United States Office of Research and

Environmental Protection Development

Agency Washington DC 20460

EPA/600/4-90/027F

August 1993

&EPA Methods for Measuring the

Acute Toxicity of

Effluents and Receiving

Waters to Freshwater and

Marine Organisms

Fourth Edition
EPA/600/4-90/027F

August 1993

METHODS FOR MEASURING THE ACUTE TOXICITY OF EFFLUENTS AND RECEIVING WATERS

TO FRESHWATER AND MARINE ORGANISMS

(Fourth Edition)

Edited by

Cornelius I. Weber

ENVIRONMENTAL MONITORING SYSTEMS LABORATORY - CINCINNATI

OFFICE OF RESEARCH AND DEVELOPMENT

U.S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268

@ Printed on Recycled Paper
DISCLAIMER

This document has been reviewed by the Environmental Monitoring Systems Laboratory-Cincinnati (EMSL-Cincinnati), U. S. Environmental Protection Agency

(USEPA), and approved for publication. The mention of trade names or

commercial products does not constitute endorsement or recommendation for use.

The results of data analyses by computer programs described in the section on

data analysis were verified using data commonly obtained from effluent

toxicity tests. However, these computer programs may not be applicable to all

data, and the USEPA assumes no responsibility for their use.

i i
FOREWORD

Environmental measurements are required to determine the quality of ambient

waters and the character of waste effluents. The Environmental Monitoring

Systems Laboratory-Cincinnati (EMSL-Cincinnati) conducts research to:

• Develop and evaluate analytical methods to identify and measure the

concentration of chemical pollutants in drinking waters, surface waters,

groundwaters, wastewaters, terrestrial ecosystems, sediments, sludges,

and solid wastes.

• Investigate methods for the identification and measurement of viruses,

bacteria and other microbiological organisms in drinking waters, surface

waters, groundwaters, wastewaters, terrestrial ecosystems, sediments,

sludges, and solid wastes to determine the response of aquatic

organisms.

• Develop and operate a quality assurance program to support the

achievement of data quality objectives in measurements of pollutants in

drinking water, surface water, groundwater, wastewater, terrestrial

ecosystems, sediment, sludges, and solid waste.

• Develop methods and models to detect and quantify exposures and

responses in aquatic and terrestrial organisms to environmental

stressors and to correlate the exposure with responses on chemical and

biological indicators.

The Federal Water Pollution Control Act Amendments of 1972 (PL 92-500), the

Clean Water Act (CWA) of 1977 (PL 95-217), and the Water Quality Act of 1987

(PL 100-4) explicitly state that it is the national policy that the discharge

of toxic substances in toxic amounts be prohibited. The detection of

chronically toxic effects, therefore, plays an important role in identifying

and controlling toxic discharges to surface waters. This manual is a fourth

edition of the acute toxicity test manual for effluents first published

(EPA/600/4-78/012) by EMSL-Cincinnati in 1978. It provides updated methods

for estimating the acute toxicity of effluents and receiving waters to

freshwater organisms for use by the U.S. Environmental Protection Agency

(USEPA) regional and state programs, and National Pollutant Discharge

Elimination System (NPDES) permittees.

Thomas A. Clark, Director

Environmental Monitoring Systems

Laboratory-Cincinnati

iii
This manual represents the fourth edition of the Agency's general purpose effluent acute toxicity test manual initially published by EMSL-Cincinnati in

January, 1978. This edition reflects changes recommended by the Toxicity

Assessment Subcommittee of EPA's Office of Research and Development Biological Advisory Committee, USEPA headquarters program offices and regional staff,

other Federal agencies, protection groups, trade associations, major industries, consulting firms, academic institutions engaged in aquatic toxicology research, and other interested parties in the private sector.

The membership of the Toxicity Assessment Subcommittee, EPA's Biological

Advisory Committee is as follows:

William Peltier, Subcommittee Chairman,

Environmental Services Division, Region 4

Peter Nolan, Environmental Services Division, Region 1

Steve Ward, Environmental Services Division, Region 2

Ronald Preston, Environmental Services Division, Region 3

Charles Steiner, Environmental Services Division, Region 5

Evan Hornig, Environmental Services Division, Region 6

Terry Hollister, Environmental Services Division, Region 6

Michael Tucker, Environmental Services Division, Region 7

Loys Parrish, Environmental Services Division, Region 8

Peter Husby, Environmental Services Division, Region 9

Joseph Cummins, Environmental Services Division, Region 10

Bruce Binkley, National Enforcement Investigations Center, Denver

Wesley Kinney, Environmental Monitoring Systems Laboratory - Las Vegas

George Morrison, Environmental Research Laboratory - Narragansett Douglas Middaugh, Environmental Research Laboratory - Gulf Breeze

Teresa Norberg-King, Environmental Research Laboratory - Duluth

Donald J. Klemm, Environmental Monitoring Systems Laboratory - Cincinnati

James M. Lazorchak, Environmental Monitoring Systems Laboratory - Cincinnati

Philip Lewis, Environmental Monitoring Systems Laboratory - Cincinnati

Cornelius I. Weber, Environmental Monitoring Systems Laboratory - Cincinnati

Richard Swartz, Environmental Research Laboratory - Newport

Margarete Heber, Health and Ecological Criteria Division, Office of Science

and Technology (OST), Office of Water (OW)

