (or as per instructions of instrument manufacturer) for standards and

calibration check (see USEPA Method 150.1, USEPA, 1979b). 

16.6.10 Laboratory quality assurance samples and standards for the above methods. 

16.6.11 Reference toxicant solutions see Section 4, Quality Assurance.

16.6.12 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). 

16.6.13 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. 
335

16.6.13.1 Saline test and dilution water -- the use of natural seawater is recommended for this test. A recipe for the

nutrients that must be added to the natural seawater is given in Table 1. The salinity of the test water must be 30‰,

and vary no more than ± 2‰ among the replicates. If effluent and receiving water tests are conducted concurrently,

the salinity of these tests should be similar.

16.6.13.2 The overwhelming majority of industrial and sewage treatment effluents entering marine and estuarine

systems contain little or no measurable salts. Therefore, exposure of the red macroalga, Champia parvula, to

effluents will usually require adjustments in the salinity of the test solutions. Although the red macroalga, Champia

parvula, cannot be cultured in 100% artificial seawater, 100% artificial seawater can be used during the two day

exposure period. This allows 100% effluent to be tested. It is important to maintain a constant salinity across all

treatments. The salinity of the effluent can be adjusted by adding hypersaline brine (HSB) prepared from natural

seawater (100‰), concentrated (triple strength) salt solution (GP2 described in Table 2), or dry GP2 salts (Table 2),

to the effluent to provide a salinity of 30‰. Control solutions should be prepared with the same percentage of

natural seawater and at the same salinity (using deionized water adjusted with dry salts, or brine) as used for the

effluent dilutions. 

16.6.13.3 Artificial seawater -- A slightly modified version of the GP2 medium (Spotte, et al, 1984) has been used

successfully to perform the red macroalga sexual reproduction test. The preparation of artificial seawater (GP2) is

described in Table 2. 
336

TABLE 1. NUTRIENTS TO BE ADDED TO NATURAL SEAWATER AND TO ARTIFICIAL 

SEAWATER (GP2) DESCRIBED IN TABLE 2. THE CONCENTRATED NUTRIENT STOCK

SOLUTION IS AUTOCLAVED FOR 15 MINIMUM (VITAMINS ARE AUTOCLAVED 

SEPARATELY FOR 2 MINIMUM AND ADDED AFTER THE NUTRIENT STOCK

SOLUTION IS AUTOCLAVED). THE pH OF THE SOLUTION IS ADJUSTED TO

APPROXIMATELY pH 2 BEFORE AUTOCLAVING TO MINIMIZE THE POSSIBILITY OF

PRECIPITATION

 

 Amount of Reagent Per Liter of Concentrated 

 Nutrient Stock Solution 

Stock Solution For Stock Solution For

 Culture Medium Test Medium 

 

Nutrient Stock Solution1

NaNO3 6.35 g 1.58 g

NaH2P04 · H2O 0.64 g 0.16 g

Na2EDTA · 2 H2O 133 mg --

Na3C6H5O7 · 2 H2O 51 mg 12.8 mg

Iron2

 9.75 mL 2.4 mL

Vitamins3 10 mL 2.5 mL

1 Add 10 mL of appropriate nutrient stock solution per liter of culture or test medium. 2 A stock solution of iron is made that contains 1 mg iron/mL. Ferrous or ferric chloride can be used. 3 A vitamin stock solution is made by dissolving 4.88 g thiamine HCl, 2.5 mg biotin, and 2.5 mg B12 in 500 mL

deionized water. Adjust approximately pH 4 before autoclaving 2 min. It is convenient to subdivide the vitamin

stock into 10 mL volumes in test tubes prior to autoclaving. 
337

TABLE 2. REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2 ARTIFICIAL 

SEAWATER FOR USE IN CONJUNCTION WITH NATURAL SEAWATER FOR THE RED 

MACROALGA, CHAMPIA PARVULA, CULTURING AND TOXICITY TESTING1,2,3,4,5,6,7

Compound Concentration Amount (g) 

 (g/L) Required for

 20 L

NaCl 21.03 420.6

Na2SO4 3.52 70.4

KCl 0.61 12.2 

KBr 0.088 1.76

Na2B4O7@10 H2O 0.034 0.68

MgCl2@6 H2O 9.50 190.0

CaCl2@@

2 H2O 1.32 26.4

SrCl2@6 H2O 0.02 0.400

NaHCO3 0.17 3.40

1 Modified GP2 from Spotte et al. (1984). 2 The constituent salts and concentrations were taken from USEPA (l990b). 3 The original formulation calls for autoclaving anhydrous and hydrated salts separately to avoid precipitation. 

However, if the sodium bicarbonate is autoclaved separately (dry), all of the other salts can be autoclaved together. 

Since no nutrients are added until needed, autoclaving is not critical for effluent testing. To minimize microalgal

contamination, the artificial seawater should be autoclaved when used for stock cultures. Autoclaving (120°C)

should be for a least 10 minimum for 1-L volumes, and 20 minimum for 10-to-20-L volumes. 4 Prepare in 10-L to 20-L batches. 5 A stock solution of 68 mg/mL sodium bicarbonate is prepared by autoclaving it as a dry powder, and then

dissolving it in sterile deionized water. For each liter of GP2, use 2.5 mL of this stock solution. 6 Effluent salinity adjustment to 30‰ can be made by adding the appropriate amount of dry salts from this

formulation, by using a triple-strength brine prepared from this formulation, or by using a 100‰ salinity brine

prepared from natural seawater. 7 Nutrients listed in Table 1 should be added to the artificial seawater in the same concentration described for natural

seawater. 

16.6.14 TEST ORGANISMS RED MACROALGA, CHAMPIA PARVULA

16.6.14.1 Cultures

16.6.14.1.1 Mature plants are illustrated in Figure 1. The adult plant body (thallus) is hollow, septate, and highly

branched. New cultures can be propagated asexually from excised branches, making it possible to maintain clonal

material indefinitely.
338

Figure 1. Life history of the red macroalga, Champia parvula. Upper left: Size and degree of branching in female 

 branch tips used for toxicity tests. From USEPA (1987f).

16.6.14.1.2 Unialgal stock cultures of both males and females are maintained in separate, aerated 1000 mL Erlenmeyer

flasks containing 800 mL of the culture medium. All culture glass must be acid-stripped in 15% HCl and rinsed in

deionized water after washing. This is necessary since some detergents can leave a residue that is toxic to the red

macroalga, Champia parvula. Periodically (at least every 6 months) culture glassware should be baked in a muffle

furnace to remove organic material that may build up on its surface. Alternately, a few mL of concentrated sulfuric acid

can be rolled around the inside of wet glassware. CAUTION: the addition of acid to the wet glassware generates heat. 

16.6.14.1.3 The culture medium is made from natural seawater to which additional nutrients are added. The nutrients

added are listed in Table 1. Almost any nutrient recipe can be used for the red macroalga, Champia parvula, cultured in

either natural seawater or a 50-50 mixture of natural and artificial seawaters. Healthy, actively growing plants are the

goal, not a standard nutrient recipe for cultures.

16.6.14.1.4 Several cultures of both males and females should be maintained simultaneously to keep a constant supply

of plant material available. To maintain vigorous growth, initial stock cultures should be started periodically with about

twenty 0.5 to l.0 cm branch tips. Cultures are gently aerated through sterile, cotton-plugged, disposable, polystyrene l

mL pipettes. Cultures are capped with foam plugs and aluminum foil and illuminated with ca. 75 μE/m2

/s (500 ft-c) of

cool-white fluorescent light on a 16:8 h light:dark cycle. Depending on the type of culture chamber or room used, i.e.,
339

the degree of reflected light, the light levels may have to be adjusted downward. The temperature is 22 to 24°C and the

salinity 28-30‰. Media are changed once a week. 

16.6.14.1.5 Prior to use in toxicity tests, stock cultures should be examined to determine their condition. Females can

be checked by examining a few branch tips under a compound microscope (100 X or greater). Several trichogynes

(reproductive hairs to which the spermatia attach) should be easily seen near the apex (Figure 2). 

16.6.14.1.6 Male plants should be visibly producing spermatia. This can be checked by placing some male tissue in a

petri dish, holding it against a dark background and looking for the presence of spermatial sori. Mature sori can also be

easily identified by looking along the edge of the thallus under a compound microscope (Figures 3 and 4). 

16.6.14.1.7 A final, quick way to determine the relative "health" of the male stock culture is to place a portion of a

female plant into some of the water from the male culture for a few seconds. Under a compound microscope numerous

spermatia should be seen attached to both the sterile hairs and the trichogynes (Figure 5).

16.6.14. Culture medium prepared from natural seawater is preferred (Table 1). However, as much as 50% of the

natural seawater may be replaced by the artificial seawater (GP2) described in Table 2. 

16.6.14.2.1 Seawater for cultures is filtered at least to 0.45 μm to remove most particulates and then autoclaved for 30

minute at 15 psi (120°C). Carbon stripping the seawater may be necessary before autoclaving to enhance its water

quality (USEPA, 1990b). This is done by adding 2 g activated carbon per liter of seawater and stirring on a stir plate for

2 h. After stirring filter through a Whatman number 2 filter, then through a 0.45 membrane filter. The culture flasks are

capped with aluminum foil and autoclaved dry, for 10 minute. Culture medium is made up by dispensing seawater into

sterile flasks and adding the appropriate nutrients from a sterile stock solution. 

Figure 2. Apex of branch of female plant, showing sterile hairs and reproductive hairs (trichogynes). Sterile

hairs are wider and generally much longer than trichogynes, and appear hollow except at the tip. Both

types of hairs occur on the entire circumference of the thallus, but are seen easiest at the "edges." 

Receptive trichogynes occur only near the branch tips. From USEPA (1987f). 
340

Figure 3. A portion of the male thallus showing spermatial sori. The sorus areas are generally slightly

thicker and somewhat lighter in color. From USEPA (1987f).

Figure 4. A magnified portion of a spermatial sorus. Note the rows of cells that protrude from the thallus

surface. From USEPA (1987f). 

Figure 5. Apex of a branch on a mature female plant that was exposed to spermatia from a male plant. The

sterile hairs and trichogynes are covered with spermatia. Note that few or no spermatia are

attached to the older hairs (those more than l mm from the apex). From USEPA (1987f). 

100μm
341

16.6.14.2.2 Alternately, 1-L flasks containing seawater can be autoclaved. Sterilization is used to prevent

microalgal contamination, and not to keep cultures bacteria free. 

16.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND STORAGE 

16.7.1 See Section 8, Effluent and Receiving Water Sampling, Sample Handling, and Sample Preparation for

Toxicity Tests.

16.8 CALIBRATION AND STANDARDIZATION 

16.8.1 See Section 4, Quality Assurance. 

16.9 QUALITY CONTROL 

16.9.1 See Section 4, Quality Assurance. 

16.10 TEST PROCEDURES 

16.10.1 TEST SOLUTIONS 

16.10.1.1 Receiving Waters 

16.10.1.1.1 The sampling point is determined by the objectives of the test. At estuarine and marine sites, samples

are usually collected at mid-depth. Receiving water toxicity is determined with samples used directly as collected or

with samples passed through a 60 μm NITEX® 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 800 mL or more of sample per test. 

16.10.1.2 Effluents 

16.10.1.2.1 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 ± 100%, and allows for

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 dilution factors are increased beyond 0.5 and

declines rapidly if smaller dilution factors are used. Therefore, USEPA recommends the use of the $ 0.5 dilution

factor.

16.10.1.2.2 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%). 

16.10.1.2.3 The volume of effluent required for the test using a 0.5 dilution series is approximately 1800 mL. 

Prepare enough test solution at each effluent concentration (approximately 800 mL) to provide 100 mL of test

solution for each of four (minimum of three) replicate test chambers and 400 mL for chemical analyses and record

data (Figure 6).

16.10.1.2.4 Effluents can be tested at 100%. A 100% concentration of effluent can be achieved if the salinity of

the effluent is adjusted to 30‰ by adding the GP2 dry salt formulation described in Table 2. 

16.10.1.2.5 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 (25 ± 1°C) and maintained at the temperature

during the addition of dilution water.
342

SITE: 

COLLECTION DATE: 

TEST DATE: 

LOCATION INITIAL

SALINITY

FINAL

SALINITY

SOURCE OF SALTS FOR1

SALINITY ADJUSTMENT

1

Natural seawater, GP2 brine, GP2 salts, etc. (include some indication of amount)

COMMENTS:

Figure 6. Data form for the red macroalga, Champia parvula, sexual reproduction test. Receiving water

summary sheet. From USEPA (1987f).
343

16.10.1.2.6 Effluent dilutions should be prepared for all replicated in each treatment in one beaker to minimize

variability among the replicates. The test chambers are labeled with the test concentration and replicate number. 

Dispense into the appropriate effluent dilution chamber.

16.10.1.3 Dilution Water 

16.10.1.3.1. The formula for the enrichment for natural seawater is listed in Table 1. Both EDTA and trace metals

have been omitted. This formula should be used for the 2-day exposure period, but it is not critical for the recovery

period. Since natural seawater quality can vary among laboratories, a more complete nutrient medium (e.g., the

addition of EDTA) may result in faster growth (and therefore faster cystocarp development) during the recovery

period. 

16.10.2 PREPARATION OF PLANTS FOR TEST 

16.10.2.1 Once cultures are determined to be usable for toxicity testing (have trichogynes and sori with spermatia),

plant cuttings should be prepared for the test, using fine-point forceps, with the plants in a little seawater in a petri

dish. For female plants, five cuttings, severed 7-10 mm from the ends of the branch, should be prepared for each

treatment chamber. Try to be consistent in the number of branch tips on each cutting. For male plants, one cutting,

severed 2.0 to 3.0 cm from the end of the branch, is prepared for each test chamber. Prepare the female cuttings

first, to minimize the chances of contaminating them with water containing spermatia from the male stock cultures. 

16.10.3 START OF TEST 

16.10.3.1 Tests should begin as soon as possible after sample collection, preferably within 24 h. 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 Test, Subsection 8.5.4).

16.10.3.2 Just prior to test initiation (approximately 1 h), the temperature of a sufficient quantity of the sample to

make the test solution should be adjusted to the test temperature (23 ± 1°C) and maintained at that temperature

during the addition of dilution water. 

16.10.3.3 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 controls) should have four (minimum of three) replicates. 

16.10.3.4 Randomize the position of test chambers at the beginning of the test.

16.10.3.5 Prepare test solutions and add to the test chambers. 

16.10.3.6 Add five female branches and one male branch to each test chamber. The toxicant must be present

before the male plant is added.

16.10.3.7 Gently hand swirl the chambers twice a day, or shake continuously at 100 rpm on a rotary shaker. 

16.10.3.8 If desired, the media can be changed after 24 h. 

16.10.4 LIGHT, PHOTOPERIOD, SALINITY, AND TEMPERATURE

16.10.4.1 The light quality and intensity should be at 75 μE/m2

/s, or 500 foot candles (ft-c) with a photoperiod of

16 h light and 8 h darkness. The water temperature in the test chambers should be maintained at 23 ± 1°C. The test

salinity should be in the range of 28 to 32‰. The salinity should vary by no more than ± 2‰ among the chambers
344

on a given day. If effluent and receiving water tests are conducted concurrently, the salinities of these tests should

be similar. 

16.10.5 DISSOLVED OXYGEN (DO) CONCENTRATION

16.10.5.1 Aeration may affect the toxicity of effluent and should be used only as a last resort to maintain a

satisfactory DO. The DO concentrations should be measured on new solutions at the start of the test (Day 0) and

should be measured before renewal of the test solution after 24 h. The DO 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 treatments and the control should be aerated. The aeration rate should not exceed 100

bubbles/minute, using a pipet with a 1-2 mm orifice, such as a 1mL KIMAX® serological pipet, or equivalent. Care

should be taken to ensure that turbulence resulting from the aeration does not occur.

16.10.6 OBSERVATIONS DURING THE TEST

16.10.6.1 Routine Chemical and Physical Observations

16.10.6.1.1 DO is measured at the beginning and end of each 24-h exposure period in one test chamber at each

concentration and in the control. 

16.10.6.1.2 Temperature, pH, and salinity are measured at the end of each 24-h exposure period in one test

chamber at each concentration and in the control. Temperature should also be monitored continuously, 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 chambers at least at the end of the test to determine temperature

variation in environmental chamber.

16.10.6.1.3 The pH is measured in the effluent sample each day before new test solutions are made.

16.10.6.1.4 Record all the measurements on the data sheet.

16.10.6.2 Routine Biological Observations

16.10.6.2.1 Protect the red macroalga from unnecessary disturbance during the test by carrying out the daily test

observations and solution renewals carefully.

16.10.7 TRANSFER OF PLANTS TO CONTROL WATER AFTER 48 H 

16.10.7.1 Label the recovery vessels. These vessels can be almost any type of container or flask containing 100 to

200 mL of seawater and nutrients (see Tables 1 and 2). Smaller volumes can be used, but should be checked to

make sure that adequate growth will occur without having to change the medium.

16.10.7.2 With forceps, gently remove the female branches from test chambers and place into recovery bottles. 

Add aeration tubes and foam stoppers.

16.10.7.3 Place the vessels under cool white light (at the same irradiance as the stock cultures) and aerate for the

5-7 day recovery period. If a shaker is used, do not aerate the solutions (this will enhance the water motion).

16.10.8 TERMINATION OF THE TEST

16.10.8.1 At the end of the recovery period, count the number of cystocarps (Figures 7, 8, and 9) per female and

record the data (Figure 10). Cystocarps may be counted by placing females between the inverted halves of a

polystyrene petri dish or other suitable containers with a small amount of seawater (to hold the entire plant in one

focal plane). Cystocarps can be easily counted under a stereomicroscope, and are distinguished from young

branches because they possess an apical opening for spore release (ostiole) and darkly pigmented spores. 
345

Figure 7. A mature cystocarp. In the controls and lower effluent concentrations, cystocarps often occur in

clusters of 10 or 12. From USEPA (1987f).