Chris Zarba, Health and Ecological Criteria Division, OST, OW

Bruce Newton, Assessment and Watershed Protection Division, Office

of Wetlands, Oceans, and Watersheds, OW

Dan Rieder, Hazard Evaluation Division, Office of Pesticide Programs

Jerry Smrchek, Health and Environmental Review Division, Office of

Toxic Substances

Gail Hansen, Office of Solid Waste

Royal Nadeau, Emergency Response Team, Edison, NJ

Teresa Norberg-King Chairman, Biological Advisory Committee

iv
ABSTRACT

This manual describes methods for measuring the acute toxicity of effluents

to freshwater, estuarine, and marine macroinvertebrates and fish. The methods

include single and multiple concentration static non-renewal, static-renewal,

and flow-through toxicity tests for effluents and receiving waters. Also

included are guidelines on laboratory safety; quality assurance; facilities

and equipment; test species selection and handling; dilution water; effluent

and receiving water sample collection, preservation, shipping, and holding;

test conditions; toxicity test data analysis; report preparation; organism

culturing; and dilutor and mobile laboratory construction.

v
Foreword

Preface . Abstract

Contents

Figures . Tables .... Acknowledgments

CONTENTS

iii

iv

v

vi

ix

x

xii

1. Introduct ion . 1

2. Types of Tests. 3

3. Health and Sa fety . . 6

General Precautions 6

Safety Equipment ... . . . . . . . . 6

General Laboratory and Field Operations 6

Di sease Prevention 7

Safety Manual s 7

Waste Disposal 7

4. Qual ity Assurance 8

Introduction. ..... . . . . . 8

Facilities, Equipment, and Test Chambers 8

Test Organisms . . . . . . . . . . . . . . . . 8

Laboratory Water Used for Culturing and Test Dilution Water 9

Effluent Sampling and Sample Handling 9

Test Condi t ions . . . . . . . . . . . . . . . 9

Quality of Test Organisms. . . . . . . . . . 9

Food Qual ity . . . . . . . . . . . . . . . . 10

Acceptability of Acute Toxicity Test Results 10

Analytical Methods . . . . . . . . . . . . 11

Calibration and Standardization. . . . . . . 11

Repl ication and Test Sensitivity . . . . . . 11

Variability in Toxicity Test Results . . . . . 11

Demonstrating Acceptable Laboratory Performance 12

Documenting Ongoing Laboratory Performance 12

Reference Toxi cants . . . . . . 18

Record Keep i ng . . . . . . . . . . . . . . 20

5. Facilities and Equipment. . . . . . . . . . . 21

General Requ irements . . . . . . . . . . . . . 21

Cleaning Test Chambers and Laboratory Apparatus . . . . 22

Apparatus and Equipment for Culturing and Toxicity Tests 23

Reagents and Consumable Materials. 24

Test Organisms . . . . 26

6. Test Organisms. . . . . . 27

Test Species . . . . . 27

Sources of Test Organisms 28

LHe Stage . . . . . 29

Laboratory Cul turi ng . . . . . 29

Holding and Handling Test Organisms 29

Transportation to the Test Site 30

vi
CONTENTS (CONTINUED)

Page

Test Organism Disposal 32

7. Dilution Water. . . . . . . . . . . . 33

Types of Dilution Water. . . . . . 33

Standard, Synthetic Dilution Water . . 33

Use of Receiving Water as Dilution Water 35

Use of Tap Water as Dilution Water 38

Dilution Water Holding . . . . . . . . . . . . . . . 39

8. Effluent and Receiving Water Sampling and Sample Handling 40

Effluent Sampling. . . . . . . . 40

Effluent Sample Types. . . . . . . . . . . . . . . . . 40

Effluent Sampling Recommendations. . . . . . . . . . . 41

Receiving Water Sampling . . . . . . . . . . . . . . . 42

Effluent and Receiving Water Sample Handling, Preservation, and

Shipping. . . . . . . . . . . . . . . . . . 43

Sample Receiving . . . . . . . . . . . . . . 44

Persistence of Effluent Toxicity During Sample Shipment and

Holding......................... 44

9. Acute Toxicity Test Procedures . . . . . . . . . . . . . . . . .. 45

Preparation of Effluent and Receiving Water Samples for Toxicity

Tests . . . . . . . . . . . . . . . . . . . 45

Preliminary Toxicity Range-finding Tests . . . . . . . . 47

Multi-Concentration (Definitive) Effluent Toxicity Tests 47

Receiving Water Tests . . 48

Static Tests . . . . . . 48

Fl ow-Through Tests 49

Number of Test Organisms 50

Replicate Test Chambers 50

Loading of Test Organisms 52

Illumination . . . . 52

Feeding . . . . . . . . . 52

Test Temperature . . . . 53

Stress .. . . . . . . . . . 53

Dissolved Oxygen Concentration 53

Test Duration . . . . . . . . . . 56

Acceptability of Test Results. . . . . . . . . . . . . . . 56

Summary of Test Conditions for the Principal Test Organisms 56

10. Test Data . . . . . . . . . . 71

Biological Data. . . . . . 71

Chemical and Physical Data 71

11. Acute Toxicity Data Analysis. . 75

Introduct ion .. . . . . . . . . . . . . . . . . . . . . 75

Determination of the LC50 from Definitive, Multi-EffluentConcentration Acute Toxicity Tests . 76

The Graphical Method . . . . . . . 78

The Spearman-Karber Method . . . . 81

The Trimmed Spearman-Karber Method 83

The Probi t Method 85

vii
CONTENTS (CONTINUED)

219

229

247

264

267

280

280

283

284

284

286

291

292

293

. 91 . . 91

101

106

119

119

119

119

120

120

120

120

120

121

127

133

134

134

151

172

191

200

E.3.