Figure 8. Comparison of a very young branch and an immature cystocarp. Both can have sterile hairs. 

Trichogynes might or might not be present on a young branch, but are never present on an immature

cystocarp. Young branches are more pointed at the apex and are made up of larger cells than

immature cystocarps, and never have ostioles. From USEPA (1987f).

Figure 9. An aborted cystocarp. A new branch will eventually develop at the apex. From USEPA (1987f).
346

16.10.8.2 One advantage of this test procedure is that if there is uncertainty about the identification of an immature

cystocarp, it is necessary only to aerate the plants a little longer in the recovery bottles. Within 24 to 48 h, the

presumed cystocarp will either look more like a mature cystocarp or a young branch, or will have changed very

little, if at all (i.e., an aborted cystocarp). No new cystocarps will form since the males have been removed, and the

plants will only get larger. Occasionally, cystocarps will abort, and these should not be included in the counts. 

Aborted cystocarps are easily identified by their dark pigmentation and, often, by the formation of a new branch at

the apex. 

16.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA 

16.11.1 A summary of test conditions and test acceptability criteria is listed in Table 3. 

16.12 ACCEPTABILITY OF TEST RESULTS 

16.12.1 The test is acceptable if (1) control survival equals or exceeds 80% and (2) control plants average 10 or

more cystocarps per plant. 

16.12.2 If plants fragment in the controls or lower exposure concentrations, it may be an indication that they are

under stress. 

16.13 DATA ANALYSIS

16.13.1 GENERAL

16.13.1.1 Tabulate and summarize the data. A sample set of reproduction data is listed in Table 4. 

16.13.1.2 The endpoints of the red macroalga, Champia parvula, toxicity test are based on the adverse effects on

sexual reproduction as the mean number of cystocarps. The LC50, the IC25, and the IC50 are calculated using

point estimation techniques (see Section 9, Chronic Toxicity Test Endpoints and Data Analysis). NOEC and LOEC

values 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 NOEC and LOEC endpoints and for the IC25 and IC50. See the Appendices for examples of the manual

computations, program listing, and example of data input and program output.

16.13.1.3 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.
347

COLLECTION DATE RECOVERY BEGAN (date) 

EXPOSURE BEGAN (date) COUNTED (date) 

EFFLUENT OR TOXICANT 

TREATMENT (% EFFLUENT, mG/L, or RECEIVING WATER SITES)

REPLICATES CONTROL

A 1

 2

 3

 4

MEAN

B 1

 2

 3

 4

MEAN

C 1

 2

 3

 4

MEAN

OVERALL

MEAN

Temperature 

Salinity 

Light 

Source of Dilution Water 

Figure 10. Data form for the red macroalga, Champia parvula, sexual reproduction test. Cystocarp data sheet. 

 From USEPA (1987f).
348

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE

RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUCTION TEST WITH

EFFLUENTS AND RECEIVING WATERS 

CAUTION: This method is not listed at 40 CFR Part 136 for nationwide use.

1. Test type: Static, non-renewal 

2. Salinity: 30‰ (± 2‰ of the selected test salinity)

3. Temperature: 23 ± 1°C 

4. Light quality: Cool-white fluorescent lights 

5. Light intensity: 75 μE/m2

/s (500 ft-c)

6. Photoperiod: 16 h light, 8 h darkness

7. Test chamber size: 200 mL polystyrene cups, or 250 mL Erlenmeyer flasks 

8. Test solution volume: 100 mL (minimum) 

9. No. organisms

 per test chamber: 5 female branch tips and 1 male plant

10. No. replicate

 per concentration: 4 (minimum of 3) 

11. No. organisms per

 concentrations: 24 (minimum of 18)

12. Dilution water: 30‰ salinity natural seawater, or a combination of 50% of 30‰

salinity natural seawater and 50% of 30‰ salinity GP2 artificial

seawater (see Section 7, Dilution Water)

13. Test concentrations: Effluents: Minimum of 5 and a control

Receiving waters: 100% receiving water or minimum of 5 and a 

control 

14. Dilution factor: Effluents: $ 0.5

Receiving waters: None or$ 0.5
349

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE

RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUCTION TEST WITH

EFFLUENTS AND RECEIVING WATERS (CONTINUED)

15. Test duration: 2 day exposure to effluent, followed by 5 to 7-day recovery period in 

control medium for cystocarp development

16. Endpoints: Reduction in cystocarp production compared to controls

17. Test acceptability criteria 80% or greater survival, and an average of 10 cystocarps per plant in 

controls

18. Sampling requirements: 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 before first use (see Section 8,

Effluent and Receiving Water Sampling, Sampling Handling, and

Sample Preparation for Toxicity Tests, Subsection 8.5.4) 

19. Sample volume required: 2 L per test

16.13.2 EXAMPLE OF ANALYSIS OF THE RED MACROALGA, CHAMPIA PARVULA, REPRODUCTION

DATA 

16.13.2.1 Formal statistical analysis of the data is outlined in Figure 11. The response used in the analysis is the

mean number of cystocarps per replicate chamber. Separate analyses are performed for the estimation of the NOEC

and LOEC endpoints and for the estimation of the IC25 endpoint and the IC50 endpoint. Concentrations that have

exhibited no sexual reproduction (less than 5% of controls) are excluded from the statistical analysis of the NOEC

and LOEC, but included in the estimation of the IC endpoints.

16.13.2.2 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. The assumptions of Dunnett's Procedures, 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. Tests for normality and homogeneity of variance are included in Appendix B. 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.
350

TABLE 4. DATA FROM THE RED MACROALGA, CHAMPIA PARVULA, EFFLUENT TOXICITY 

TEST. CYSTOCARP COUNTS FOR INDIVIDUAL PLANTS AND MEAN COUNT PER 

TEST CHAMBER FOR EACH EFFLUENT CONCENTRATION1

 Effluent Replicate Plant Mean 

Concentration Test 1 2 3 4 5 Cystocarp 

 (%) Chamber Count 

Control A 19 20 24 7 18 17.60 

B 19 12 21 11 23 17.20 

C 17 25 18 20 16 19.20 

0.8 A 10 16 11 12 11 12.00 

B 12 10 6 9 10 9.40 

C 12 9 9 13 8 10.20 

1.3 A 10 0 3 5 4 4.40 

B 6 4 4 8 4 5.20 

C 4 4 2 6 4 4.00 

2.2 A 1 2 5 4 0 2.40 

B 7 9 9 4 6 7.00 

C 3 2 2 0 0 1.40 

3.6 A 2 1 1 5 0 1.80 

B 3 4 6 4 2 3.80 

C 0 4 3 1 3 2.20 

6.0 A 1 0 0 0 0 0.20 

B 1 2 1 0 0 0.80 

C 0 4 3 1 3 2.20 

10.0 A 0 0 0 0 - 0.00 

B 1 0 0 0 0 0.20 

C 2 1 0 0 0 0.60 

1 Data provided by the ERL-N, USEPA, Narragansett, RI. 
351

Figure 11. Flowchart for statistical analysis of the red macroalga, Champia parvula, data
352

6.13.2.3 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

(Appendix D). The Wilcoxon Rank Sum Test with the Bonferroni adjustment is the nonparametric alternative.

16.13.2.4 Example of Analysis of Reproduction Data 

16.13.2.4.1 In this example, the data, mean and standard deviation of the observations at each concentration

including the control are listed in Table 5. The data are plotted in Figure 12. As can be seen from the data in the

table, mean reproduction per chamber in the 10% effluent concentration is less than 5% of the control. Therefore

the 10% effluent concentration is not included in the subsequent analysis. 

TABLE 5. RED MACROALGA, CHAMPIA PARVULA, SEXUAL REPRODUCTION DATA 

Effluent Concentration (%) 

Replicate Control 0.8 1.3 2.2 3.6 6.0 10.0 

A 17.60 12.00 4.40 2.40 1.80 0.20 0.00 

B 17.20 9.40 5.20 7.00 3.80 0.80 0.20 

C 19.20 10.20 4.00 1.40 2.20 2.20 0.60 

_

Mean(Yi

) 18.00 10.53 4.53 3.60 2.60 1.07 0.27 

S 1.12 1.77 0.37 8.92 1.12 1.05 0.09 2

i

i 1 2 3 4 5 6 7 

16.13.2.5 Test for Normality 

16.13.2.5.1 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 6. 

354

X ' 1

8

(0.01) ' 0.00

16.13.2.5.3 For this set of data, n = 18

D = 28.7201 

16.13.2.5.4 Order the centered observations from smallest to largest 

X(1) # X(2) # ... # X(n)

Where X(i) is the ith ordered observation. These ordered observations are listed in Table 7. 

TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

I X(i) i X(i)

1 -2.20 10 -0.33 

2 -1.20 11 -0.27 

3 -1.13 12 -0.13 

4 -0.87 13 0.67 

5 -0.80 14 1.13 

6 -0.80 15 1.20 

7 -0.53 16 1.20 

8 -0.40 17 1.47 

9 -0.40 18 3.40 

16.13.2.5.5 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-1)/2 if n is odd. For the data in this example, n = 18 and k = 9. The ai

 values are listed in Table 8.

TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE 

i ai X(n-i+1) - X(i)

1 0.4886 5.60 X(18) - X(1)

2 0.3253 2.67 X(17) - X(2)

3 0.2553 2.33 X(17) - X(3)

4 0.2027 2.07 X(15) - X(4)

5 0.1587 1.93 X(14) - X(5)

6 0.1197 1.47 X(13) - X(6)

7 0.0837 0.40 X(12) - X(7)

8 0.0496 0.13 X(11) - X(8)

9 0.0163 0.07 X(10) - X(9)
355

W ' 1

D[j

k

i'1

ai(X (n&i%1)&X (i)]

2

B '

[(j

p

i'1

Vi) lnS ̄2

&j

p

i'1

VilnS 2

i ]

C

S ̄2 '

(j

p

i'1

ViS 2

i )

j

p

i'1

Vi

C ' 1%[3(p&1)]&1[j

p

i'1

1/Vi

&(j

p

i'1

Vi)

&1

]

16.13.2.5.6 Compute the test statistic, W, as follows:

The differences X(n-i+1) - X(i) are listed in Table 8. For the data, 

 

 W = 1 (5.1425)2

 = 0.921 

 28.7201 

16.13.2.5.7 The decision rule for this test is to compare W as calculated in Subsection 16.3.2.5.6 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

= 0.921 is greater than the critical value, conclude of the test is that the data are normally distributed. 

16.13.2.6 Test for Homogeneity of Variance 

16.13.2.6.1 The test used to examine whether the variation in mean cystocarp production is the same across all effluent

concentrations including the control, is Bartlett's Test (Snedecor and Cochran, 1980). The test statistic is as follows: 

Where: Vi = degrees of freedom for each effluent concentration and control, Vi

 = (ni - 1)

p = number of levels of effluent concentration including the control

ni

 = the number of replicates for concentration i

ln = loge

i = 1, 2, ..., p where p is the number of concentrations 

16.13.2.6.2 For the data in this example (See Table 5) all effluent concentrations including the control have the same

number of replicates (ni

 = 3 for all i). Thus, Vi = 2 for all i. 
356

B ' [(12)ln(2.3917)&2j

p

i'1

ln(S 2

i )]/1.194

16.13.2.6.3 Bartlett's statistic is therefore: 

= [12(0.8720) - 2(1n(1.12)+ln(1.77)+...+ln(1.05))]/1.1944 

= (10.4640 - 4.0809)/1.1944 

= 5.34

16.13.2.6.4 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 = 5.34 is less than the critical value of 15.09, conclude that the variances are not different.

16.13.2.7 Dunnett's Procedure 

16.13.2.7.1 To obtain an estimate of the pooled variance for the Dunnett's Procedure, construct an ANOVA table as

described in Table 9. 

TABLE 9. ANOVA TABLE 

Source df Sum of Squares Mean Square(MS) 

(SS) (SS/df) 

Between p - 1 SSB = SSB/(p - 1) S 2

B

Within N - p SSW = SSW/(N - p) S 2

W

 

Total N - 1 SST

Where: p = number effluent concentrations including the control 

N = total number of observations n1 + n2 ... + np

ni

 = number of observations in concentration i

SSB ' j Between Sum of Squares

p

i'1

T 2

i /ni

&G 2 /N

SST ' j Total Sum of Squares

p

i'1

j

nj

j'1

Y 2

ij &G 2 /N

SSW ' SST&SSB Within Sum of Squares 
357

G ' j

p

i'1

Ti

SST ' j

p

i'1

j

nj

j'1

Y 2

ij &G 2 /N

SSW ' SST&SSB

G = the grand total of all sample observations,

Ti

 = the total of the replicate measurements for concentration i

Yi j = the jth observation for concentration i (represents the mean (across plants) number of cystocarps for 

effluent concentration i in test chamber j) 

16.13.2.7.2 For the data in this example: 

n1 = n2 = n3 = n4 = n5 = n6 = 3 

N = 18 

T1 = Y11 + Y12 + Y13 = 17.6 + 17.2 + 19.2 = 54 

T2 = Y21 + Y22 + Y23 = 12.0 + 9.4 + 10.2 = 31.6 

T3 = Y31 + Y32 + Y33 = 4.4 + 5.2 + 4.0 = 13.6 

T4 = Y41 + Y42 + Y43 = 2.4 + 7.0 + 1.4 = 10.8 

T5 = Y51 + Y52 + Y53 = 1.8 + 3.8 + 2.2 = 7.8 

T6 = Y61 + Y62 + Y63 = 0.2 + 0.8 + 2.2 = 3.2 

G = T1 + T2 + T3 + T4 + T5 + T6 = 121.0 

SSB ' j

p

i'1

T 2

i /ni

&G 2 /N

= 1 (4287.24) - (121.0)2

 = 615.69 

3 18 

= 1457.8 - (121.0)2

 = 644.41 

18

= 644.41 - 615.69 = 28.72 

S = SSB/(p-1) = 615.69/(6-1) = 123.14 2

B

S = SSW/(N-p) = 28.72/(18-6) = 2.39 2

W

16.13.2.7.3 Summarize these calculations in the ANOVA table (Table 10).
358

t

i ' (Y ̄

1&Y ̄

i

)

SW (1/n1)%(1/ni

)

t

2 ' (18&10.53)

[1.55 (1/3)%(1/3)]

' 5.9

TABLE 10. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE

Source df Sum of Squares Mean Square(MS) 

(SS) (SS/df) 

Between 5 615.69 123.14

Within 12 28.72 2.39

Total 17 644.41

16.13.2.7.4 To perform the individual comparisons, calculate the t statistic for each concentration, and control

combination as follows: 

 Where: = mean number of cystocarps for effluent concentration i Y ̄

i

Y ̄ = mean number of cystocarps for the control 1

SW = square root of the within mean square 

n1 = number of replicates for the control 

ni

 = number of replicates for concentration i

16.13.2.7.5 Table 11 includes the calculated t values for each concentration and control combination. In this example,

comparing the 0.8% concentration with the control the calculation is as follows:

16.13.2.7.6 Since the purpose of this test is to detect a significant reduction in cystocarp production, 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. Mean cystocarp production for concentration i is considered significantly less control if ti

 is greater than the

critical value. Therefore, mean cystocarp productions for all effluent concentrations in this example have

significantly lower cystocarp production than the control. Hence the NOEC is 0.8% and the LOEC is 0.8%.
359

MSD ' dSW (1/n1)%(1/n)

MSD ' 2.50(1.55) (1/3)%(1/3)

TABLE 11. CALCULATED T VALUES 

Effluent Concentration(%) i ti

0.8 2 5.90

1.3 3 10.64

2.2 4 11.38

3.6 5 12.17

6.0 6 13.38

16.13.2.7.7 To quantify the sensitivity of the test, the minimum significant difference (MSD) that can be

statistically detected may be calculated: 

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) 

n1 = the number of replicates in the control. 

16.13.2.7.8 In this example, 

= 2.50 (1.55)(.8165) 

= 3.16

16.13.2.7.9 Therefore, for this set of data, the minimum difference that can be detected as statistically significant is

3.16 cystocarps. 

16.13.2.7.10 This represents a 17.6% reduction in cystocarp production from the control. 

16.13.2.8 Calculation of the ICp

16.13.2.8.1 The sexual reproduction data in Table 5 are utilized in this example. Table 12 contains the mean

number of cystocarps for each effluent concentration. As can be seen, the observed means are monotonically nonincreasing with respect to concentration. Therefore, it is not necessary to smooth the means prior to calculating the

ICp. Refer to Figure 12 for a plot of the response curve. 
360

ICp ' Cj

%[M1 (1&p/100)&Mj

] (C(j%1)&Cj

)

(M(j%1)&Mj

)

IC25 ' 0.0%[18.00(1&25/100)&18.00] (0.8&0.0)

(10.53&18.00)

ICp ' Cj

%[M1 (1&p/100)&Mj

] (C(j%1)&Cj

)

(M(j%1)&Mj

)

IC50'0.8%[18.00(1&50/100)&10.53] (1.3&0.8)

(4.53&10.53)

TABLE 12. RED MACROALGA, CHAMPIA PARVULA, MEAN NUMBER OF CYSTOCARPS

Effluent Response Smoothed

Conc. Means Means

(%) i (mg) Mi Y ̄ (mg) i

Control 1 18.00 18.00

0.8 2 10.53 10.53

1.3 3 4.53 4.53

2.2 4 3.60 3.60

3.6 5 2.60 2.60

6.0 6 1.07 1.07

10.0 7 0.27 0.27

16.13.2.8.2 An IC25 and IC50 can be estimated using the Linear Interpolation Method. A 25% reduction in mean

number of cystocarps, compared to the controls, would result in a mean number of 13.50 cystocarps, where M1(1-

p/100) = 18.00(1-25/100). A 50% reduction in mean number of cystocarps, compared to the controls, would result

in a mean number of 9.00 cystocarps. Examining the means and their associated concentrations (Table 12), the

response, 13.50, is bracketed by C1 = 0.0% effluent and C2 = 0.8% effluent. The response, 9.00, is bracketed by C2

= 0.8% effluent and C3 = 1.3% effluent.

16.13.2.8.3 Using the equation from Section 4.2 in Appendix L, the estimate of the IC25 is calculated as follows:

= 0.5%.

16.13.2.8.4 Using the equation from Section 4.2 from Appendix L, the estimate of the IC50 is calculated as

follows:

= 0.9%
361

16.13.2.8.5 When the ICPIN program was used to analyze this set of data, requesting 80 resamples, the estimate of

the IC25 was 0.4821%. The empirical 95.0% confidence interval for the true mean was 0.4013% to 0.6075%. The

computer program output for the IC25 for this data set is shown in Figure 13.

16.13.2.8.6 When the ICPIN program was used to analyze this set of data, requesting 80 resamples, the estimate of

the IC50 was 0.9278%. The empirical 95.0% confidence interval for the true mean was 0.7893% and 1.0576%. 

The computer program output for the IC50 for this data set is shown in Figure 14.

16.14 PRECISION AND ACCURACY 

16.14.1 PRECISION 

16.14.1.1 Single-Laboratory Precision

16.14.1.1.1 The single-laboratory precision data from six tests with copper sulfate (Cu) and six tests with sodium

dodecyl sulfate (SDS) are listed in Tables 13-16. The NOECs with Cu differed by only one concentration interval

(factor of two), showing good precision. The precision of the first four tests with SDS was somewhat obscured by

the choice of toxicant concentrations, but appeared similar to that of Cu in the last two tests. The IC25 and IC50 are

indicated in Tables 13-16. The coefficient of variation, based on the IC25 for these two reference toxicants in

natural seawater and a mixture of natural seawater and GP2, ranged from 59.6% to 69.0%, and for the IC50, ranged

from 22.9% to 43.7%. 

16.14.1.1.2 EPA evaluated single-laboratory (within-laboratory) precision of the Red Macroalga, Champia parvula,

Reproduction Test using a database of routine reference toxicant test results from two laboratories (USEPA, 2000b). 

The database consisted of 23 reference toxicant tests conducted in 2 laboratories using reference toxicants

including: copper and sodium dodecyl sulfate. The within-laboratory CVs calculated for routine reference toxicant

tests at these 2 laboratories were 58% and 59% for the IC25 reproduction endpoint. 

16.14.1.2 Multilaboratory Precision

16.14.1.2.1 The multilaboratory precision of the test has not yet been determined. 

16.14.2 ACCURACY 

16.14.2.1 The accuracy of toxicity tests cannot be determined. 
362

Conc. ID 1 2 3 4 5 6 7

Conc. Tested 0 .8 1.3 2.2 3.6 6 10

Response 1 19 10 10 1 2 1 0

Response 2 20 16 0 2 10 0

Response 3 24 11 3 5 10 0

Response 4 7 12 5 4 50 0

Response 5 18 11 4 0 00 1

Response 6 19 12 6 7 31 0

Response 7 12 10 4 9 42 0

Response 8 21 6 4 9 61 0

Response 9 11 9 8 4 40 0

Response 10 23 10 4 6 20 2

Response 11 17 12 4 3 00 1

Response 12 25 9 4 2 44 0

Response 13 18 9 2 2 33 0

Response 14 20 13 6 0 11 0

Response 15 16 8 4 0 33 0

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: effluent Test Start Date: Test Ending Date: 

Test Species: RED MACROALGA, Champia parvula

Test Duration: DATA FILE: champia.icp OUTPUT FILE: champia.i25

Conc. Number Concentration Response Standard. Pooled

 ID Replicates % Means Dev. Response Means

 1 15 0.000 18.000 4.928 18.000

 2 15 0.800 10.533 2.356 10.533

 3 15 1.300 4.533 2.356 4.533

 4 15 2.200 3.600 3.066 3.600

 5 15 3.600 2.600 1.805 2.600

 6 15 6.000 1.067 1.335 1.067

 7 15 10.000 0.267 0.594 0.267

The Linear Interpolation Estimate: 0.4821 Entered P Value: 25

Number of Resamplings: 80 The Bootstrap Estimates Mean: 0.4947 Standard Deviation: 0.0616

Original Confidence Limits: Lower: 0.4013 Upper: 0.6075

Resampling time in Seconds: 3.68 Random Seed: 703617166

Figure 13. ICPIN program output for the IC25.
363

Conc. ID 1 2 3 4 5 6 7

Conc. Tested 0 .8 1.3 2.2 3.6 6 10

Response 1 19 10 10 1 2 1 0

Response 2 20 16 0 2 10 0

Response 3 24 11 3 5 10 0

Response 4 7 12 5 4 50 0

Response 5 18 11 4 0 00 1

Response 6 19 12 6 7 31 0

Response 7 12 10 4 9 42 0

Response 8 21 6 4 9 61 0

Response 9 11 9 8 4 40 0

Response 10 23 10 4 6 20 2

Response 11 17 12 4 3 00 1

Response 12 25 9 4 2 44 0

Response 13 18 9 2 2 33 0

Response 14 20 13 6 0 11 0

Response 15 16 8 4 0 33 0

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: effluent Test Start Date: Test Ending Date: 

Test Species: RED MACROALGA, Champia parvula

Test Duration: DATA FILE: champia.icp OUTPUT FILE: champia.i50

Conc. Number Concentration Response Standard. Pooled

 ID Replicates % Means Dev. Response Means

 1 15 0.000 18.000 4.928 18.000

 2 15 0.800 10.533 2.356 10.533

 3 15 1.300 4.533 2.356 4.533

 4 15 2.200 3.600 3.066 3.600

 5 15 3.600 2.600 1.805 2.600

 6 15 6.000 1.067 1.335 1.067

 7 15 10.000 0.267 0.594 0.267

The Linear Interpolation Estimate: 0.9278 Entered P Value: 50

Number of Resamplings: 80

The Bootstrap Estimates Mean: 0.9263 Standard Deviation: 0.0745

Original Confidence Limits: Lower: 0.7893 Upper: 1.0576

Resampling time in Seconds: 3.63 Random Seed: -1255453122

Figure 14. ICPIN program output for the IC50.
364

TABLE 13. SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAMPIA PARVULA,

REPRODUCTION TEST PERFORMED IN A 50/50 MIXTURE OF NATURAL SEAWATER

AND GP2 ARTIFICIAL SEAWATER, USING GAMETES FROM ADULTS CULTURED IN

NATURAL SEAWATER. THE REFERENCE TOXICANT USED WAS COPPER (CU)

SULFATE1,2,3,4,5

Test NOEC IC25 IC50

Number (μg/L) (μg/L) (μg/L)

1 1.0 1.67 2.37 

2 1.0 1.50 1.99

3 1.0 0.69 1.53

4 1.0 0.98 1.78 

5 0.5 0.38 0.76

6 0.5 0.38 0.75

n: 6 6 6

Mean: NA 0.93 1.5 

CV(%): NA 59.6 43.7 

1 Data from USEPA (1991a). 2 Tests performed by Glen Thursby and Mark Tagliabue, ERL-N, USEPA, Narragansett, RI. Tests were 

conducted at 22°C, in 50/50 GP2 and natural seawater at a salinity of 30‰. 3 Copper concentrations were: 0.5, 1.0, 2.5, 5.0, 7.5, and 1.0 μg/L. 4 NOEC Range: 0.5 - 1.0 μg/L (this represents a difference of one exposure concentration). 5 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 
365

TABLE 14. SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAMPIA PARVULA,

REPRODUCTION TEST PERFORMED IN A 50/50 MIXTURE OF NATURAL SEAWATER

AND GP2 ARTIFICIAL SEAWATER, USING GAMETES FROM ADULTS CULTURED IN

NATURAL SEAWATER. THE REFERENCE TOXICANT USED WAS SODIUM DODECYL

SULFATE (SDS)1,2,3,4,5

Test NOEC IC25 IC50

Number (mg/L) (mg/L) (mg/L)

1 < 0.80 0.6 0.3

2 0.48 0.7 0.6

3 < 0.48 0.4 0.2

4 < 0.48 0.2 0.4

5 0.26 0.2 0.5

6 0.09 0.1 0.3

7 0.16 0.2 0.3

8 0.09 0.1 0.2

9 < 0.29 0.3 0.4

n: 5 9 9

Mean: NA 0.31 0.36

CV(%): NA 69.0 37.0

1 Data from USEPA (1991a). 2 Tests performed by Glen Thursby and Mark Tagliabue, ERL-N, USEPA, Narragansett, RI. Tests were 

conducted at 22°C, in 50/50 GP2 and natural seawater at a salinity of 30‰. 3 SDS concentrations for Test 1 were: 0.8, 1.3, 2.2, 3.6, 6.0, and 10.0 mg/L. SDS concentrations for Tests 2, 3,

and 4 were: 0.48, 0.8, 1.3, 2.2, 3.6, and 6.0 mg/L. SDS concentrations for Tests 5 and 6 were: 0.09, 0.16, 2.26,

0.43, 0.72, and 1.2 mg/L. 4 NOEC Range: 0.09 - 0.48 mg/L (this represents a difference of two exposure concentrations). 5 For a discussion of the precision of data from chronic toxicity tests see Section 4, Quality Assurance. 
366

TABLE 15. SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAMPIA PARVULA,

REPRODUCTION TEST IN NATURAL SEAWATER (30‰ SALINITY). THE REFERENCE

TOXICANT USED WAS COPPER (CU) SULFATE1,2,3

 Cu (μg/L)

Test NOEC IC25 IC50

1 1.00 2.62 4.02

2 0.50 0.71 1.66

3 0.50 2.83 3.55

4 0.50 0.99 4.15

n: 4 4 4

Mean: NA 1.79 3.35

CV(%): NA 61.09 34.45 

1 Data from USEPA (1991a). 2 Copper concentrations were 0.5, 1.0, 2.5, 5.0, 7.5, and 10 μg/L. Concentrations of Cu were made from a 100

μg/mL CuSO4 standard obtained from Inorganic Ventures, Inc., Brick, NJ. 3 Prepared by Steven Ward and Glen Thursby, Environmental Research Laboratory, USEPA, Narragansett, RI.

TABLE 16. SINGLE-LABORATORY PRECISION OF THE RED MACROALGA, CHAMPIA PARVULA,

REPRODUCTION TEST IN NATURAL SEAWATER (30‰ SALINITY). THE REFERENCE

TOXICANT USED WAS SODIUM DODECYL SULFATE (SDS)1,2,3

 SDS (mg/L) 

Test NOEC IC25 IC50

1 0.60 0.05 0.50

2 0.60 0.48 0.81

3 0.30 0.69 0.89

4 0.15 0.60 0.81

n: 4 4 4

 Mean: NA 0.46 0.75

CV(%): NA 62.29 22.92 

1 Data from USEPA (1991a). 2 SDS concentrations were 0.0375, 0.075, 0.15, 0.03, 0.60, and 1.20 mg/L. Concentrations of SDS were made

from a 44.64 ± 3.33 mg/mL standard obtained from the EMSL-USEPA, Cincinnati, OH. 3 Prepared by Steven Ward and Glen Thursby, Environmental Research Laboratory, USEPA, Narragansett, RI.
367

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APPENDICES

Page

A. Independence, Randomization, and Outliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

1. Statistical Independence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

2. Randomization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

3. Outliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

B. Validating Normality and Homogeneity of Variance Assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390

2. Tests for Normal Distribution of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390

3. Test for Homogeneity of Variance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

4. Transformations of the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399

C. Dunnett's Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

1. Manual Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

2. Computer Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408

D. T test with Bonferroni’s Adjustment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414

E. Steel's Many-one Rank Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420

F. Wilcoxon Rank Sum Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425

G. Single Concentration Toxicity Test - Comparison of Control with

100% Effluent or Receiving Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432

H. Probit Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436

I. Spearman-Karber Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439

J. Trimmed Spearman-Karber Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444

K. Graphical Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448

L. Linear Interpolation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

1. General Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

 2. Data Summary and Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

 3. Monotonicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

 4. Linear Interpolation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

 5. Confidence Intervals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453

 6. Manual Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

 7. Computer Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458

Cited References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
384

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 insure 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 Sheepshead 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.1. 

Note that the double digits 00 and 97 through 99 were not used.
385

TABLE A.1. RANDOM ASSIGNMENT OF FISH TO REPLICATE CHAMBERS

EXAMPLE ASSIGNED NUMBERS FOR EACH REPLICATE CHAMBER

Assigned Numbers Replicate Chamber

01, 25, 49, 73 Control, replicate chamber 1 

02, 26, 50, 74 Control, replicate chamber 2

03, 27, 51, 75 Control, replicate chamber 3

04, 28, 52, 76 Control, replicate chamber 4

05, 29, 53, 77 6.25% effluent, replicate chamber 1

06, 30, 54, 78 6.25% effluent, replicate chamber 2

07, 31, 55, 79 6.25% effluent, replicate chamber 3

08, 32, 56, 80 6.25% effluent, replicate chamber 4

09, 33, 57, 81 12.5% effluent, replicate chamber 1

10, 34, 58, 82 12.5% effluent, replicate chamber 2

11, 35, 59, 83 12.5% effluent, replicate chamber 3

12, 36, 60, 84 12.5% effluent, replicate chamber 4

13, 37, 61, 85 25.0% effluent, replicate chamber 1

14, 38, 62, 86 25.0% effluent, replicate chamber 2

15, 39, 63, 87 25.0% effluent, replicate chamber 3

16, 40, 64, 88 25.0% effluent, replicate chamber 4

17, 41, 65, 89 50.0% effluent, replicate chamber 1

18, 42, 66, 90 50.0% effluent, replicate chamber 2

19, 43, 67, 91 50.0% effluent, replicate chamber 3

20, 44, 68, 92 50.0% effluent, replicate chamber 4

21, 45, 69, 93 100.0% effluent, replicate chamber 1

22, 46, 70, 94 100.0% effluent, replicate chamber 2

23, 47, 71, 95 100.0% effluent, replicate chamber 3

24, 48, 72, 96 100.0% effluent, replicate chamber 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.1, 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.
386

TABLE A.2. TABLE OF RANDOM NUMBERS (Dixon and Massey, 1983) 

 