E.4.

E. 5.

B.

C.

D.

Determination of No-Observed-Adverse-Effect Concentration

(NOAEC) from Multi-Concentration Tests, and Determination of

Pass or Fail (Pass/Fail) for Single-Concentration (Paired)

Tests . General Procedure . Single Concentration Test

Multi-Concentration Test

12. Report Preparation . Introduction . Plant Operations . Source of Effluent, Receiving Water, and Dilution Water

Test Conditions . Test Organisms . Quality Assurance . Resul ts . . . . . . . . Conclusions and Recommendations

Cited References . Bibliography . Appendi ces . . . . . . . . . . . . . . . . . . A. Distribution, Life Cycle, Taxonomy, and Culture Methods

A.I. Ceriodaphni a dubi a . . . . . . .

A.2. Daphnia (D. magna and D. pu7ex)

A.3. Mysids (Mysidopsis bahia)

A.4. Brine Shrimp (Artemia sa7ina) . A.5. Fathead Minnow (Pimepha7es prome7as) . . . .. .. A.6. Rainbow Trout, (Oncorhynchus mykiss) and Brook Trout

(Sa7ve7inus fontina7is) . . . . .. . . A.7. Sheepshead Minnow (Cyprinodon variegatus) . A.8. Silversides: Inland Silverside (Mendia bery77ina),

Atlantic Silverside (M. menidia) , and Tidewater

Silverside (M. peninsu7ae) . Supplemental List of Acute Toxicity Test Species Dilutor Systems . Plans for Mobile Toxicity Test Laboratory

0.1. Tandem-Axle Trailer . 0.2. Fifth Wheel Trailer . E. Check Lists and Information Sheets . E.1. Toxicity Test Field Equipment List. . . . . . . .. . .. E.2. Information Check List for On-Site Industrial or Municipal Toxicity Test . Daily Events Log . Oil utor Cali brat i on Form . . . . Daily Dilutor Calibration Check

viii
FIGURES

Number

1. Control (cusum) charts . 19

2. Approximate times required to replace water in test chambers in

flow-through tests . . . . . . . . . . . . . . . . . . . . 51

3. Rawson's nomograph for obtaining oxygen saturation values

in freshwater at different temperatures at sea level 55

4. Example of data sheet for effluent toxicity tests 72

5. Check list on back of effluent toxicity data sheet 73

6. Flowchart for determination of the LC50 for multi-effluentconcentration acute toxicity tests . . . . . . . . . ... 77

7. Plotted data and fitted line for graphical method, using

all-or-nothing data . 80

8. Example of input for computer program for Trimmed Spearman-Karber Method . . . .. . .. .... 86

9. Example of output from computer program for Trimmed Spearman-Karber Method " . . . .. 87

10. Example of input for computer program for Probit Method 89

11. Example of output for computer program for Probit Method 90

12. Flowchart for analysis of single-effluent-concentration test data 93

13. Flowchart for analysis of multi-effluent-concentration test data 94

14. Plot of mean survival proportion data in Table 27

ix

107
14

TABLES

Number Page

1. Intra-laboratory preclslon of LC50s from static acute toxicity

tests with aquatic organisms using reference toxicants. 13

2. Intra- and inter-laboratory precision of acute toxicity

tests with Daphnia magna, using a standard effluent

3. Inter-laboratory precision of acute toxicity tests with aquatic

organisms, using reference toxicants. . . . . . . . . . 15

4. Inter-laboratory study of acute toxicity test precision, 1990:

Summary of responses using KCL as the reference toxicant 16

5. National inter-laboratory study of acute toxicity test

precision, 1991: Summary of responses using reference toxicants 17

6. Preparation of synthetic freshwater using reagent grade chemicals 36

7. Preparation of synthetic freshwater using mineral water 36

8. Preparation of synthetic seawater using reagent grade chemicals 37

9. Percent unionized NH3 in aqueous ammonia solutions:

Temperatures between 15-26°C and pH's 6.0-8.9 ... .... 46

10. Oxygen solubility (mg/L) in water at equilibrium with air at

760 mm Hg . . . . . . . . . . . . . . . . . . . . . . .. . .. 54

11. Summary of test conditions and test acceptability criteria for

Ceriodaphnia dubia acute toxicity tests with effluents

and receiving waters . . . . . . . . . . . . . . . . . .. . .. 57

12. Summary of test conditions and test acceptability criteria for

Daphnia pu7ex and D. magna acute toxicity tests with effluents

and recei vi ng waters . . . . . . . . . . . . . . . . . . . . . . . . 59

13. Summary of test conditions and test acceptability criteria for

fathead minnow, Pimepha7es prome7as, acute toxicity tests with

effluents and receiving waters. . . . . . . . . . . . . . .. .. 61

14. Summary of test conditions and test acceptability criteria for

rainbow trout, Oncorhynchus mykiss, and brook trout, Sa7ve7inus

fontina7is, acute toxicity tests with effluents and receiving

waters 63

15. Summary of test conditions and test acceptability criteria for

mysid, Mysidopsis bahia, acute toxicity tests with effluents and

receiving waters 65

x
TABLES (CONTINUED)