 10 09 73 25 33 76 52 01 35 86 34 67 35 43 76 80 95 90 91 17 39 29 27 49 45

 37 54 20 48 05 64 89 47 42 96 24 80 52 40 37 20 63 61 04 02 00 82 29 16 65

 08 42 26 89 53 19 64 50 93 03 23 20 90 25 60 15 95 33 47 64 35 08 03 36 06

 99 01 90 25 29 09 37 67 07 15 38 31 13 11 65 88 67 67 43 97 04 43 62 76 59

 12 80 79 99 70 80 15 73 61 47 64 03 23 66 53 98 95 11 68 77 12 27 17 68 33

 66 06 57 47 17 34 07 27 68 50 36 69 73 61 70 65 81 33 98 85 11 19 92 91 70

 31 06 01 08 05 45 57 18 24 06 35 30 34 26 14 86 79 90 74 39 23 40 30 97 32

 85 26 97 76 02 02 05 16 56 92 68 66 57 48 18 73 05 38 52 47 18 62 38 85 79

 63 57 33 21 35 05 32 54 70 48 90 55 35 75 48 28 46 82 87 09 83 49 12 56 24

 73 79 64 57 53 03 52 96 47 78 35 80 83 42 82 60 93 52 03 44 35 27 38 84 35

 98 52 01 77 67 14 90 56 86 07 22 10 94 05 58 60 97 09 34 33 50 50 07 39 98

 11 80 50 54 31 39 80 82 77 32 50 72 56 82 48 29 40 52 42 01 52 77 56 78 51

 83 45 29 96 34 06 28 89 80 83 13 74 67 00 78 18 47 54 06 10 68 71 17 78 17

 88 68 54 02 00 86 50 75 84 01 36 76 66 79 51 90 36 47 64 93 29 60 91 10 62

 99 59 46 73 48 87 51 76 49 69 91 82 60 89 28 93 78 56 13 68 23 47 83 41 13

 65 48 11 76 74 17 46 85 09 50 58 04 77 69 74 73 03 95 71 86 40 21 81 65 44

 80 12 43 56 35 17 72 70 80 15 45 31 82 23 74 21 11 57 82 53 14 38 55 37 63

 74 35 09 98 17 77 40 27 72 14 43 23 60 02 10 45 52 16 42 37 96 28 60 26 55

 69 91 62 68 03 66 25 22 91 48 36 93 68 72 03 76 62 11 39 90 94 40 05 64 18

 09 89 32 05 05 14 22 56 85 14 46 42 75 67 88 96 29 77 88 22 54 38 21 45 98

 91 49 91 45 23 68 47 92 76 86 46 16 28 35 54 94 75 08 99 23 37 08 92 00 48

 80 33 69 45 98 26 94 03 68 58 70 29 73 41 35 53 14 03 33 40 42 05 08 23 41

 44 10 48 19 49 85 15 74 79 54 32 97 92 65 75 57 60 04 08 81 22 22 20 64 13

 12 55 07 37 42 11 10 00 20 40 12 86 07 46 97 96 64 48 94 39 28 70 72 58 15

 63 60 64 93 29 16 50 53 44 84 40 21 95 25 63 43 65 17 70 82 07 20 73 17 90

 61 19 69 04 46 26 45 74 77 74 51 92 43 37 29 65 39 45 95 93 42 58 26 05 27

 15 47 44 52 66 95 27 07 99 53 59 36 78 38 48 82 39 61 01 18 33 21 15 94 66

 94 55 72 85 73 67 89 75 43 87 54 62 24 44 31 91 19 04 25 92 92 92 74 59 73

 42 48 11 62 13 97 34 40 87 21 16 86 84 87 67 03 07 11 20 59 25 70 14 66 70

 23 52 37 83 17 73 20 88 98 37 68 93 59 14 16 26 25 22 96 63 05 52 28 25 62

 04 49 35 24 94 75 24 63 38 24 45 86 25 10 25 61 96 27 93 35 65 33 71 24 72

 00 54 99 76 54 64 05 18 81 59 96 11 96 38 96 54 69 28 23 91 23 28 72 95 29

 35 96 31 53 07 26 89 80 93 45 33 35 13 54 62 77 97 45 00 24 90 10 33 93 33

 59 80 80 83 91 45 42 72 68 42 83 60 94 97 00 13 02 12 48 92 78 56 52 01 06

 46 05 88 52 36 01 39 09 22 86 77 28 14 40 77 93 91 08 36 47 70 61 74 29 41

 32 17 90 05 97 87 37 92 52 41 05 56 70 70 07 86 74 31 71 57 85 39 41 18 38

 69 23 46 14 06 20 11 74 52 04 15 95 66 00 00 18 74 39 24 23 97 11 89 63 38

 19 56 54 14 30 01 75 87 53 79 40 41 92 15 85 66 67 43 68 06 84 96 28 52 07

 45 15 51 49 38 19 47 60 72 46 43 66 79 45 43 59 04 79 00 33 20 82 66 95 41

 94 86 43 19 94 36 16 81 08 51 34 88 88 15 53 01 54 03 54 56 05 01 45 11 76

 98 08 62 48 26 45 24 02 84 04 44 99 90 88 96 39 09 47 34 07 35 44 13 18 80

 33 18 51 62 32 41 94 15 09 49 89 43 54 85 81 88 69 54 19 94 37 54 87 30 43

 80 95 10 04 06 96 38 27 07 74 20 15 12 33 87 25 01 62 52 98 94 62 46 11 71

 79 75 24 91 40 71 96 12 82 96 69 86 10 25 91 74 85 22 05 39 00 38 75 95 79

 18 63 33 25 37 98 14 50 65 71 31 01 02 46 74 05 45 56 14 27 77 93 89 19 36

 74 02 94 39 02 77 55 73 22 70 97 79 01 71 19 52 52 75 80 21 80 81 45 17 48

 54 17 84 56 11 80 99 33 71 43 05 33 51 29 69 56 12 71 92 55 36 04 09 03 24

 11 66 44 98 83 52 07 98 48 27 59 38 17 15 39 09 97 33 34 40 88 46 12 33 56

 48 32 47 79 28 31 24 96 47 10 02 29 53 68 70 32 30 75 75 46 15 02 00 99 94

 69 07 49 41 38 87 63 79 19 76 35 58 40 44 01 10 51 82 16 15 01 84 87 69 38

 
387

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.1, 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.

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 fish taken from tank 25.0% effluent, replicate chamber 1

Second fish taken from tank 6.25% effluent, replicate chamber 2

Third fish taken from tank 50.0% effluent, replicate chamber 4

Fourth fish taken from tank 100.0% effluent, replicate chamber 4

Fifth fish taken from tank 6.25% effluent, replicate chamber 1

Sixth fish taken from tank 25.0% effluent, replicate chamber 4

Seventh fish taken from tank 50.0% effluent, replicate chamber 1

Eighth fish taken from tank 100.0% effluent, replicate chamber 3

Ninth fish taken from tank 50.0% effluent, replicate chamber 2

Tenth fish taken from tank 100.0% effluent, replicate chamber 4

2.3.5 Four double-digit numbers were assigned to each replicate chamber (instead of one, two, or three doubledigit 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.1. 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.
388

TABLE A.4. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS: EXAMPLE

LABELING THE POSITIONS WITHIN THE WATER BATH

12 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 23 24

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.

TABLE A.5. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS: EXAMPLE

ASSIGNED NUMBERS FOR EACH POSITION

Assigned Numbers Position

01, 25, 49, 73 1

02, 26, 50, 74 2

03, 27, 51, 75 3

04, 28, 52, 76 4

05, 29, 53, 77 5

06, 30, 54, 78 6

07, 31, 55, 79 7

08, 32, 56, 80 8

09, 33, 57, 81 9

10, 34, 58, 82 10

11, 35, 59, 83 11

12, 36, 60, 84 12

13, 37, 61, 85 13

14, 38, 62, 86 14

15, 39, 63, 87 15

16, 40, 64, 88 16

17, 41, 65, 89 17

18, 42, 66, 90 18

19, 43, 67, 91 19

20, 44, 68, 92 20

21, 45, 69, 93 21

22, 46, 70, 94 22

23, 47, 71, 95 23

24, 48, 72, 96 24
389

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 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.

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 100.0% 6.25% 6.25% 6.25% 12.5% 

Control 12.5% Control 25.0% 12.5% 25.0% 

100.0% 50.0% 100.0% Control 100.0% 25.0% 

50.0% 50.0% 25.0% 50.0% 12.5% 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 doubledigit 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

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). 
390

TABLE B.1. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL GROWTH

 DATA (WEIGHT IN MG) FOR THE SHAPIRO-WILK'S TEST 

 Effluent Concentration (%) 

Replicate Control 6.25 12.5 25.0 50.0 

1 1.017 1.157 0.998 0.837 0.715 

2 0.745 0.914 0.793 0.935 0.907 

3 0.862 0.992 1.021 0.839 1.044 

Mean(Yi) 0.875 1.021 0.937 0.882 0.889 

Si

2 0.019 0.015 0.016 0.0031 0.027 

i 1 2 3 4 5 

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 judgement 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 Sheepshead 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.1. 
391

TABLE B.2. EXAMPLE OF SHAPIRO-WILK'S TEST: CENTERED OBSERVATIONS

 Effluent Concentration (%) 

 

Replicate Control 6.25 12.5 25.0 50.0 

1 0.142 0.136 0.061 - 0.009 - 0.174 

2 - 0.130 - 0.107 - 0.144 0.053 0.018 

3 - 0.013 - 0.029 0.084 - 0.043 0.155 

D ' '

n

i'1

(Xi

&X)

2

2.3 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 listed in Table

B.2. 

2.4 Calculate the denominator, D, of the test statistic:

Where: Xi

 = the centered observations, X& is the overall mean of the centered observations, and n is the total

number of the centered observations. For this set of data, X& = 0, and D = 0.1589.

2.4.1 For this set of data,

n = 15

X ̄ = 1/50 (0) = 0.0

D = 0.1589

2.5 Order the centered observations from smallest to largest, 

X(i) # X(2) # . . . # X(n)

where X(i) denote the ith order statistic. The ordered observations are listed in Table B.3. 
392

 TABLE B.3. EXAMPLE OF THE SHAPIRO-WILK'S TEST: ORDERED OBSERVATIONS 

i X(i)

1 - 0.174 

2 - 0.144 

3 - 0.130 

4 - 0.107 

5 - 0.043 

6 - 0.029 

7 - 0.013 

8 - 0.009 

9 0.018 

10 0.053 

11 0.061 

12 0.084 

13 0.136 

14 0.142 

15 0.155 

W' 1

D['

k

i'1

ai(X (n&i%1)&X (i))]

2

2.6 From Table B.4,for the number of observations, n, obtain the coefficients a1, a2, ..., ak, where k is n/2 if n is

even, and (n-1)/2 if n is odd. For the data in this example, n = 15, k = 7, and the ai

 values are listed in Table B.5.

The differences, X(n-i+1) - X(i), are listed in Table B.5. 

2.7 Compute the test statistic, W, as follows: 
393

TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (Conover, 1980)

Number of Observations

i±n 2 3 4 5 6 7 8 9 10

 1 0.7071 0.7071 0.6872 0.6646 0.6431 0.6233 0.6052 0.5888 0.5739

 2 - 0.0000 0.1667 0.2413 0.2806 0.3031 0.3164 0.3244 0.3291

 3 - - - 0.0000 0.0875 0.1401 0.1743 0.1976 0.2141

 4 - - - - - 0.0000 0.0561 0.0947 0.1224

 5 - - - - - - - 0.0000 0.0399

Number of Observations 

i±n 11 12 13 14 15 16 17 18 19 20

 1 0.5601 0.5475 0.5359 0.5251 0.5150 0.5056 0.4968 0.4886 0.4808 0.4734

 2 0.3315 0.3325 0.3325 0.3318 0.3306 0.3209 0.3273 0.3253 0.3232 0.3211

 3 0.2260 0.2347 0.2412 0.2460 0.2495 0.2521 0.2540 0.2553 0.2561 0.2565

 4 0.1429 0.1586 0.1707 0.1802 0.1878 0.1939 0.1988 0.2027 0.2059 0.2085

 5 0.0695 0.0922 0.1099 0.1240 0.1353 0.1447 0.1524 0.1587 0.1641 0.1686

 6 0.0000 0.0303 0.0539 0.0727 0.0880 0.1005 0.1109 0.1197 0.1271 0.1334

 7 - - 0.0000 0.0240 0.0433 0.0593 0.0725 0.0837 0.0932 0.1013

 8 - - - - 0.0000 0.0196 0.0359 0.0496 0.0612 0.0711

 9 - - - - - - 0.0000 0.0163 0.0303 0.0422

10 - - - - - - - - 0.0000 0.0140

 

Number of Observations

i±n 21 22 23 24 25 26 27 28 29 30

 1 0.4643 0.4590 0.4542 0.4493 0.4450 0.4407 0.4366 0.4328 0.4291 0.4254

 2 0.3185 0.3156 0.3126 0.3098 0.3069 0.3043 0.3018 0.2992 0.2968 0.2944

 3 0.2578 0.2571 0.2563 0.2554 0.2543 0.2533 0.2522 0.2510 0.2499 0.2487

 4 0.2119 0.2131 0.2139 0.2145 0.2148 0.2151 0.2152 0.2151 0.2150 0.2148

 5 0.1736 0.1764 0.1787 0.1807 0.1822 0.1836 0.1848 0.1857 0.1864 0.1870

 6 0.1399 0.1443 0.1480 0.1512 0.1539 0.1563 0.1584 0.1601 0.1616 0.1630

 7 0.1092 0.1150 0.1201 0.1245 0.1283 0.1316 0.1346 0.1372 0.1395 0.1415

 8 0.0804 0.0878 0.0941 0.0997 0.1046 0.1089 0.1128 0.1162 0.1192 0.1219

 9 0.0530 0.0618 0.0696 0.0764 0.0923 0.0876 0.0923 0.0965 0.1002 0.1036

10 0.0263 0.0368 0.0459 0.0539 0.0610 0.0672 0.0728 0.0778 0.0822 0.0862

11 0.0000 0.0122 0.0228 0.0321 0.0403 0.0476 0.0540 0.0598 0.0650 0.0697

12 - - 0.0000 0.0107 0.0200 0.0284 0.0358 0.0424 0.0483 0.0537

13 - - - - 0.0000 0.0094 0.0178 0.0253 0.0320 0.0381

14 - - - - - - 0.0000 0.0084 0.0159 0.0227

15 - - - - - - - - 0.0000 0.0076
394

TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (CONTINUED) 

Number of Observations

i±n 31 32 33 34 35 36 37 38 39 40 

1 0.4220 0.4188 0.4156 0.4127 0.4096 0.4068 0.4040 0.4015 0.3989 0.3964

2 0.2921 0.2898 0.2876 0.2854 0.2834 0.2813 0.2794 0.2774 0.2755 0.2737

3 0.2475 0.2462 0.2451 0.2439 0.2427 0.2415 0.2403 0.2391 0.2380 0.2368

4 0.2145 0.2141 0.2137 0.2132 0.2127 0.2121 0.2116 0.2110 0.2104 0.2098

5 0.1874 0.1878 0.1880 0.1882 0.1883 0.1883 0.1883 0.1881 0.1880 0.1878

6 0.1641 0.1651 0.1660 0.1667 0.1673 0.1678 0.1683 0.1686 0.1689 0.1691

7 0.1433 0.1449 0.1463 0.1475 0.1487 0.1496 0.1505 0.1513 0.1520 0.1526

8 0.1243 0.1265 0.1284 0.1301 0.1317 0.1331 0.1344 0.1356 0.1366 0.1376

9 0.1066 0.1093 0.1118 0.1140 0.1160 0.1179 0.1196 0.1211 0.1225 0.1237

10 0.0899 0.0931 0.0961 0.0988 0.1013 0.1036 0.1056 0.1075 0.1092 0.1108

11 0.0739 0.0777 0.0812 0.0844 0.0873 0.0900 0.0924 0.0947 0.0967 0.0986

12 0.0585 0.0629 0.0669 0.0706 0.0739 0.0770 0.0798 0.0824 0.0848 0.0870

13 0.0435 0.0485 0.0530 0.0572 0.0610 0.0645 0.0677 0.0706 0.0733 0.0759

14 0.0289 0.0344 0.0395 0.0441 0.0484 0.0523 0.0559 0.0592 0.0622 0.0651

15 0.0144 0.0206 0.0262 0.0314 0.0361 0.0404 0.0444 0.0481 0.0515 0.0546

16 0.0000 0.0068 0.0131 0.0187 0.0239 0.0287 0.0331 0.0372 0.0409 0.0444

17 - - 0.0000 0.0062 0.0119 0.0172 0.0220 0.0264 0.0305 0.0343

18 - - - - 0.0000 0.0057 0.0110 0.0158 0.0203 0.0244

19 - - - - - - 0.0000 0.0053 0.0101 0.0146

20 - - - - - - - - 0.0000 0.0049

Number of Observations

i±n

 41 42 43 44 45 46 47 48 49 50 

1 0.3940 0.3917 0.3894 0.3872 0.3850 0.3830 0.3808 0.3789 0.3770 0.3751

2 0.2719 0.2701 0.2684 0.2667 0.2651 0.2635 0.2620 0.2604 0.2589 0.2574

3 0.2357 0.2345 0.2334 0.2323 0.2313 0.2302 0.2291 0.2281 0.2271 0.2260

4 0.2091 0.2085 0.2078 0.2072 0.2065 0.2058 0.2052 0.2045 0.2038 0.2032

5 0.1876 0.1874 0.1871 0.1868 0.1865 0.1862 0.1859 0.1855 0.1851 0.1847

6 0.1693 0.1694 0.1695 0.1695 0.1695 0.1695 0.1695 0.1693 0.1692 0.1691

7 0.1531 0.1535 0.1539 0.1542 0.1545 0.1548 0.1550 0.1551 0.1553 0.1554

8 0.1384 0.1392 0.1398 0.1405 0.1410 0.1415 0.1420 0.1423 0.1427 0.1430

9 0.1249 0.1259 0.1269 0.1278 0.1286 0.1293 0.1300 0.1306 0.1312 0.1317

10 0.1123 0.1136 0.1149 0.1160 0.1170 0.1180 0.1189 0.1197 0.1205 0.1212

11 0.1004 0.1020 0.1035 0.1049 0.1062 0.1073 0.1085 0.1095 0.1105 0.1113

12 0.0891 0.0909 0.0927 0.0943 0.0959 0.0972 0.0986 0.0998 0.1010 0.1020

13 0.0782 0.0804 0.0824 0.0842 0.0860 0.0876 0.0892 0.0906 0.0919 0.0932

14 0.0677 0.0701 0.0724 0.0745 0.0765 0.0783 0.0801 0.0817 0.0832 0.0846

15 0.0575 0.0602 0.0628 0.0651 0.0673 0.0694 0.0713 0.0731 0.0748 0.0764

16 0.0476 0.0506 0.0534 0.0560 0.0584 0.0607 0.0628 0.0648 0.0667 0.0685

17 0.0379 0.0411 0.0442 0.0471 0.0497 0.0522 0.0546 0.0568 0.0588 0.0608

18 0.0283 0.0318 0.0352 0.0383 0.0412 0.0439 0.0465 0.0489 0.0511 0.0532

19 0.0188 0.0227 0.0263 0.0296 0.0328 0.0357 0.0385 0.0411 0.0436 0.0459

20 0.0094 0.0136 0.0175 0.0211 0.0245 0.0277 0.0307 0.0335 0.0361 0.0386

21 0.0000 0.0045 0.0087 0.0126 0.0163 0.0197 0.0229 0.0259 0.0288 0.0314

22 - - 0.0000 0.0042 0.0081 0.0118 0.0153 0.0185 0.0215 0.0244

23 - - - - 0.0000 0.0039 0.0076 0.0111 0.0143 0.0174

24 - - - - - - 0.0000 0.0037 0.0071 0.0104

25 - - - - - - - - 0.0000 0.0035
395

TABLE B.5. EXAMPLE OF THE SHAPIRO-WILK'S TEST: TABLE OF COEFFICIENTS AND

DIFFERENCES

i ai X(n-i+1) - X(i)

1 0.4734 0.181 X(20) - X(1)

2 0.3211 0.128 X(19) - X(2)

3 0.2565 0.105 X(18) - X(3)

4 0.2085 0.097 X(17) - X(4)

5 0.1686 0.076 X(16) - X(5)

6 0.1334 0.048 X(15) - X(6)

7 0.1013 0.034 X(14) - X(7)

8 0.0711 0.025 X(13) - X(8)

9 0.0422 0.008 X(12) - X(9)

10 0.0140 0.005 X(11) - X(10)
396

TABLE B.6. QUANTILES OF THE SHAPIRO WILK'S TEST STATISTIC (Conover, 1980)

n 0.01 0.02 0.05 0.10 0.50 0.90 0.95 0.98 0.99

3 0.753 0.756 0.767 0.789 0.959 0.998 0.999 1.000 1.000

4 0.687 0.707 0.748 0.792 0.935 0.987 0.992 0.996 0.997

5 0.686 0.715 0.762 0.806 0.927 0.979 0.986 0.991 0.993

6 0.713 0.743 0.788 0.826 0.927 0.974 0.981 0.986 0.989

7 0.730 0.760 0.803 0.838 0.928 0.972 0.979 0.985 0.988

8 0.749 0.778 0.818 0.851 0.932 0.972 0.978 0.984 0.987

9 0.764 0.791 0.829 0.859 0.935 0.972 0.978 0.984 0.986

10 0.781 0.806 0.842 0.869 0.938 0.972 0.978 0.983 0.986

11 0.792 0.817 0.850 0.876 0.940 0.973 0.979 0.984 0.986

12 0.805 0.828 0.859 0.883 0.943 0.973 0.979 0.984 0.986

13 0.814 0.837 0.866 0.889 0.945 0.974 0.979 0.984 0.986

14 0.825 0.846 0.874 0.895 0.947 0.975 0.980 0.984 0.986

15 0.835 0.855 0.881 0.901 0.950 0.975 0.980 0.984 0.987

16 0.844 0.863 0.887 0.906 0.952 0.976 0.981 0.985 0.987

17 0.851 0.869 0.892 0.910 0.954 0.977 0.981 0.985 0.987

18 0.858 0.874 0.897 0.914 0.956 0.978 0.982 0.986 0.988

19 0.863 0.879 0.901 0.917 0.957 0.978 0.982 0.986 0.988

20 0.868 0.884 0.905 0.920 0.959 0.979 0.983 0.986 0.988

21 0.873 0.888 0.908 0.923 0.960 0.980 0.983 0.987 0.989

22 0.878 0.892 0.911 0.926 0.961 0.980 0.984 0.987 0.989

23 0.881 0.895 0.914 0.928 0.962 0.981 0.984 0.987 0.989

24 0.884 0.898 0.916 0.930 0.963 0.981 0.984 0.987 0.989

25 0.888 0.901 0.918 0.931 0.964 0.981 0.985 0.988 0.989

26 0.891 0.904 0.920 0.933 0.965 0.982 0.985 0.988 0.989

27 0.894 0.906 0.923 0.935 0.965 0.982 0.985 0.988 0.990

28 0.896 0.908 0.924 0.936 0.966 0.982 0.985 0.988 0.990

29 0.898 0.910 0.926 0.937 0.966 0.982 0.985 0.988 0.990

30 0.900 0.912 0.927 0.939 0.967 0.983 0.985 0.988 0.990

31 0.902 0.914 0.929 0.940 0.967 0.983 0.986 0.988 0.990

32 0.904 0.915 0.930 0.941 0.968 0.983 0.986 0.988 0.990

33 0.906 0.917 0.931 0.942 0.968 0.983 0.986 0.989 0.990

34 0.908 0.919 0.933 0.943 0.969 0.983 0.986 0.989 0.990

35 0.910 0.920 0.934 0.944 0.969 0.984 0.986 0.989 0.990

36 0.912 0.922 0.935 0.945 0.970 0.984 0.986 0.989 0.990

37 0.914 0.924 0.936 0.946 0.970 0.984 0.987 0.989 0.990

38 0.916 0.925 0.938 0.947 0.971 0.984 0.987 0.989 0.990

39 0.917 0.927 0.939 0.948 0.971 0.984 0.987 0.989 0.991

40 0.919 0.928 0.940 0.949 0.972 0.985 0.987 0.989 0.991

41 0.920 0.929 0.941 0.950 0.972 0.985 0.987 0.989 0.991

42 0.922 0.930 0.942 0.951 0.972 0.985 0.987 0.989 0.991

43 0.923 0.932 0.943 0.951 0.973 0.985 0.987 0.990 0.991

44 0.924 0.933 0.944 0.952 0.973 0.985 0.987 0.990 0.991

45 0.926 0.934 0.945 0.953 0.973 0.985 0.988 0.990 0.991

46 0.927 0.935 0.945 0.953 0.974 0.985 0.988 0.990 0.991

47 0.928 0.936 0.946 0.954 0.974 0.985 0.988 0.990 0.991

48 0.929 0.937 0.947 0.954 0.974 0.985 0.988 0.990 0.991

49 0.929 0.937 0.947 0.955 0.974 0.985 0.988 0.990 0.991

50 0.930 0.938 0.947 0.955 0.974 0.985 0.988 0.990 0.991
397

B '

[('

P

i'1

Vi) ln S2 & '

P

i'1

Vi ln S 2

i ]

C

S2

'

('

P

i'1

ViS 2

i )

'

P

i'1

Vi

C'1%[3(p&1)]&1['

P

i'1

1/Vi

&('

P

i'1

Vi)

&1

]

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 15 observations (n) is 0.835. The calculated value, 0.9516, 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 Wilk's Test for normality. 

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. 

3.2 The data used in this example are growth data from a Sheepshead Minnow Larval Survival and Growth Test,

and are the same data used in Appendices C and D. These data are listed in Table B.7, together with the calculated

variance for the control and each toxicant concentration. 

3.3 The test statistic for Bartlett's Test (Snedecor and Cochran, 1980) is as follows: 

Where: Vi

 = degrees of freedom for each effluent concentration and control, (Vi = ni - 1)

p = number of levels of toxicant concentration including the control

ln = loge

i = 1, 2, ..., p where p is the number of concentrations including the control

ni

 = the number of replicates for concentration i.
398

TABLE B.7. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL GROWTH DATA

(WEIGHT IN MG) USED FOR BARTLETT'S TEST FOR HOMOGENEITY OF

VARIANCE 

Effluent Concentration (%)

Replicate Control 6.25 12.5 25.0 50.0 

1 1.017 1.157 0.998 0.873 0.715 

2 0.745 0.914 0.793 0.935 0.907 

3 0.862 0.992 1.021 0.839 1.044 

Mean 0.875 1.021 0.937 0.882 0.889 

Si

2 0.019 0.015 0.016 0.0024 0.027

i 1 2 3 4 5 

B'

2[5(ln 0.0158)&'

i

ln(S 2

i )]

1.2

' 2.3103

' 2[5(& 4.1477) & (&22.1247)]

1.2

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, Vi

 = 2, p = 5, S&2 = 0.0158, and C = 1.2. The calculated B value is: 

 

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 13.3 for a significance level of 0.01. Since B is less than 13.3, the

conclusion is that the variances are not different. 
399

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 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, the Shapiro-Wilk'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 Pi

 (1 - Pi), where Pi 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 Pi is

close to zero or one, the normality assumption may be invalid. The arc sine square root (arc sine %&P)

transformation is commonly used for such data to stabilize the variance and satisfy the normality requirement. 

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 determined, and the angle (in radians) for the square root of the

sine value is determined. Whenever the proportion dead is 0 or 1, a special modification of the arc sine square root

transformation must be used (Bartlett, 1937). An explanation of the arc sine square root transformation and the

modification is provided below. 

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: 

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 RP

Example: If RP = 0.60:

Angle = arc sine 0.60

= arc sine 0.7746

= 0.8861 radians

4.2.4.2 Modification of the arc sine square root when RP = 0. 
400

Angle (in radians) = arc sine 1/4N

Where: N = Number of animals/treatment replicate 

Example: If 20 animals are used: 

Angle = arc sine 1/80

= arc sine 0.1118 

= 0.1120 radians 

4.2.4.3 Modification of the arc sine square root when RP = 1

Angle = 1.5708 radians - (radians for RP = 0) 

Example: Using above value: 

 Angle = 1.5708 - 0.1120 

= 1.4588 radians 
401

TABLE C.1. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL GROWTH DATA

(WEIGHT IN MG) USED FOR DUNNETT'S PROCEDURE 

Effluent Replicate Test Vessel Total Mean 

Conc (%)

i 1 2 3 (Ti

) (&Yi) 

Control 1 1.017 0.745 0.862 2.624 0.875 

6.25 2 1.157 0.914 0.992 3.063 1.021 

12.5 3 0.998 0.793 1.021 2.812 0.937 

25.0 4 0.873 0.935 0.839 2.647 0.882 

50.0 5 0.715 0.907 1.044 2.666 0.889 

APPENDIX C

DUNNETT'S PROCEDURE

1. MANUAL CALCULATIONS

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, the t test with Bonferroni's adjustment is used

(see Appendix D). 

1.2 The data used in this example are growth data from a Sheepshead Minnow Larval Survival and Growth Test,

and are the same data used in Appendices B and D. These data are listed in Table C.1. 

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: 

Where: p = number of effluent concentrations including the control:

N = the total sample size; N''

i

ni

ni = the number of replicates for concentration "i" 

 Total Sum of Squares SST''

ij

Yij

2

&G 2/N
402

SST''

ij

Yij

2

&G 2

/N

SSW'SST&SSB

 Between Sum of Squares SSB''

i

Ti

2

/ni&G 2/N

SSW'SST&SSB Within Sum of Squares

 G = the grand total of all sample observations; G' '

P

i'1

Ti

 Ti = the total of the replicate measurements for concentration i

 N = the total sample size; N''

i

ni

 ni = the number of replicates for concentration i

 Yij = the jth observation for concentration i

1.4 For the data in this example:

n1 = n2 = n 3 = n 4 = n 5 = 3

N = 20

T1 = Y11 + Y12 + Y 13 = 2.624

T2 = Y21 + Y22 + Y 23 = 3.063

T3 = Y31 + Y32 + Y 33 = 2.812

T4 = Y41 + Y42 + Y 43 = 2.647

T5 = Y51 + Y52 + Y 53 = 2.666

G = T1 + T2 + T 3 + T 4 + T 5 = 13.812

 

= 12.922 - (13.812)2

/15 

= 0.204 

= 12.763 - (13.812)2

/15

= 0.045

 = 0.204 - 0.045 
403

 TABLE C.2. ANOVA TABLE FOR DUNNETT'S PROCEDURE

Source df Sum of Mean Square (MS)

Squares (SS) (SS/df)

 

Between p - 1 SSB SB

2

 = SSB/(p-1)

Within N - p SSW SW

2 = SSW/(N-p)

Total N - 1 SST

 TABLE C.3. COMPLETED ANOVA TABLE FOR DUNNETT'S PROCEDURE

Source df SS Mean Square

Between 5 - 1 = 4 0.045 0.011

Within 15 - 5 = 10 0.159 0.016

Total 14 0.204

t

i

' (Y1&Yi

)

Sw (1/n1)%(1/ni

)

= 0.159 

1.5 Summarize these data in the ANOVA table (Table C.2).

1.6 Summarize data for ANOVA (Table C.3). 

1.7 To perform the individual comparisons, calculate the t statistic for each concentration and control combination,

as follows:

 Where: &Yi

 = mean for each concentration i.
404

TABLE C.4. CALCULATED T VALUES 

Effluent

Concentration i ti

(%) 

6.25 2 - 1.414 

12.5 3 - 0.600 

25.0 4 - 0.068 

50.0 5 - 0.136 

MSD'd Sw (1/n1)%(1/n)

&Y1 = mean for the control

Sw = square root of the within mean square 

n1 = number of replicates in the control. 

ni

 = number of replicates for concentration i.

1.8 Table C.4 includes the calculated t values for each concentration and control combination.

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 (2.47), with an overall alpha level of 0.05, 10 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). Comparing each of

the calculated t values in Table C.4 with the critical value, no decreases in growth from the control were detected. 

Thus the NOEC is 50.0%.

1.10 To quantify the sensitivity of the test, the minimum significant difference (MSD) may be calculated. The

formula is as follows: 

 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 

n1 = number of replicates in the control
405

MSD'2.47(0.126)[ (1/3)%(1/3)]'2.47(0.126)( 2/3)

For example:

= 2.47 (0.126)(0.816) 

= 0.254 
406

TABLE C.5. DUNNETT'S "T" VALUES (Miller, 1981) 

(One-tailed)dαk

v

k α = .05 α = 0.1

12345678912345678 9

5 2.02 2.44 2.58 2.85 2.98 3.08 3.16 3.24 3.30 3.37 3.90 4.21 4.43 4.50 4.73 4.85 4.94 5.036 1.94 2.34 2.56 2.71 2.83 2.92 3.00 3.07 3.12 3.14 3.61 4.88 4.07 4.21 4.33 4.43 4.51 4.397 1.89 2.27 2.48 2.62 2.73 2.82 2.89 2.95 3.01 3.00 3.42 3.56 3.83 3.96 4.07 4.15 4.23 4.308 1.86 2.22 2.42 2.55 2.66 2.74 2.81 2.87 2.92 2.90 3.20 3.51 3.67 3.79 3.88 3.96 4.03 4.099 1.83 2.18 2.37 2.50 2.60 2.68 2.75 2.81 2.86 2.82 3.19 3.40 3.55 3.64 3.75 3.82 3.89 3.9410 1.81 2.15 2.34 2.47 2.56 2.64 2.70 2.76 2.81 2.76 3.11 3.31 3.45 3.56 3.64 3.71 3.78 3.8311 1.80 2.13 2.31 2.44 2.53 2.60 2.67 2.72 2.77 2.72 3.06 3.25 3.38 3.46 3.56 3.63 3.69 3.7412 1.78 2.11 2.29 2.41 2.50 2.58 2.64 2.69 2.74 2.68 3.01 3.19 3.32 3.42 3.50 3.56 3.62 3.6713 1.77 2.09 2.27 2.39 2.48 2.55 2.61 2.68 2.71 2.65 2.97 3.15 3.27 3.37 3.44 3.91 3.56 3.6114 1.76 2.08 2.25 2.37 2.46 2.53 2.59 2.64 2.69 2.62 2.94 3.11 3.23 3.32 3.40 3.46 3.51 3.5615 1.75 2.07 2.24 2.36 2.44 2.51 2.57 2.62 2.67 2.60 2.91 3.08 3.20 3.29 3.36 3.42 3.47 3.5216 1.75 2.06 2.23 2.34 2.43 2.50 2.56 2.61 2.65 2.58 2.38 3.05 3.17 3.28 3.33 3.39 3.44 3.4817 1.74 2.05 2.22 2.33 2.42 2.49 2.54 2.59 2.64 2.57 2.86 3.03 3.14 3.23 3.30 3.36 3.41 3.4518 1.73 2.04 2.21 2.32 2.41 2.48 2.53 2.58 2.62 2.55 2.84 3.01 3.12 3.21 3.27 3.33 3.38 3.4219 1.73 2.03 2.20 2.31 2.40 2.47 2.52 2.57 2.61 2.54 2.83 2.99 3.10 3.18 3.25 3.31 3.36 3.4020 1.72 2.03 2.19 2.30 2.38 2.46 2.51 2.56 2.60 2.53 2.81 2.97 3.08 3.17 3.23 3.29 3.34 3.3824 1.71 2.01 2.17 2.28 2.36 2.43 2.48 2.53 2.57 2.40 2.77 2.92 3.03 3.11 3.17 3.22 3.27 3.3130 1.70 1.99 2.15 2.25 2.33 2.40 2.45 2.50 2.54 2.46 2.72 2.87 2.97 3.05 3.11 3.16 3.21 3.2440 1.68 1.97 2.13 2.23 2.31 2.37 2.42 2.47 2.51 2.42 2.68 2.82 2.92 2.99 3.06 3.10 3.14 3.1860 1.67 1.95 2.10 2.21 2.28 2.35 2.39 2.44 2.48 2.39 2.64 2.78 2.87 2.94 3.08 3.04 3.06 3.12120 1.86 1.93 2.08 2.18 2.26 2.32 2.37 2.41 2.45 2.36 2.60 2.73 2.82 2.90 2.94 2.90 3.03 3.06α 1.64 1.92 2.06 2.16 2.23 2.29 2.34 2.33 2.42 2.33 2.56 2.68 2.77 2.34 2.90 2.83 2.97 3.00
407

Percent Reduction ' MSDu

Controlu

x 100

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 0.254 mg. This represents a decrease in growth of 29% 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. 

MSDu = control u - D u

Where: MSDu = the minimum significant difference for untransformed data 

Controlu = the untransformed control mean 

Du = the untransformed difference 

1.11.2.2 Calculate the percent reduction from the control that MSDu represents as: 

 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.1

were transformed by the arc sine square root transformation. Thus: 

0.875 - 0.254 = 0.621 

Step 2. Obtain untransformed values for the control mean (0.875) and the difference (0.621) obtained in Step 1,

above. 

[ Sine (0.875)]2

 = 0.589 

[ Sine (0.621)]2

 = 0.339 

Step 3. The untransformed MSD (MSDu) is determined by subtracting the untransformed values obtained in Step

2. 

MSDu = 0.589 - 0.339 = 0.250 

In this case, the MSD would represent a 42% decrease in survival from the control [(0.250/0.589)(100)]. 
408

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 (1955). 

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. 

2.4 If the number of replicates at each toxicant concentration and control are not equal, a t test with the Bonferroni

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 version of the program can be obtained from EMSL-Cincinnati by sending a

diskette with a written request.

2.6 DATA INPUT AND OUTPUT

2.6.1 Data on the number of surviving mysids, Mysidopsis bahia, from a survival, growth and fecundity test (Table C.6)

are used to illustrate the data input and output for this program. 

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 analyzed:

 1. Response proportions, like survival or fertilization proportions data.

 2. Counts and measurements, like offspring counts, cystocarp and algal cell counts, weights, chlorophyll

measurements or turbidity measurements.

2.6.2.2 After the type of analysis for the 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 response proportions, the program prompts the user for the

following information:

1. Number of concentrations, including control

2. For each concentration and replicate:

 - number of organisms exposed per replicate

- number of organisms responding per replicate (organisms surviving, eggs fertilized, etc.)

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 main menu

(see below).
409

2.6.2.5 Sample data input is shown in Figure C.1. 

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 C.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.
410

TABLE C.6. SAMPLE DATA FOR DUNNETT'S PROGRAM FOR SURVIVING MYSIDS,

MYSIDOPSIS BAHIA

Treatment Replicate Total No.