Number

16. Summary of test conditions and test acceptability criteria for

sheepshead minnow, Cyprinodon variegatus, acute toxicity tests with

effluents and receiving waters 67

17. Summary of test conditions and test acceptability criteria for

silverside, Menidia beryl1ina, M. menidia, and M. peninsulae, acute

toxi city tests with effl uents and recei vi ng waters . . . . . . . . . 69

18. Mortality data (number of dead organisms) from acute toxicity tests

used in examples of LC50 determinations (20 organisms in the

control and all test concentrations) . . 79

19, Coefficients for the Shapiro-Wilk's test. . . . 96

20. Quantiles of the Shapiro-Wilk's test statistic 98

21. Critical values for Wilcoxon's rank sum test at the five percent significance level . . . . . . . . . . . . . . . . . . . 101

22. Data from an acute single-concentration toxicity test with

Ceriodaphnia . . . . . . . . . . . . . . . . . . . . . 102

23. Example of Shapiro-Wilk's test: Centered observations 102

24. Example of Shapiro-Wilk's test: Ordered observations. 103

25. Example of Shapiro-Wilk's test: Table of coefficients and

differences . . . . . . . . . . . . . . . . . 104

26. Example of Wilcoxon's rank sum test: Assigning ranks to the

control and 100% effluent concentrations. 105

27. Fathead minnow survival data. . . . . . 108

28. Centered observations for Shapiro-Wilk's example 108

29. Ordered centered observations for the Shapiro-Wilk's example 109

30. Coefficients and differences for the Shapiro-Wilk's example 110

31. ANOVA table

32. ANOVA table for Dunnett's Procedure example

33. Calculated t values

34. Dunnett's !It !I values

xi

112

114

115

117
ACKNOWLEDGMENTS

The principal authors of this document are Cornelius I. Weber, Donald J.

Klemm, Philip A. Lewis, Quentin H. Pickering, Florence Fulk, Mark E. Smith,

and James M. Lazorchak, Environmental Monitoring Systems Laboratory, Cincinnati, OH; Teresa J. Norberg-King, Environmental Research Laboratory, Duluth, MN; George E. Morrison, and David A. Bengtson, Environmental Research

Laboratory, Narragansett, RI; Douglas P. Middaugh, Environmental Research

Laboratory, Gulf Breeze, FL; Margarete A. Heber, Office of Science and

Technology, Office of Water, Washington, D.C.; Stephan H. Ward, Environmental

Sciences Division, Region 2, Edison, NJ; William H. Peltier, Environmental

Services Division, Region 4, Athens, GA; Laura Gast, Technology Applications,

Inc., Cincinnati, OH; and Cathy Poore, Computer Sciences Corporation,

Cincinnati, OH. Contributors to specific sections and appendices of this

manual are listed below.

1. Sections 1-10 and 12: General Guidelines

Margarete A. Heber

James M. Lazorchak

Teresa J. Norberg-King George E. Morrison

William H. Peltier

Cornelius I. Weber

2. Section 11: Data Analysis Florence Fulk

Laura Gast

Cathy Poore

3. Appendices

A.l. Ceriodaphnia dubia - Philip A. Lewis and James M. Lazorchak

A.2. Daphnia (D. magna and D. pulex) - Philip A. Lewis and James

M. Lazorchak

A.3. Mysids (Mysidopsis bahia) - Stephan H. Ward

A.4. Brine Shrimp (Artemia salina) - Philip A. Lewis and David A.

Bengtson

A.5. Fathead Minnow, (Pimephales promelas) - Donald J. Klemm, Quentin

H. Pickering, and Mark E. Smith

A.6. Rainbow Trout, (Oncorhynchus mykiss) and Brook Trout,

(Salvelinius fontinalis) - Donald J. Klemm

A.7. Sheepshead Minnow, (Cyprinodon variegatus) - Donald J. Klemm

A.a. Silversides: Inland Silverside (Mendia beryllina),

Atlantic Silverside (M. menidia) , and Tidewater Silverside

(M. peninsulae) - Douglas P. Middaugh and Donald J. Klemm

B. Supplemental List of Acute Toxicity Test Species - Margarete A.

Heber

C. Diluter Systems - William H. Peltier

D. Plans for Mobile Toxicity Test Laboratory - William H. Peltier

E. Check Lists and Information Sheets - William H. Peltier

xii
Review comments from the following persons are gratefully acknowledged:

Barbara Albrecht, Environmental Research Laboratory, U.S. Environmental

Protection Agency, Gulf Breeze, FL

Robert Burm, Environmental Services Division, U.S. Environmental Protection

Agency, Region 8, Denver, CO

Randy Crawford, Missouri Department of Natural Resources, Jefferson City,

MO

Geri Cripe, Environmental Research Laboratory, U.S. Environmental Protection

Agency, Gulf Breeze, FL

Philip Crocker, Environmental Services Division, U.S. Environmental Protection

Agency, Region 6, Dallas, TX

Joseph Cummins, Environmental Services Division, U.S. Environmental Protection

Agency, Region 10, Seattle, WA

Robert Donaghy, Environmental Services Division, U.S. Environmental Protection

Agency, Region 3, Wheeling, WV

Lee Dunbar, Connecticut Department of Environmental Protection, Hartford, CT

William Gidley, Nebraska Department of Environmental Control, Lincoln, NE

James Green, Environmental Services Division, U.S. Environmental Protection

Agency, Region 3, Wheeling, WV

Steve Haslouer, Kansas Department of Health and Environment, Topeka, KS

Thom Haze, Connecticut Department of Environmental Protection, Hartford, CT

Michael Henebry, Illinois Environmental Protection Agency, Springfield, IL

Terry Hollister, Environmental Services Division, U.S. Environmental

Protection Agency, Region 6, Houston, TX

Jack Kennedy, University of Iowa Hygienic Laboratory, Iowa City, IA

Alfred Korndorfer, New Jersey Department of Environmental Protection,

Trenton, NJ

Robert Masnado, Wisconsin Department of Natural Resources, Madison, WI

Ann McGinley, Texas Water Commission, Austin, TX

Mary Moffett, Environmental Services Division, U.S. Environmental Protection

Agency, Kansas City, KS

Michael Morton, Environmental Services Division, U.S. Environmental Protection

Agency, Region 6, Dallas, TX

Peter Nolan, Environmental Services Division, U.S. Environmental Protection

Agency, Region 1, Lexington, MA

Loys Parrish, Environmental Sciences Division, U.S. Environmental Protection

Agency, Region 8, Denver, CO

Glen Rodriguez, Environmental Services Division, U.S. Environmental Protection

Agency, Region 6, Denver, CO

Janice Smithson, West Virginia Division of Natural Resources, Charleston, WV

Charles Steiner, Environmental Services Division, U.S. Environmental

Protection Agency, Region 5, Chicago, IL

Donald Thurston, New Hampshire Department of Environmental Services, Concord,

NH

Michael Tucker, Environmental Services Division, U.S. Environmental Protection

Agency, Region 7, Kansas City, KS

Bruce Walker, Michigan Department of Natural Resources, Lansing, MI

Audrey Weber, Virginia State Water Quality Control Board, Richmond, VA

Charles Webster, Ohio Environmental Protection Agency, Columbus, OH

xiii
ACKNOWLEDGEMENTS (CONTINUED)

Many, useful public comments on the third edition of the acute toxicity

test methods (EPA/600/4-85/013) were received in response to the proposed

rule, published in the Federal Register, December 4, 1989 [FR 54(231):50216-

50224], regarding the Agency's intent to include the acute toxicity tests in

Table lA, 40 CFR Part 136. These comments were considered in the preparation

of the fourth edition of the manual, and are included in the Public Docket for

the rulemaking, located at room 2904, EPA Headquarters, Washington, D.C.

xiv
SECTION 1

INTRODUCTION

1.1 This manual describes acute toxicity tests for use in the National

Pollutant Discharge Elimination System (NPDES) Permits Program to identify

effluents and receiving waters containing toxic materials in acutely toxic

concentrations. The methods included in this manual are referenced in Table

lA, 40 CFR Part 136 regulations and, therefore, constitute approved methods

for acute toxicity tests. They are also suitable for determining the toxicity

of specific compounds contained in discharges. The tests may be conducted in

a central laboratory or on-site, by the regulatory agency or the permittee.

1.2 The data are used for NPDES permits development and to determine

compliance with permit toxicity limits. Data can also be used to predict

potential acute and chronic toxicity in the receiving water, based on the LC50

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 acute 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, 1988a; USEPA, 1988b; USEPA, 1989a; USEPA, 1989b;

USEPA, 1991a).

1.4 This methods manual serves as a companion to the short-term chronic

toxicity test methods manuals for freshwater and marine organisms (USEPA,

1993a; USEPA, 1993b), the NPDES compliance inspection manual (1988c), and the

manual for evaluation of laboratories performing aquatic toxicity tests

(USEPA, 1991b).

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, 1991c).

1.6 The use of any test species or test conditions other than those described

in Tables 11-17 in this manual and referenced in Table lA, 40 CFR 136.3, shall

be considered a major modification to the method and subject to application

and approval of alternate test procedures under 40 CFR 136.4 and 40 CFR 136.5.

1.7 These methods are restricted to use by, or under the supervision of,

analysts experienced in the use or conduct of, and interpretation of data

from, aquatic toxicity tests. Each analyst must demonstrate the ability to

generate acceptable test results with the methods using the procedures described in this methods manual.

1
1.8 This manual was prepared in the established EMSL-Cincinnati format

(USEPA, 1983a).

2
SECTION 2

TYPES OF TESTS

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 Effluent acute toxicity is generally measured using a multiconcentration, 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 is lethal to

50% of the test organisms (LC50) within the prescribed period of time

(24-96h), or the highest effluent concentration in which survival is not

statistically significantly different from the control.

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%, the effluent concentrations used in the

toxicity test would be 100%, 75%, 50%, 25%, and 12.5%.

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.

2.5 A negative result from an acute toxicity test does not preclude the

presence of chronic 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 acute (or chronic) toxicity.

2.6 The frequency with which acute 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.

2.7 Tests may be static (static non-renewal or static renewal), or flowthrough.

2.7.1 STATIC TESTS

2.7.1.1 Static non-renewal tests - The test organisms are exposed to the same

test solution for the duration of the test.

3
2.7.1.2 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.

2.7.2 FLOW-THROUGH TESTS

2.7.2.1 Two types of flow-through tests are in common use: (1) sample is

pumped continuously from the sampling point directly to the dilutor system; and (2) grab or composite samples are collected periodically, placed in a tank

adjacent to the test laboratory, and pumped continuously from the tank to the

dilutor system. The flow-through method employing continuous sampling is the

preferred method for on-site tests. Because of the large volume (often 400

L/day) of effluent normally required for flow-through tests, it is generally

considered too costly and impractical to conduct these tests off-site at a

central laboratory.

2.8 Advantages and disadvantages of the types of tests are as follows:

2.8.1 STATIC NON-RENEWAL TESTS

2.8.1.1 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 flowthrough tests.

2.8.1.2 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 or flow-through tests,

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.