Chamber Mysids Alive

1 Control 1 5 4

 2 5 4

35 5

45 5

55 5

65 5

75 5

85 4

2 50 ppb 1 5 4

25 5

35 4

45 4

55 5

65 5

75 4

85 5

3 100 ppg 1 5 3

 25 5

 35 5

 45 5

 55 5

 65 3

 75 4

 8 4 4

4 210 ppb 1 5 5

25 4

35 1

45 4

55 3

65 4

75 4

85 4

5 450 ppb 1 5 0

 25 1

 35 0

45 1

 55 0

 65 0

 75 0

85 2
411

EMSL Cincinnati Dunnett Software

Version 1.5

1) Create a data file

2) Edit a data file

3) Analyze an existing data set

4) Stop

Your choice ? 3 

Number of concentrations, including control ? 5

Number of replicates for conc. 1 (the control) ? 8

replicate number of organisms exposed number of organisms responding 

(organisms surviving, eggs fertilized, etc.)

 1 5 4

 2 5 4

 3 5 5

 4 5 5

 5 5 5

 6 5 5

 7 5 5

 8 5 4

 

Number of replicates for conc. 2 ? 8 

Do you wish to save the data on disk ? y

Disk file for output ? mysidsur.dat

Figure C.1. Sample Data Input for Dunnett's Program for Survival Data from Table C.6.
412

EMSL Cincinnati: Dunnett Software

Version 1.5

 

 1) Create a data file

 2) Edit a data file

 3) Analyze an existing data set

 4) Stop

Your choice ? 3 

File name ? mysidsur.dat

Available Transformations

 1) no transform

 2) square root

 3) log10

 4) arcsine square root

Your choice ? 4 

Dunnett's test as implemented in this program is 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 concentrations to be less than or greater than the mean for the control

concentration.

Direction for Dunnetts test : L=less than, G=greater than ? l 

Summary Statistics for Raw Data 

 Conc. n Mean s.d. cv%

 1 = control 8 .9250 .1035 11.2

 2 8 .9000 .1069 11.9

 3 8 .8500 .1773 20.9

 4 8 .7250 .2375 32.8

 5 8 .1000 .1512 151.2

Mysid Survival Example with Data in Table C.6

Figure C.2. Example of Choosing Option 3 from the Main Menu of the Dunnett Program.
413

Mysid Survival Example with Data in Table C.6

Summary Statistics and ANOVA

Transformation = Arcsine Square Root 

Conc. n Mean s.d. cv%

1 = control 8 1.2560 .1232 9.8

2 8 1.2262 .1273 10.4

3 8 1.1709 .2042 17.4

4* 8 1.0288 .2593 25.2

5* 8 .3424 .1752 51.2

*) the mean for this conc. is significantly less than 

 the control mean at alpha = 0.05 (1-sided) by Dunnett's test

Minimum detectable difference for Dunnett's test = -.208074

This corresponds to a difference of -.153507 in original units

This difference corresponds to -16.98 percent of control

Between concentrations

sum of squares = 4.632112 with 4 degrees of freedom.

Error mean square = .034208 with 35 degrees of freedom.

Bartlett's test p-value for equality of variances = .257

Do you wish to restart the program ? 

Figure C.3. Example of Program Output for the Dunnett’s Program Using the Survival Data in Table C.6.
414

TABLE D.1. SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, LARVAL GROWTH DATA

(WEIGHT IN MG) USED FOR THE T TEST WITH BONFERRONI'S ADJUSTMENT 

Effluent Replicate Test Vessel Total Mean 

Conc (%)

i 1 2 3 (Ti

) (&Yi) 

Control 1 1.017 0.745 0.862 2.624 0.875 

6.25 2 1.157 0.914 0.992 3.063 1.021 

12.5 3 0.998 0.793 1.021 2.812 0.937 

 25.0 4 0.873 0.935 0.839 2.647 0.882 

 50.0 5 0.715 0.907 (Lost) 1.622 0.811 

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 of distribution 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 50% effluent treatment is presumed to have been lost. 

Thus, Dunnett's Procedure cannot be used. The weight data are presented in Table D.1. 

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''

i

ni

ni = the number of replicates for concentration i

Total Sum of Squares SST''

ij

Yij

2

&G 2

/N
415

 Between Sum of Squares SSB''

i

Ti

2

/ni&G 2/N

 Within Sum of Squares SSW'SST&SSB

Where: G = The grand total of all sample observations; G' '

P

i'1

Ti

Ti = The total of the replicate measurements for concentration i

 

Yij = The jth observation for concentration i

 3.2 For the data in this example:

n1 = n2 = n3 = n 4 = 3

N = 20

 T1 = Y11 + Y12 + Y 13 = 2.624

T2 = Y21 + Y22 + Y 23 = 3.063

T3 = Y31 + Y32 + Y 33 = 2.812

T4 = Y41 + Y42 + Y 43 = 2.647

T5 = Y51 + Y52 + Y 53 = 1.622

G = T1 + T2 + T3 + T 4 + T 5 = 12.768

 SSB''

i

Ti

2

/ni

&G 2

/N

= 11.709 - (12.768)2/14 

= 0.064 

 SST''

ij

Yij

2

&G 2/N

= 11.832 - (12.768)2/14

= 0.188 

 SSW'SST&SSB

= 0.188 - 0.064 

= 0.124 
416

 TABLE D.3. COMPLETED ANOVA TABLE FOR THE T-TEST WITH BONFERRONI'S ADJUSTMENT

Source df SS Mean Square

Between 5 - 1 = 4 0.064 0.016

Within 14 - 5 = 9 0.124 0.014

Total 13 0.188

ti

' (Y1&Yi

)

Sw (1/n1)%(1/ni

)

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 SB

2 = SSB/(p-1)

Within N - p SSW SW

2 = SSW/(N-p)

Total N - 1 SST

3.4 Summarize these calculations 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: 

Where: &Yi

 = mean for concentration i 

&Y1 = mean for the control 

Sw = square root of the within mean square

n1 = number of replicates in the control.
417

TABLE D.4. CALCULATED T VALUES 

Effluent 

Concentration i ti

(%) 

6.25 2 - 1.511 

12.5 3 - 0.642 

25.0 4 - 0.072 

50.0 5 0.592 

ni = number of replicates for concentration i.

3.6 Table D.4 includes the calculated t values for each concentration and control combination. 

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 (2.686), with an overall alpha level of 0.05, nine degrees of freedom and

four concentrations excluding the control, was obtained from Table D.5. Comparing each of the calculated t values in

Table D.4 with the critical value, no decreases in growth from the control were detected. Thus the NOEC is 50.0%.
418

TABLE D.5. CRITICAL VALUES FOR "T" FOR THE T TEST WITH BONFERRONI'S ADJUSTMENT 

P = 0.05 CRITICAL LEVEL, ONE TAILED 

 df K = 1 K = 2 K = 3 K = 4 K = 5 K = 6 K = 7 K = 8 K = 9 K = 10

 1 6.314 12.707 19.002 25.452 31.821 38.189 44.556 50.924 57.290 63.657

 2 2.920 4.303 5.340 6.206 6.965 7.649 8.277 8.861 9.408 9.925 3 2.354 3.183 3.741 4.177 4.541 4.857 5.138 5.392 5.626 5.841 4 2.132 2.777 3.187 3.496 3.747 3.961 4.148 4.315 4.466 4.605 5 2.016 2.571 2.912 3.164 3.365 3.535 3.681 3.811 3.927 4.033 6 1.944 2.447 2.750 2.969 3.143 3.288 3.412 3.522 3.619 3.708 7 1.895 2.365 2.642 2.842 2.998 3.128 3.239 3.336 3.422 3.500 8 1.860 2.307 2.567 2.752 2.897 3.016 3.118 3.206 3.285 3.356 9 1.834 2.263 2.510 2.686 2.822 2.934 3.029 3.111 3.185 3.250 10 1.813 2.229 2.406 2.634 2.764 2.871 2.961 3.039 3.108 3.170 11 1.796 2.301 2.432 2.594 2.719 2.821 2.907 2.981 3.047 3.106 12 1.783 2.179 2.404 2.561 2.681 2.730 2.863 2.935 2.998 3.055 13 1.771 2.161 2.380 2.533 2.651 2.746 2.827 2.897 2.950 3.013 14 1.762 2.145 2.360 2.510 2.625 2.718 2.797 2.864 2.924 2.977 15 1.754 2.132 2.343 2.490 2.603 2.694 2.771 2.837 2.895 2.947 16 1.746 2.120 2.329 2.473 2.584 2.674 2.749 2.814 2.871 2.921 17 1.740 2.110 2.316 2.459 2.567 2.655 2.729 2.793 2.849 2.899 18 1.735 2.101 2.305 2.446 2.553 2.640 2.712 2.775 2.830 2.879 19 1.730 2.094 2.295 2.434 2.540 2.626 2.697 2.759 2.813 2.861 20 1.725 2.086 2.206 2.424 2.528 2.613 2.684 2.745 2.798 2.846 21 1.721 2.080 2.278 2.414 2.518 2.602 2.672 2.732 2.785 2.832 22 1.718 2.074 2.271 2.406 2.509 2.592 2.661 2.721 2.773 2.819 23 1.714 2.069 2.264 2.398 2.500 2.583 2.651 2.710 2.762 2.808 24 1.711 2.064 2.258 2.391 2.493 2.574 2.642 2.701 2.752 2.797 25 1.709 2.060 2.253 2.385 2.486 2.566 2.634 2.692 2.743 2.788 26 1.706 2.056 2.248 2.379 2.479 2.559 2.627 2.684 2.734 2.779 27 1.704 2.052 2.243 2.374 2.473 2.553 2.620 2.677 2.727 2.771 28 1.702 2.049 2.239 2.369 2.468 2.547 2.613 2.670 2.720 2.764
419

 TABLE D.5. CRITICAL VALUES FOR "T" FOR THE T TEST WITH BONFERRONI'S ADJUSTMENT

P = 0.05 CRITICAL LEVEL, ONE TAILED (CONTINUED)

df K = 1 K = 2 K = 3 K = 4 K = 5 K = 6 K = 7 K = 8 K = 9 K = 10 29 1.700 2.046 2.235 2.364 2.463 2.541 2.607 2.664 2.713 2.757 30 1.698 2.043 2.231 2.360 2.458 2.536 2.602 2.658 2.707 2.750 31 1.696 2.040 2.228 2.356 2.453 2.531 2.597 2.652 2.701 2.745 32 1.694 2.037 2.224 2.352 2.449 2.527 2.592 2.647 2.696 2.739 33 1.693 2.035 2.221 2.349 2.445 2.523 2.587 2.643 2.691 2.734 34 1.691 2.033 2.219 2.346 2.442 2.519 2.583 2.638 2.686 2.729 35 1.690 2.031 2.216 2.342 2.438 2.515 2.579 2.634 2.682 2.724 36 1.689 2.029 2.213 2.340 2.435 2.512 2.575 2.630 2.678 2.720 37 1.688 2.027 2.211 2.337 2.432 2.508 2.572 2.626 2.674 2.716 38 1.686 2.025 2.209 2.334 2.429 2.505 2.568 2.623 2.670 2.712 39 1.685 2.023 2.207 2.332 2.426 2.502 2.565 2.619 2.667 2.708 40 1.684 2.022 2.205 2.329 2.424 2.499 2.562 2.616 2.663 2.705 50 1.676 2.009 2.189 2.311 2.404 2.478 2.539 2.592 2.638 2.678 60 1.671 2.001 2.179 2.300 2.391 2.463 2.324 2.576 2.621 2.661 70 1.667 1.995 2.171 2.291 2.381 2.453 2.513 2.564 2.609 2.648 80 1.665 1.991 2.166 2.285 2.374 2.446 2.505 2.556 2.600 2.639 90 1.662 1.987 2.162 2.280 2.369 2.440 2.499 2.549 2.593 2.632 100 1.661 1.984 2.158 2.276 2.365 2.435 2.494 2.544 2.588 2.626 110 1.659 1.982 2.156 2.273 2.361 2.432 2.490 2.540 2.583 2.622 120 1.658 1.980 2.153 2.270 2.358 2.429 2.487 2.536 2.580 2.618Infinite 1.645 1.960 2.129 2.242 2.327 2.394 2.450 2.498 2.540 2.576 d.f. = Degrees of freedom for MSE (Mean Square Error) from ANOVA. K = Number of concentrations to be compared to the control.
420

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 the

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. 

3. An example of the use of this test is provided below. The test employs survival data from a mysid 7-day, chronic test. 

The data are listed in Table E.1. Throughout the test, the control data are taken from the site water control. Since there is

0% survival for all eight replicates for the 50% concentration, it is not included in this analysis and is considered a

qualitative mortality effect. 

4. 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, 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 3.12% 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.

6. For this set of data, determine if the survival in any of the effluent concentrations is significantly lower than the

survival of 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 survival at each of the various effluent concentrations with

some "minimum" or critical rank sum, at or below which the survival would be considered to be significantly lower than

the control. At a probability level of 0.05, the critical rank sum in a test with four concentrations and eight replicates per

concentration, is 47 (see Table E.5).

7. Of the rank sums in Table E.4, none are less than 47. Therefore, due to the qualitative effect at the 50% effluent

concentration, the NOEC is 25% effluent and the LOEC is 50% effluent. 
421

TABLE E.1. EXAMPLE OF STEEL'S MANY-ONE RANK TEST: DATA FOR MYSID, MYSIDOPSIS

BAHIA, 7-DAY CHRONIC TEST

Effluent Replicate Number of Number of

Concentration Chamber Mysids at Live Mysids

Start of Test at End of Test

154

254

355

Control 4 5 4

(Site Water) 5 5 5

654

754

855

153

255

353

Control 4 5 3

(Brine & 5 5 4

Dilution Water) 6 5 4

753

853

154

254

354

3.12% 4 5 5

554

654

755

853

153

254

355

6.25% 4 5 4

554

654

755

855

155

254

355

12.5% 4 5 3

555

654

754

853

155

255

355

25.0% 4 5 5

553

655

754

854

150

250

350

50.0% 4 5 0

550

650

750

850
422

TABLE E.2. EXAMPLE OF STEEL'S MANY-ONE RANK TEST: ASSIGNING

 RANKS TO THE CONTROL AND 3.12% EFFLUENT CONCENTRATIONS

 

Rank Number of Live Control or % Effluent

Mysids, Mysidopsis bahia

1 3 3.12

6.5 4 Control

6.5 4 Control

6.5 4 Control

6.5 4 Control

6.5 4 Control

6.5 4 3.12

6.5 4 3.12

6.5 4 3.12

6.5 4 3.12

6.5 4 3.12

14 5 Control

14 5 Control

14 5 Control

14 5 3.12

14 5 3.12
423

TABLE E.3. TABLE OF RANKS 

Replicate Effluent Concentration (%) 

Chamber Control1 3.12 6.25 12.5 25.0

1 4 (6.5,6,6.5,5) 4 (6.5) 3 (1) 5 (13.5) 5 (12.5)

 2 4 (6.5,6,6.5,5) 4 (6.5) 4 (6) 4 (6.5) 5 (12.5)

3 5 (14,13.5,13.5,12.5) 4 (6.5) 5 (13.5) 5 (13.5) 5 (12.5)

4 4 (6.5,6,6.5,5) 5 (14) 4 (6) 3 (1.5) 5 (12.5)

 5 5 (14,13.5,13.5,12.5) 4 (6.5) 4 (6) 5 (13.5) 3 (1)

6 4 (6.5,6,6.5,5) 4 (6.5) 4 (6) 4 (6.5) 5 12.5)

7 4 (6.5,6,6.5,5) 5 (14) 5 (13.5) 4 (6.5) 4 (5) 

8 5 (14,13.5,13.5,12.5) 3 (1) 5 (13.5) 3 (1.5) 4 (5) 

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

(%)

3.12 61.5

6.25 65.5

12.50 63.0

25.00 73.5
424

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 (excluding control)

n 23 4567 89

 

4 11 10 10 10 10 -- -- --

-- -- -- -- -- -- -- --

5 18 17 17 16 16 16 16 15

15 -- -- -- -- -- -- --

6 27 26 25 25 24 24 24 23

23 22 21 21 -- -- -- --

7 37 36 35 35 34 34 33 33

32 31 30 30 29 29 29 29

8 49 48 47 46 46 45 45 44

43 42 41 40 40 40 39 39

9 63 62 61 60 59 59 58 58

56 55 54 53 52 52 51 51

10 79 77 76 75 74 74 73 72

71 69 68 67 66 66 65 65

11 97 95 93 92 91 90 90 89

87 85 84 83 82 81 81 80

12 116 114 112 111 110 109 108 108

105 103 102 100 99 99 98 98

13 138 135 133 132 130 129 129 128

125 123 121 120 119 118 117 117

14 161 158 155 154 153 152 151 150

147 144 142 141 140 139 138 137

15 186 182 180 178 177 176 175 174

170 167 165 164 162 161 160 160

16 213 209 206 204 203 201 200 199

196 192 190 188 187 186 185 184

17 241 237 234 232 231 229 228 227

223 219 217 215 213 212 211 210

18 272 267 264 262 260 259 257 256

252 248 245 243 241 240 239 238

19 304 299 296 294 292 290 288 287

282 278 275 273 272 270 268 267

20 339 333 330 327 325 323 322 320

315 310 307 305 303 301 300 299
425

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. The use of this test may be illustrated with fecundity data from the mysid test in Table F.1. The site water control and

the 12.5% effluent concentration each have seven replicates for the proportion of females bearing eggs, while there are

eight replicates for each of the remaining three concentrations.

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. 

4. An example of assigning ranks to the combined data for the control and effluent concentration 3.12% 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 fecundity in any of the test concentrations is significantly lower than the fecundity

in the control. If this occurs, the rank sum at that concentration would be significantly lower than the rank sum. Thus,

compare the rank sums for fecundity of each of the various effluent concentrations with some "minimum" or critical rank

sum, at or below which the fecundity 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 seven replicates in the control is 44 for those concentrations

with eight replicates, and 34 for those concentrations with seven replicates (see Table F.5, for K = 4).

6. Comparing the rank sums in Table F.4 to the appropriate critical rank, only the 25% effluent concentration does not

exceed its critical value of 44. Thus, the NOEC and LOEC for fecundity are 12.5% and 25%, respectively. 
426

TABLE F.1. EXAMPLE OF WILCOXON'S RANK SUM TEST: FECUNDITY DATA FOR MYSID,

MYSIDOPSIS BAHIA, 7-DAY CHRONIC TEST

Effluent Replicate Number of Number of Proportion

Concentration Chamber Mysids at Live Mysids of Females

Start of Test at End of Test with Eggs

Control 1 5 4 0.50

(Site Water) 2 5 4 ----

3 5 5 0.75

4 5 4 0.67

5 5 5 0.67

6 5 4 0.50

7 5 4 1.00

8 5 5 1.00

Control 1 5 3 1.00

(Brine & 2 5 5 1.00

Dilution Water) 3 5 3 1.00

4 5 3 1.00

5 5 4 1.00

6 5 4 0.50

7 5 3 0.50

8 5 3 0.50

3.12% 1 5 4 1.00

2 5 4 0.50

3 5 4 0.67

4 5 5 1.00

5 5 4 0.50

6 5 4 1.00

7 5 5 1.00

8 5 3 0.00

6.25% 1 5 3 0.50

2 5 4 0.00

3 5 5 0.75

4 5 4 1.00

5 5 4 1.00

6 5 4 1.00

7 5 5 0.67

8 5 5 0.67

1 5 5 0.33

12.5% 2 5 4 0.50

3 5 5 1.00

4 5 3 ----

5 5 5 1.00

6 5 4 0.00

7 5 4 0.33

8 5 3 0.50

25.0% 1 5 5 0.00

2 5 5 0.50

3 5 5 0.13

4 5 5 0.00

5 5 3 0.50

6 5 5 0.00

7 5 4 0.50

8 5 4 0.50

50.0% 1 5 0 ----

2 5 0 ----

3 5 0 ----

4 5 0 ----

5 5 0 ----

6 5 0 ----

7 5 0 ----

8 5 0 ----
427

TABLE F.2. EXAMPLE OF WILCOXON'S RANK SUM TEST: ASSIGNING RANKS TO THE CONTROL

AND 3.12% EFFLUENT CONCENTRATIONS

Rank Proportion of Site Water Control

Females W/Eggs or Effluent % 

 1 0.00 3.12

3.5 0.50 Control

3.5 0.50 Control

3.5 0.50 3.12

3.5 0.50 3.12

 7 0.67 Control

7 0.67 Control

7 0.67 3.12

9 0.75 Control

12.5 1.00 Control

12.5 1.00 Control

12.5 1.00 3.12

12.5 1.00 3.12

12.5 1.00 3.12

12.5 1.00 3.12
428

TABLE F.3. TABLE OF RANKS1

Rep Proportion Site Water Effluent Concentration (%) 

Control Rank 3.12 6.25 12.5 25.0

1 0.50 (3.5,3,5.5,7.5) 1.00 (12.5) 0.50 (3) 0.33 (2.5) 0.00 (2)

2 - - - - 0.50 (3.5) 0.00 (1) 0.50 (5.5) 0.50 (7.5)

3 0.75 (9,9.5,10,13) 0.67 (7) 0.75 (9.5) 1.00 (12.5) 0.33 (4)

4 0.67 (7,6.5,8.5,11.5) 1.00 (12.5) 1.00 (13) -- 0.00 (2)

5 0.67 (7,6.5,8.5,11.5) 0.50 (3.5) 1.00 (13) 1.00 (12.5) 0.50 (7.5)

6 0.50 (3.5,3,5.5,7.5) 1.00 (12.5) 1.00 (13) 0.00 (1) 0.00 (2)

7 1.00 (12.5,13,12.5,14.5) 1.00 (12.5) 0.67 (6.5) 0.33 (2.5) 0.50 (7.5)

8 1.00 (12.5,13,12.5,12.5) 0.00 (1) 0.67 (6.5) 0.50 (5.5) 0.50 (7.5)

 1Control ranks are given in the order of the concentration with which they were ranked.

TABLE F.4. RANK SUMS

Effluent Rank Sum No. of Critical

Concentration Replicates Rank Sum

(%) 

3.12 65 8 44

6.25 65.5 8 44

12.50 42 7 34

25.00 40 8 44
429

 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. Replicates No. of Replicates Per Effluent Concentration

in Control 

3 4 5 6 7 8 9 10 

1 3 6 10 16 23 30 39 49 59

4 6 11 17 24 32 41 51 62

5 7 12 19 26 34 44 54 66

6 8 13 20 28 36 46 57 69

7 8 14 21 29 39 49 60 72

8 9 15 23 31 41 51 63 72

9 10 16 24 33 43 54 66 79

10 10 17 26 35 45 56 69 82

2 3 -- -- 15 22 29 38 47 58

4 -- 10 16 23 31 40 49 60

5 6 11 17 24 33 42 52 63

6 7 12 18 26 34 44 55 66

7 7 13 20 27 36 46 57 69

8 8 14 21 29 38 49 60 72

9 8 14 22 31 40 51 62 75

10 9 15 23 32 42 53 65 78

3 3 -- -- -- 21 29 37 46 57

4 -- 10 16 22 30 39 48 59

5 -- 11 17 24 32 41 51 62

6 6 11 18 25 33 43 53 65

7 7 12 19 26 35 45 56 68

8 7 13 20 28 37 47 58 70

9 7 13 21 29 39 49 61 73

10 8 14 22 31 41 51 63 76
430

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 Replicates Per Effluent Concentration

in Control 

3 4 5 6 7 8 9 10

4 3 -- -- -- 21 28 37 46 56

4 -- -- 15 22 30 38 48 59

5 -- 10 16 23 31 40 50 61

6 6 11 17 24 33 42 52 64

7 6 12 18 26 34 44 55 67

8 7 12 19 27 36 46 57 69

9 7 13 20 28 38 48 60 72

10 7 14 21 30 40 50 62 75

5 3 -- -- -- -- 28 36 46 56

4 -- -- 15 22 29 38 48 58

5 -- 10 16 23 31 40 50 61

6 -- 11 17 24 32 42 52 63

7 6 11 18 25 34 43 54 66

8 6 12 19 27 35 45 56 68

9 7 13 20 28 37 47 59 71

10 7 13 21 29 39 49 61 74

6 3 -- -- -- -- 28 36 45 56

4 -- -- 15 21 29 38 47 58

5 -- 10 16 22 30 39 49 60

6 -- 11 16 24 32 41 51 63

7 6 11 17 25 33 43 54 65

8 6 12 18 26 35 45 56 68

9 6 12 19 27 37 47 58 70

10 7 13 20 29 38 49 60 73

7 3 -- -- -- -- -- 36 45 56

4 -- -- -- 21 29 37 47 58

5 -- -- 15 22 30 39 49 60

6 -- 10 16 23 32 41 51 62

7 -- 11 17 25 33 43 53 65

8 6 11 18 26 35 44 55 67

9 6 12 19 27 36 46 58 70

10 7 13 20 28 38 48 60 72
431

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 Replicate Per Effluent Concentration

in Control 

3 4 5 6 7 8 9 10 

8 3 -- -- -- -- -- 36 45 55 

4 -- -- -- 21 29 37 47 57 

5 -- -- 15 22 30 39 49 59 

6 -- 10 16 23 31 40 51 62 

7 -- 11 17 24 33 42 53 64 

8 6 11 18 25 34 44 55 67 

9 6 12 19 27 36 46 57 69 

10 6 12 19 28 37 48 59 72 

9 3 -- -- -- -- -- -- 45 55 

4 -- -- -- 21 28 37 46 57 

5 -- -- 15 22 30 39 48 59 

6 -- 10 16 23 31 40 50 62 

7 -- 10 17 24 33 42 52 64 

8 -- 11 18 25 34 44 55 66 

9 6 11 18 26 35 46 57 69 

10 6 12 19 28 37 47 59 71 

10 3 -- -- -- -- -- -- 45 55 

4 -- -- -- 21 28 37 46 57 

5 -- -- 15 22 29 38 48 59 

6 -- 10 16 23 31 40 50 61 

7 -- 10 16 24 32 42 52 64 

8 -- 11 17 25 34 43 54 66 

9 6 11 18 26 35 45 56 68 

10 6 12 19 27 37 47 58 71 
432

F ' S 2

1

S 2

2

where S 2

1 >S 22

F ' 0.00131

0.000861 ' 1.52

 APPENDIX G

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. 

5. Compare F with the 0.005 level of a tabled F value with n1 - 1 and n2 - 1 degrees of freedom, where n1 and n2 are

the number of replicates for each of the two groups. 

6. A set of mysid growth data from an effluent (single concentration) 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 G.1. 

7. Since the variability of the 100% effluent is greater than the variability of the control, S2

 for the 100% effluent

concentration is placed in the numerator of the F statistic and S2

 for the control is placed in the denominator. 

8. There are 8 replicates for the effluent concentration and 8 replicates for the control. Thus, both numerator and

denominator degrees of freedom are equal to 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 distribution (Snedecor and Cochran, 1980). The critical F value for this test

is 8.89. Since 1.52 is not greater than 8.89, the conclusion is that the variances of the control and 100% effluent are

homogeneous. 
433

t ' Y ̄

1&Y ̄2

Sp

1

n1

% 1

n2

Sp ' (n1&1) S 2

1 %(n2&1) S 22

n1%n2&2

t ' 0.1950.132

0.0329 1

8

% 1

8

' 3.83

TABLE G.1. MYSID, MYSIDOPSIS BAHIA, GROWTH DATA FROM AN EFFLUENT (SINGLE

CONCENTRATION) TEST

Replicate 

1 234 5 6 7 8 S  ̄ 2 X

Control 0.183 0.148 0.216 0.199 0.176 0.243 0.213 0.180 0.195 0.000861

100%

Effluent 0.153 0.117 0.085 0.153 0.086 0.193 0.137 0.129 0.132 0.00131

9. Equal Variance T Test. 

9.1 To perform the t test, calculate the following test statistic: 

Where: = mean for the control Y ̄

1

Y ̄ = mean for the effluent concentration 2

S = estimate of the variance for the control 2

1

S = estimate of the variance for the effluent 2

2

concentration 

n1 = 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 control, such as a decrease in survival or a

decrease in reproduction, a one-tailed test is appropriate. Thus, you would compare the calculated t with a critical t,

where the critical t is at the 5% level of significance with n1 + n2 - 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 G.1 to illustrate the t test, the calculation of t is as follows: 
434

Sp ' (8&1)0.000861%(8&1)0.00131

8%8&2 ' 0.0329

t ' Y ̄

1&Y ̄2

S 2

1

n1

% S 2

2

n2

dfN ' (n1&1) (n2&1)

(n2&1) C2%(1&C)

2 (n1&1)

Where: 

9.4 For an 0.05 level of significance test with 14 degrees of freedom, the critical t is 1.762 (Note: Table D.5 for 

K = 1 includes the critical t values for comparing two groups). Since 3.83 is greater than 1.762, the conclusion is

that the growth for the 100% effluent concentration is significantly lower than growth for the control. 

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 follows:

Where: = mean for the control Y ̄

1

Y ̄ = mean for the effluent concentration 2

S = estimate of the variance for the control 2

1

S = estimate of the variance for the effluent concentration 2

2

n1 = number of replicates for the control 

n2 = number of replicates for the effluent concentration 

10.2 Additionally, the degrees of freedom for the test are adjusted using the following formula: 

Where:

C '

S 2

1

n1

S 2

1

n1

% S 2

2

n2
435

10.3 The modified degrees of freedom is usually not an integer. Common practice is to round down to the nearest

integer. 

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. 
436

APPENDIX H

PROBIT ANALYSIS

1. This program calculates the EC1 and EC50 (or LC1 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 and supporting

documentation 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 mortality data (Figure H.1) from a sheepshead minnow embryo-larval

survival and teratogenicity test. The program begins with a request for the following information: 

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 H.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. The estimated LC1 and LC50 values and associated 95% confidence intervals (see Figure H.2). 
437

EPA PROBIT ANALYSIS PROGRAM

USED FOR CALCULATING LC/EC VALUES

 Version 1.5

Do you wish abbreviated (A) or full (F) input/output? A

Output to printer (P) or disk file (D)? P

Title ? Example of Probit Analysis

Number responding in the control group = ? 17

Number of animals exposed in the concurrent control group = ? 100

Number of exposure concentrations, exclusive of controls ? 5

Input data starting with the lowest exposure concentration

Concentration = ? 6.25

Number responding = ? 14

Number exposed = ? 100

Concentration = ? 12.5

Number responding = ? 16

Number exposed = ? 102

Concentration = ? 25.0

Number responding = ? 35

Number exposed = ? 100

Concentration = ? 50.0

Number responding = ? 72

Number exposed = ? 99

Concentration = ? 100

Number responding = ? 99

Number exposed = ? 99

Number Number

Number Conc. Resp. Exposed

1 6.2500 14 100

2 12.5000 16 102

3 25.0000 35 100

4 50.0000 72 99

5 100.0000 99 99

Do you wish to modify your data ? N

The number of control animals which responded = 17

The number of control animals exposed = 100

Do you wish to modify these values ? N

Figure H.1. Sample Data Input for USEPA Probit Analysis Program, Version 1.5. 
438

Example of Probit Analysis

Proportion

Observed Responding

Number Number Proportion Adjusted for

Conc. Exposed Resp. Responding Controls

 Control 100 17 0.1700 0.0000

6.2500 100 14 0.1400 0.0201

12.5000 102 16 0.1569 0.0001

25.0000 100 35 0.3500 0.2290

50.0000 99 72 0.7273 0.6765

100.0000 99 99 1.0000 1.0000

Chi - Square for Heterogeneity (calculated) = 3.472

Chi - Square for Heterogeneity 

 (tabular value at 0.05 level) = 7.815

Example of Probit Analysis

 Estimated LC/EC Values and Confidence Limits

Exposure Lower Upper

Point Conc. 95% Confidence Limits

LC/EC 1.00 12.917 8.388 16.888

LC/EC 50.00 37.667 32.898 42.081

Figure H.2. USEPA Probit Analysis Program used for Calculating LC/EC Values, Version 1.5.
439

p s

i&1 ' p si ' (pi

%pi&1)/2

p s

0 'p s1 'p s2 ' 0.05%0.05%0.00%0.00

4 ' 0.104 ' 0.025

APPENDIX I

SPEARMAN-KARBER METHOD

1. The Spearman-Karber Method is a nonparametric statistical 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 log10 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.

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.

6. The Spearman-Karber Method is illustrated below using a set of mortality data from a Sheepshead Minnow

Larval Survival and Growth test. These data are listed in Table I.1.

7. Let p0, p1, ..., pk denote the observed response proportion mortalities for the control and k effluent

concentrations. The first step is to smooth the pi

 if they do not satisfy p 0 # p 1 # ... # p k. The smoothing process

replaces any adjacent pi

's that do not conform to p 0 # p 1 # ... # p k with their average. For example, if pi is less

than pi-1 then:

Where: = the smoothed observed proportion mo p rtality for effluent concentration i. 2

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:
440

p a

i ' (p si &p

2

o )/(1&p so )

TABLE I.1. EXAMPLE OF SPEARMAN-KARBER METHOD: MORTALITY DATA FROM A 

SHEEPSHEAD MINNOW LARVAL SURVIVAL AND GROWTH TEST (40 ORGANISMS

PER CONCENTRATION)

Effluent Number of Mortality

Concentration Mortalities Proportion

%

Control 2 0.05

6.25 2 0.05

12.5 0 0.00

25.0 0 0.00

50.0 26 0.65

100.0 40 1.00

 

 

7.2 Since p4 = 0.65 is larger than , set = 0.65. Similarly, p5 p = 1.00 is larger than so set = 1.00. s

3 p s4 p5 p4

Additional smoothing is not necessary. The smoothed observed proportion mortalities are shown in Table I.2.

TABLE I.2. EXAMPLE OF SPEARMAN-KARBER METHOD: SMOOTHED, ADJUSTED 

MORTALITY DATA FROM A SHEEPSHEAD MINNOW LARVAL SURVIVAL AND

GROWTH TEST

 

 

Smoothed,

Effluent Smoothed Adjusted

Concentration Mortality Mortality Mortality

% Proportion Proportion Proportion

 

Control 0.05 0.025 0.000

6.25 0.05 0.025 0.000

12.5 0.00 0.025 0.000

25.0 0.00 0.025 0.000

50.0 0.65 0.650 0.641

100.0 1.00 1.000 1.000

 

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 : = the smoothed observed pr p oportion mortality for the control s

o

p = the smoothed observed proportion mortality for effluent concentration i. s

i
441

p a

o 'p a1 'p a2 'p a3 ' p s

1 &p so

1&p s

o

' 0.025&0.025

1&0.025 ' 0.00.975 '0.0

p a

5 ' p s

5 &p so

1&p s

o

' 1.000&0.025

1&0.025 ' 0.9750.975 '1.000

p a

4 ' p s

4 &p so

1&p s

o

' 0.650&0.025

1&0.025 ' 0.06250.975 '0.641

m ' j

k

i'1

&1

(p a

i%1) (Xi

%Xi%1)

2

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:

The smoothed, adjusted response proportions for the effluent concentrations are shown in Table I.2. 

A plot of the smoothed, adjusted data is shown in Figure I.1.

9. Calculate the log10 of the estimated LC50, m, as follows:

Where: = the smoothed adjusted propor p tion mortality at concentration i a

i

Xi = the log10 of concentration i

k = the number of effluent concentrations tested, not including the control.