2.8.2 STATIC-RENEWAL, ACUTE TOXICITY TESTS

2.8.2.1 Advantages:

1. Reduced possibility of dissolved oxygen (DO) depletion from high

chemical oxygen demand (COD) and/or biological oxygen demand (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.

4
3. Test organisms that rapidly deplete energy reserves are fed when the

test solutions are renewed, and are maintained in a healthier state.

2.8.2.2 Disadvantages:

10 Require greater volume of effluent that non-renewal tests.

2. Generally less sensitive than flow-through tests, 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.

2.8.3 FLOW-THROUGH TESTS

2.8.3.1 Advantages:

1. Provide a more representative evaluation of the acute toxicity of the

source, especially if sample is pumped continuously directly from the

source and its toxicity varies with time.

2. DO concentrations are more easily maintained in the test chambers.

3. A higher loading factor (biomass) may be used.

4. The possibility of loss of toxicant due to volatilization, adsorption,

degradation, and uptake is reduced.

2.8.3.2 Disadvantages:

1. Large volumes of sample and dilution water are required.

2. Test equipment is more complex and expensive, and requires more

maintenance and attention.

3. More space is required to conduct tests.

4. Because of the resources required, it would be very difficult to

perform multiple or overlapping sequential tests.

5
SECTION 3

HEALTH AND SAFETY

3.1 GENERAL PRECAUTIONS

3.1.1 Development and maintenance of an effective health and safety program in the laboratory requires an ongoing commitment by laboratory management, and

includes (1) the appointment of a laboratory health and safety officer with

the responsibility and authority to develop and maintain a safety program, (2)

the preparation of a formal, written, health and safety plan, which is

provided to each laboratory staff member, (3) an ongoing training program on

laboratory safety, and (4) regularly scheduled, documented, safety

inspections.

3.1.2 Collection and use of effluents in toxicity tests may involve

significant risks to personal safety and health. Personnel collecting

effluent samples and conducting toxicity tests should take all safety precautions necessary for the prevention of bodily injury and illness which

might result from ingestion or invasion of infectious agents, inhalation or

absorption of corrosive or toxic substances through skin contact, and

asphyxiation due to lack of oxygen or presence of noxious gases.

3.1.3 Prior to sample collection and laboratory work, personnel must

determine that all required 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.

3.2.2 LABORATORY SAFETY EQUIPMENT

3.2.2.1 Each laboratory (including mobile laboratories) must be provided with

safety equipment such as first aid kits, fire extinguishers, fire blankets,

emergency showers, 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 Guidance in Material Safety Data Sheets should be followed for

reagents and other chemicals purchased from supply houses. Incompatible materials should not be stored together.

6
3.3.2 Work with effluents must be performed in compliance with accepted rules

pertaining to the handling of hazardous materials (see Safety Manuals,

Subsection 3.5). Personnel collecting samples and performing toxicity tests

should not work alone.

3.3.3 Because the chemical composition of effluents is usually only poorly known, they must be considered as potential health hazards, and exposure to

them should be minimized. Fume and canopy hoods over the test areas must be

used whenever necessary.

3.3.4 It is advisable to cleanse exposed parts of the body immediately after

collecting effluent samples.

3.3.5 All containers must be adequately labeled to indicate their contents.

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 Good housekeeping contributes to safety and reliable results.

3.3.8 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.

3.3.9 Mobile laboratories must 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 hepatitis B, tetanus, typhoid fever,

and polio.

3.5 SAFETY MANUALS

3.5.1 For further guidance on safe practices when collecting effluent samples and conducting toxicity tests, check with the permittee and consult general industrial safety manuals, including USEPA (1986) 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 testing activities. Local fire officials

should be notified of any potentially hazardous conditions.

7
SECTION 4

QUALITY ASSURANCE

4.1 INTRODUCTION

4.1.1 Development and maintenance of a toxicity test laboratory quality

assurance (QA) program requires an ongoing commitment by laboratory

management, and includes the following: (1) appointment of a laboratory quality assurance officer with the responsibility and authority to develop and

maintain a QA program; (2) preparation of a quality assurance plan with data

quality objectives; (3) preparation of written descriptions of laboratory

standard operating procedures (SOP's) for test organism culturing, toxicity testing, instrument calibration, sample chain-of-custody, laboratory sample tracking system, etc.; and (4) provision of adequate, qualified technical

staff and suitable space and equipment to assure reliable data.

4.1.2 QA practices within an aquatic toxicology laboratory 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 and operation 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 related to toxicity testing,

see: FDA, 1978; USEPA, 1975; USEPA, 1979a; USEPA, 1980a; USEPA, 1980b; USEPA,

1991b; DeWoskin, 1984; and Taylor, 1987.

4.1.4 Guidance for the evaluation of laboratories performing toxicity tests

and laboratory evaluation criteria may be found in USEPA, 1991b.

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 toxicity testing areas, and from

toxicity test laboratories 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 and Equipment).

d... _ • < T~~T _.." nDr.AN1~M~ "'.,,.,............ ...,11...,

4.3.1 The test organisms used in the procedures described in this manual are

listed in Section 6, Test Organisms. The organisms should appear healthy,

8
behave normally, feed well, and have low mortality in cultures, during

holding, and in test controls. Test organisms should be positively identified

to species.

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 in toxicity tests is extremely important. Water for these two uses

should come from the same source. The dilution water used in effluent

toxicity tests will depend in part on the objectives of the study and

logistical constraints, as discussed in detail in Section 7, Dilution Water.

For tests performed to meet NPDES objectives, synthetic, moderately hard water

should be used. The dilution water used for internal quality assurance tests

with organisms, food, and reference toxicants should be the water routinely

used with success in the laboratory. Types of water are discussed in

Section 5, Facilities and Supplies. Water used for culturing and test

dilution should be analyzed at least annually or whenever difficulty is

encountered in meeting minimum acceptability criteria for control survival and

reproduction or growth for toxic metals and organics. The concentration of

the metals, Al, As, Cr, Co, Cu, Fe, Pb, Ni, Zn, expressed as total metal,

should not exceed 1 ~g/L each, and Cd, Hg, and Ag, expressed as total metal,

should not exceed 100 ng/L each. Total organochlorine pesticides plus PCBs

should be less than 50 ng/L (APHA 1992). Individual pesticide concentrations

should not exceed EPA's Ambient ,Water Quality chronic criteria values where

available.

4.5 EFFLUENT SAMPLING AND SAMPLE HANDLING

4.5.1 Sample holding times and temperatures must conform to conditions

described in Section 8, Effluent Sampling and Sample Handling.

4.6 TEST CONDITIONS

4.6.1 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 must be maintained within the limits specified for each

test. DO concentration and pH in test chambers should be checked daily

throughout the test period, as prescribed in Section 9, Acute Toxicity Test

Procedures.

4.7 QUALITY OF TEST ORGANISMS

4.7.1 Where acute or short-term chronic toxicity tests are performed with

effluents or receiving waters using test organisms obtained from outside the

test laboratory, concurrent toxicity tests of the same type must be performed

with a reference toxicant, unless the test organism supplier provides control

chart data from at least the last five monthly acute toxicity tests using the

same reference toxicity and test conditions.

9
4.7.2 The supplier should also 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.

4.7.3 If the laboratory performing toxicity tests maintains its own stock

cultures, the sensitivity of the offspring should be determined in a toxicity

test performed with a reference toxicant at least once each month (see

Subsection 4.15). If preferred, this reference toxicant test may be performed

concurrently with each effluent toxicity test. However, if a given species of

test organism produced by inhouse cultures is used only monthly, or less

frequently, in effluent toxicity tests, a reference toxicant test must be

performed concurrently with the effluent toxicity test.

4.7.4 If a routine reference toxicant test fails to meet acceptability

criteria, the test must be immediately repeated. If the failed reference

toxicant test was being performed concurrently with an effluent or receiving

water toxicity test, both tests must be repeated (for exception, see

Subsection 4.16.5).

4.8 FOOD QUALITY

4.8.1 The nutritional quality of the food used in culturing and testing fish

and invertebrates is an important factor in the quality of the toxicity test

data. This is especially true for the unsaturated fatty acid content of brine

shrimp nauplii, Artemia. Suitable trout chow, Artemia, and other foods must

be obtained as described in this manual.

4.8.2 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. 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 effect 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 acute toxicity tests can be compared with a food of known quality

in side-by-side, multi-concentration acute 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 also 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 organic chlorine

exceeds 0.15 ~g/g wet weight, or the total concentration of organochlorine

pesticides plus PCBs exceeds 0.30 ~g/g wet weight, or toxic metals exceed 20 ~g/g wet weight, the food should not be used (for analytical methods see AOAC,

1990; USDA, 1989).

4.9 ACCEPTABILITY OF ACUTE TOXICITY TEST RESULTS

4.9.1 For the test results to be acceptable, control survival must equal or

exceed 90%.

10
4.9.2 An individual test may be conditionally acceptable if temperature, DO,

and other specified conditions fall outside specifications, depending on the

degree of the departure and the objectives of the tests (see test condition

summaries). The acceptability of the test will depend on the experience and

professional judgment of the laboratory analyst and the reviewing staff of the

regulatory authority. Any deviation from test specifications must be noted

when reporting data from a test.

4.10 ANALYTICAL METHODS

4.10.1 Routine chemical and physical analyses for culture and dilution water,

food, and test solutions must include established quality assurance practices

outlined in Agency methods manuals (USEPA, 1979a; USEPA, 1993c).

4.10.2 Reagent containers should be dated 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, salinity, alkalinity,

and hardness must be calibrated and standardized prior to use each day according to the instrument manufacturer's procedures as indicated in the

general section on quality assurance (see EPA Methods 150.1, 360.1, 170.1, and

120.1; USEPA, 1979b). Calibration data are recorded in a permanent log.

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 EPA methods (see EPA Methods 130.2 and 310.1;

USEPA 1979b).

4.12 REPLICATION AND TEST SENSITIVITY

4.12.1 The sensitivity of toxicity 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 preC1Slon include: the

experience and skill of the laboratory analyst; test organism age, condition,

and sensitivity; dilution water quality; temperature control; 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

11
sample size (numbers of fish) and the standard error of the test, and

considered 20 fish per concentration as optimum for Probit Analysis.

4.13.2 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. The single-laboratory (intra-laboratory) and

multi-laboratory (inter-laboratory) precision of acute toxicity tests with

several common test species and reference toxicants are listed in Tables 1-4.

Intra- and inter-laboratory precision are described by the mean, standard

deviation, and relative standard deviation (percent coefficient of variation,

or CV) of the calculated endpoints from the replicated tests.