9.1 For this example, the log10 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)]/2

 = 1.656527 
442

0.00 6.25 12.50 25.00 50.00 

0.00.10.20.30.40.50.60.70.80.91.0

EFFLUENT CONCENTRATION (%)

PROPORTIONMORTALITY

SMOOTHED PROPORTION MORTALITYOBSERVED PROPORTIONMORTALITYSMOOTHED ADJUSTED PROPORTION MORTALITY

Figure I.1. Plot of observed, smoothed, and adjusted response proportions for sheepshead minnow, Cyprinodon variegatus, survi
443

V(m) ' j

k

i'2

&1

p a

i (1&p ai ) (Xi%1%Xi&1)

2

4(ni

&1)

1.656527 ± 2 0.00053477 '(1.610277, 1.702777)

10. Calculate the estimated variance of m as follows:

Where: Xi = the log10 of concentration i

ni

 = the number of organisms tested at effluent concentration i

p = the smoothed adjusted observed proportion mortality at effluent concentration i a

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 follows:

V(m) = (0.000)(1.000)(1.3979 - 0.7959)2

/4(39) +

(0.000)(1.000)(1.6990 - 1.0969)2

/4(39) +

(0.641)(0.359)(2.0000 - 1.3979)2

/4(39)

 = 0.00053477

11. Calculate the 95% confidence interval for m: m ± 2.0 V(m)

11.1 For this example, the 95% confidence interval for m is calculated as follows:

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(1.610277) = 40.8%

 upper limit: antilog(1.702777) = 50.4%
444

APPENDIX J

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). 

The Trimmed Spearman-Karber Method estimates the trimmed mean of the distribution of the log10 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 Method is recommended only when the requirements for the Probit

Analysis 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, p1, ..., pk denote the observed proportion mortalities for the control and the k effluent concentrations. 

The first step is to smooth the pi

 if they do not satisfy p 0# p 1# ...# p k. The smoothing process replaces any

adjacent pi

's that do not conform to p 0 # p 1 # ...# p k, with their average. For example, if pi is less than pi-1 then:

Where: pi s

&1s ' p s

i ' (pi

%pi&1)/2

 = the smoothed observed p proportion mortality for effluent concentration i. s

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 a

i ' (p si &p so )/(1&p so )

p = the smoothed observed proportion mortality for the control s

o

p = the smoothed observed proportion mortality for effluent concentration i. s

i

8. Calculate the amount of trim to use in the estimation of the LC50 as follows:

Where: Trim = maximum p a

1 ,(1&p ak )

p = the smoothed, adjusted proportion mortality for the lowest effluent concentration, exclusive of a

1

the control

p = the smoothed, adjusted proportion mortality for the highest effluent concentration a

k
445

k = the number of effluent concentrations, exclusive of the control.

The minimum trim should be calculated for 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 through the EMSL, 3411 Church Street, Cincinnati, OH 45244. The

program can be obtained from EMSL-Cincinnati by sending a written request to the above address.

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 Sheepshead Minnow Larval Survival and Growth test will be used. The data are listed in

Table J.1.

12.1 The program requests the following input (Figure J.1):

a. Output destination (D = disk file or P = printer).

b. Control data.

c. Data for each toxicant concentration.

12.2 The program output includes the following (Figure J.2):

a. A table of the concentrations tested, number of organisms exposed, and the mortalities.

b. The amount of trim used in the calculation.

c. The estimated LC50 and the associated 95% confidence interval.

TABLE J.1. EXAMPLE OF TRIMMED SPEARMAN-KARBER METHOD: MORTALITY DATA FROM A

SHEEPSHEAD MINNOW LARVAL SURVIVAL AND GROWTH TEST (40 ORGANISMS PER

CONCENTRATION)

 Effluent Number of Mortality

Concentration Mortalities Proportion

%

Control 2 0.05

6.25 0 0.00

12.5 2 0.05

25.0 0 0.00

50.0 0 0.00

100.0 32 0.80
446

A:>TSK

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: 

Sheepshead minnow

ENTER TOXICANT NAME: 

effluent 

ENTER UNITS FOR EXPOSURE CONCENTRATION OF TOXICANT : 

% 

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; MAXIMUM = 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)? 

y 

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 J.1. Example input for Trimmed Spearman-Karber Method.
447

TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5 

DATE: 1 TEST NUMBER: 2 DURATION: 7 Days TOXICANT: 

effluent 

SPECIES: sheepshead minnow

RAW DATA: Concentration Number Mortalities

 --- ---- (%) Exposed 

.00 40 2

6.25 40 0

12.50 40 2

25.00 40 0

50.00 40 0

100.00 40 32

SPEARMAN-KARBER TRIM: 20.41% 

SPEARMAN-KARBER ESTIMATES: 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 J.2. Example output for Trimmed Spearman-Karber Method.
448

APPENDIX K

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 (log10)

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.

3. The Graphical Method is illustrated below using a set of mortality data from an Inland Silverside Larval

Survival and Growth test. These data are listed in Table K.1.

TABLE K.1. EXAMPLE OF GRAPHICAL METHOD: MORTALITY DATA FROM AN INLAND 

SILVERSIDE LARVAL SURVIVAL AND GROWTH TEST (40 ORGANISMS PER 

CONCENTRATION)

Effluent Number of Mortality

Concentration Mortalities Proportion

 %

 Control 2 0.05

6.25 0 0.00 

12.5 0 0.00 

25.0 0 0.00

50.0 40 1.00

100.0 40 1.00 

4. Let p0, p1, ..., pk denote the observed proportion mortalities for the control and the k effluent concentrations. 

The first step is to smooth the pi

 if they do not satisfy p 0 # p 1 # ... # p k. The smoothing process replaces any

adjacent pi

's that do not conform to p 0 # p 1 # ... # p k with their average. For example, if pi is less than pi-1 then:

Where: p s

s&1 ' p si ' (pi

%pi&1)/2

p = the smoothed observed proportion mortality for effluent s

i

concentration i.
449

p s

o ' p si ' p s2 ' p s3 ' 0.05%0.00%0.00%0.00

4 ' 0.054 ' 0.0125

p a

o ' p a1 ' p a2 ' p a3 ' P s

1 &P so

1&p s

o

' 0.0125&0.125

1&0.0125 ' 0.00.9875 '0.0

p a

4 ' p a5 ' P s

4 &p so

1&p s

o

' 1.00&0.0125

1&0.0125 ' 0.98750.9875 ' 1.00

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 observed response proportion for the control, the values for these four

groups must be averaged:

4.2 Since p4 = p5 = 1.00 are larger then 0.0125, set = = 1.00. p Additional smoothing is not necessary. The s

4 p s5

smoothed observed proportion mortalities are shown in Table K.2.

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: p a

1 '(p si &p so )/(1&p so )

p = the smoothed observed proportion mortality for the control s

o

p = the smoothed observed proportion mortality for effluent s

i

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:

A table of the smoothed, adjusted response proportions for the effluent concentrations are shown in Table K.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 K.1.
450

TABLE K.2. EXAMPLE OF GRAPHICAL METHOD: SMOOTHED, ADJUSTED MORTALITY DATA 

FROM AN INLAND SILVERSIDE LARVAL SURVIVAL AND GROWTH TEST

Smoothed

 Effluent Smoothed Adjusted

Concentration Mortality Mortality Mortality

 % Proportion Proportion Proportion

 Control 0.05 0.0125 0.00

6.25 0.00 0.0125 0.00

12.5 0.00 0.0125 0.00

25.0 0.00 0.0125 0.00

50.0 1.00 1.0000 1.00

100.0 1.00 1.0000 1.00

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.
451

Figure K.1. Plot of the smoothed adjusted response proportions for inland silverside, Menidia beryllina,

survival data.
452

APPENDIX L

LINEAR INTERPOLATION METHOD

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 IC). 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) are 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 non-increasing, 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.

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 should stay the same (Y ̄ i

)

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 . If the mean observed response at the lo (Y ̄ west toxicant concentration is equal to or smaller 1) (Y ̄2)

than the control mean , it is used as the response. If it (Y ̄ is larger than the control mean, it is averaged with the 1)

control, and this average is used for both the control response (M1) and the lowest toxicant concentration response

(M2). This mean is then compared to the mean observed response for the next higher toxicant concentration . (Y ̄3)

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 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 decrease monotonically, the become Mi Y ̄ without smoothing. i Y ̄ i

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.
453

ICp ' CJ % [M1 (1&1p/100)&M )

J] (CJ%1&CJ

)

(MJ%1&MJ

)

4.2 To obtain the estimate, determine the concentrations CJ and CJ+1 which bracket the response M1 (1 - p/100),

where M1 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:

Where: CJ = tested concentration whose observed mean response is greater than M1(1 - p/100).

CJ + 1 = tested concentration whose observed mean response is less than M1(1 - p/100).

M1 = 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 CJ. 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.

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 Yji is randomly resampled with replacement to produce a new set of data Yji*, 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 times, 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 IC estimate is in question. However, narrow intervals do
454

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 mysid growth data used in the example in Section 14. The data is

presented as the mean weight per original number of organisms. Table L.1 includes the raw data and the mean

growth for each concentration. A plot of the data is provided in Figure L.1.
455

Figure L.1. Plot of raw data, observed means, and smoothed means for the mysid, Mysidopsis bahia, growth data.
456

TABLE L.1. MYSID, MYSIDOPSIS BAHIA, GROWTH DATA

Toxicant Concentration (ppb) 

Replicate Control 50 100 210 450

1 0.146 0.154 0.114 0.153 0

2 0.118 0.193 0.172 0.094 0.012

3 0.216 0.190 0.160 0.017 0

4 0.199 0.190 0.199 0.122 0.002

5 0.176 0.256 0.165 0.052 0

6 0.243 0.191 0.145 0.154 0

7 0.213 0.122 0.207 0.110 0

8 0.144 0.177 0.186 0.103 0.081

Mean 0.182 0.184 0.168 0.101 0.012 (Y ̄ i

)

i1 2 3 4 5

6.2 MONOTONICITY

6.2.1 As can be seen from the plot in Figure L.1, the observed means are not monotonically non-increasing with

respect to concentration. Therefore, the means must be smoothed prior to calculating the IC.

6.2.2 Starting with the control mean = 0.186 and = 0.184, we see that . Calculate the smoothed means: Y ̄

1 Y ̄2 Y ̄1<Y ̄2

M1 ' M2 ' (Y ̄

1%Y ̄2)/2 ' 0.193

6.2.3 Since and and Y ̄

5'0.025<Y ̄4'0.101<Y ̄3'0.168<M2, set M3'0.168 M4'0.101, M5'0.025.

Table L.2 contains the smoothed means and Figure L.1 gives a plot of the smoothed response curve.

6.3 LINEAR INTERPOLATION

6.3.1 Estimates of the IC25 and IC50 can be calculated using the Linear Interpolation Method. A 25% reduction in

mean weight, compared to the controls, would result in a mean weight of 0.139, where M1(1-p/100) =

0.185(1-25/100). A 50% reduction in mean weight, compared to the controls, would result in a mean weight of

0.093 mg. Examining the smoothed means and their associated concentrations (Table L.2), the two effluent

concentrations bracketing the mean weight per original of 0.139 mg are C3 = 100 ppb and C4 = 210 ppb. The two

effluent concentrations bracketing a response of 0.093 mg per total original number of organisms are C4 = 210 ppb

and C5 = 450 ppb.
457

ICp ' Cj % [M1 (1&1p/100)&M )

j ] (Cj%1&Cj

)

(Mj%1&Mj

)

IC25'100%[0.93(1&25/100)&0.164] (210&100)

(0.101&0.164)

ICp ' Cj % [M1 (1&1p/100)&M )

j ] (Cj%1&Cj

)

(Mj%1&Mj

)

IC50'210%[210%[0.193(1&50/100)&0.101] (450&210)

(0.028&0.101)

TABLE L.2. MYSID, MYSIDOPSIS BAHIA, MEAN GROWTH RESPONSE AFTER SMOOTHING

 

Toxicant Smoothed

Conc. Mean 

(ppb) i Mi

 (mg)

 

Control 1 0.183

 50 2 0.183

100 3 0.168

210 4 0.101

450 5 0.025

 

6.3.2 Using the equation from section 4.2, the estimate of the IC25 is calculated as follows:

= 151 ppb

6.3.3 Using Equation 1 from 4.2, the estimate of the IC50 is calculated as follows:

= 239 ppb

6.4 CONFIDENCE INTERVALS

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.
458

7. COMPUTER CALCULATIONS

7.1 The computer program, ICPIN, prepared for the Linear Interpolation Methods 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). A compiled, executable version of the

program and supporting documentation can be obtained by sending a written request to EMSL-Cincinnati, 3411

Church Street, Cincinnati, OH 45244.

7.2 The ICPIN.EXE program performs the following functions: 1) it calculates the observed response means (I

)

(response means); 2) it calculates the standard deviations; 3) checks the responses for monotonicity; 4) calculates

smoothed means (Mi

) (pooled response means) if necessary; 5) uses the means, Mi, 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 input (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%.

7.4 DATA INPUT

7.4.1 Data is entered directly into the program onscreen. A sample data entry screen in shown in Figure L.2. The

program documentation provides guidance on the entering and analysis of data for the Linear Interpolation Method. 

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).
459

ICp Data Entry/Edit Screen Current File:

Conc. ID 1 2 3 4 5 6

Conc. 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

F10 for Command Menu Use Arrow Keys to Switch Fields

Figure L.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.
460

7.5 DATA OUTPUT

7.5.1 The program output includes the following (Figures L.3 and L.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 (Yi

),

standard deviations for each response mean, and the pooled response means (smoothed means;

Mi

).

3. The linear interpolation estimate of the ICp using the means (Mi

). 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 mysid growth data in Table L.1 is provided in Figures L.3 and

L.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 133.5054 (ppb). The empirical 95% confidence intervals for the true mean was 96.8623 to 186.6383

(ppb).

7.6.2 When the ICPIN program was used to analyze this set of data, requesting 80 resamples, the estimate of the

IC50 was 234.6761 (ppb). The empirical 95% confidence intervals for the true mean were 184.8692 to 283.3965

(ppb).
461

Conc. ID 1 2 3 4. 5

-------------------------------------------------------------------------------------------------------------------------------------------

Conc. Tested 0 50 100 210 450

-------------------------------------------------------------------------------------------------------------------------------------------

Response 1 .146 .154 .114 .153 0

Response 2 .118 .193 .172 .094 .012

Response 3 .216 .190 .160 .017 0

Response 4 .199 .190 .199 .122 .002

Response 5 .176 .256 .165 .052 0

Response 6 .243 .191 .145 .154 0

Response 7 .213 .122 .207 .110 0

Response 8 .144 .177 .186 .103 .081

-------------------------------------------------------------------------------------------------------------------------------------------

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent:

Test Start Date: Test Ending Date: 

Test Species: MYSID SHRIMP, Mysidopsis bahia

Test Duration: growth test

DATA FILE: mysidwt.icp

OUTPUT FILE: mysid.i25

-------------------------------------------------------------------------------------------------------------------------------------------

Conc. Number Concentration Response Standard. Pooled

 ID Replicates μg/l Means Dev. Response Means

-------------------------------------------------------------------------------------------------------------------------------------------

1 8 0.000 0.182 0.043 0.183

2 8 50.000 0.184 0.038 0.183

3 8 100.000 0.168 0.030 0.168

4 8 210.000 0.101 0.047 0.101

5 8 450.000 0.012 0.028 0.012

-------------------------------------------------------------------------------------------------------------------------------------------

The Linear Interpolation Estimate: 133.5054 Entered P Value: 25

-------------------------------------------------------------------------------------------------------------------------------------------

Number of Resamplings: 80

The Bootstrap Estimates Mean: 147.1702 Standard Deviation: 23.7984

Original Confidence Limits: Lower: 96.8623 Upper: 186.6383

Resampling time in Seconds: 0.16 Random Seed: -1623038650

Figure L.3. Example of ICPIN program output for the IC25.
462

Conc. ID 1 2 3 4. 5

-------------------------------------------------------------------------------------------------------------------------------------------

Conc. Tested 0 50 100 210 450

-------------------------------------------------------------------------------------------------------------------------------------------

Response 1 .146 .154 .114 .153 0

Response 2 .118 .193 .172 .094 .012

Response 3 .216 .190 .160 .017 0

Response 4 .199 .190 .199 .122 .002

Response 5 .176 .256 .165 .052 0

Response 6 .243 .191 .145 .154 0

Response 7 .213 .122 .207 .110 0

Response 8 .144 .177 .186 .103 .081

-------------------------------------------------------------------------------------------------------------------------------------------

*** Inhibition Concentration Percentage Estimate ***

Toxicant/Effluent: 

Test Start Date: Test Ending Date: 

Test Species: MYSID SHRIMP, Mysidopsis bahia

Test Duration: growth test

DATA FILE: mysidwt.icp

OUTPUT FILE: mysidwt.i50

-------------------------------------------------------------------------------------------------------------------------------------------

Conc. Number Concentration Response Standard. Pooled

 ID Replicates μg/L Means Dev. Response Means

-------------------------------------------------------------------------------------------------------------------------------------------

 1 8 0.000 0.182 0.043 0.183

 2 8 50.000 0.184 0.038 0.183

 3 8 100.000 0.168 0.030 0.168

 4 8 210.000 0.101 0.047 0.101

 5 8 450.000 0.012 0.028 0.012

-------------------------------------------------------------------------------------------------------------------------------------------

The Linear Interpolation Estimate: 234.6761 Entered P Value: 50

-------------------------------------------------------------------------------------------------------------------------------------------

Number of Resamplings: 80

The Bootstrap Estimates Mean: 233.3311 Standard Deviation: 28.9594

Original Confidence Limits: Lower: 184.8692 Upper: 283.3965

Resampling time in Seconds: 0.11 Random Seed: 1103756486

Figure L.4. Example ICPIN program output for the IC50.
463

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