4.13.3 Intra-laboratory precision data from 268 acute toxicity tests with

four species and five reference toxicants are listed in Tables 1 and 2. The

precision, expressed as CV%, ranged from 3% to 86%. More recent CV values

reported by Jop et al. (1986), Dorn and Rogers (1989), Hall et al. (1989), and

Cowgill et al. (1990), fell in a somewhat lower range (8% to 41%).

4.13.4 Inter-laboratory precision of acute toxicity tests from 253 reference

toxicant tests with seven species, listed in Tables 2, 3, 4, and 5 (expressed

as CV%), ranged from 11% to 167%.

4.13.5 No clear pattern of differences were noted in the intra- or interlaboratory test precision with the species listed, although the test results

with some toxicants, such as cadmium, appear to be more variable than those

with other reference toxicants.

4.13.6 Additional information on toxicity test preclslon is provided in the

Technical Support Document for Water Quality-Based Toxics Control (see pp.

2-4, and 11-15; USEPA, 1991c).

4.14 DEMONSTRATING ACCEPTABLE LABORATORY PERFORMANCE

4.14.1 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 intra-laboratory 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 the same data analysis

methods. A reference toxicant concentration series (0.5 or higher) should be

selected that will consistently provide partial mortalities at two or more

concentrations.

4.15 DOCUMENTING ONGOING LABORATORY PERFORMANCE

4.15.1 Satisfactory laboratory performance is demonstrated by performing at

least one acceptable test per month with a reference toxicant for each

toxicity test method commonly used in the laboratory. For a given test

method, successive tests must be performed with the same reference toxicant,

12
TABLE 1. INTRA-LABORATORY PRECISION OF LC50S FROM STATIC ACUTE

TOXICITY TESTS WITH AQUATIC ORGANISMS USING REFERENCE

TOXICANTS

TEST ORGANISM

Pimephales promelas

Daphnia magna

Daphnia magna

Daphnia magna

Daphnia magna

Daphnia pulex

Daphnia pulex

Daphnia pulex

Daphnia pulex

Mysidopsis bahia

(96 h,

(24 h,

(24 h,

(48 h,

(48 h,

(24 h,

(24 h,

(48 h,

(48 h,

(96 h,

21'C)3

20'C)4

26'C)4

20'C)4

26'C)4

20'C)4

26'C)4

20'C)4

26' C)4

2S' C)5

REFERENCE TOXICANT2

SOS NAPCP CO

N LC50 CV(%) N LCSO CV(%) N LCSO CV(%)

9 8.6 20 12 0.14 40 9 0.15 120

8 20.9 28 10 0.69 14 11 0.121 49

10 12.9 48 9 0.67 25 9 0.026 77

10 13.5 29 10 0.42 21 9 0.038 58

9 10.8 33 9 0.48 23 8 0.009 35

9 18.4 23 9 0.64 15 5 0.147 30

10 13.9 25 9 0.62 2S 10 0.063 4S

10 12.6 32 9 0.48 16 10 0.042 45

9 10.2 36 8 0.47 32 6 0.006 14

13 0.346 9

'Precision expressed as percent coefficient of variation, where

CV% =(standard deviation X 100)/mean.

2SDS =Sodium dodecyl (lauryl) sulfate; NAPCP =Sodium pentachlorophenate; CD =Cadmium;

N = Number of tests; toxicant concentration in mg/L.

3Pimephales promelas tests were performed in soft, synthetic freshwater; total hardness,

40·48 mg/L as CaCOg , by J. Dryer, Aquatic Biology Section, EMSL-Cincinnati.

4Daphnia data from Lewis and Horning, 1991. Tests with Q. magna used hard reconstituted

water (total hardness, 180-200 mg/L as CaCOg ); tests with Q. pulex used moderately-hard

reconstituted water (total hardness, 80-100 mg/L as CaCOg ).

5Mys id tests were performed in 25 ppt salinity, natural seawater. Data were provided by

Steve Ward, Environmental Services Division, U.S. Environmental Protection Agency,

Edison, New Jersey. Personal communication, November 14, 1990.

13
TABLE 2. INTRA- AND INTER-LABORATORY PRECISION OF ACUTE

TOXICITY TESTS WIT~ DAPHNIA MAGNA, USING A

STANDARD EFFLUENT'

INTER· LABORATORY PRECISION: INTRA-LABORATORY

LABORATORY LC50s FROM REPLICATE TESTS PRECI SIONa

24 H 48 H

INDUSTRY

14.4 4.2

11.4 4.9

2 13.9 6.8

16.6 6.1

13.7 6.1 6.4

3 11.7 3.5

17.4 7.1

GOVERNMENT

14.0 4.4

10.0 4.4

10.8 4.1 4.0

2 13.2 4.5

14.1 4.5

3 11.6 4.2

COMMERCIAL

20.1 4.9

20.1 4.7

2 8.9 3.7

12.3 5.6

3 14.8 9.0

25.4 9.1

26.4 8.6 3.0

N 20 20 3

MEAN 15.0 5.52 4.47

SO 4.75 1. 75 1. 75

CV% 31.6 31.6 39.1

'From Table 2, p. 191, Grothe and Kimerle, 1985. Tests performed at

20·C! 2·C; dilution water hardness, 100 mg/L as CaCOa; dilution water

alkalinity, 76 mg/L as CaCOa; effluent hardness, approx. 1000 mg/L as

CaCOa; effluent alkalinity, 310 mg/L as CaCOa; effluent dilutions -

56%,32%, 18%, 10%,5.6%,3.1%, 1.7"1..

2LC50 expressed in percent effluent.

alntra-laboratory precision expressed as the weighted mean CV(%).

14
TABLE 3. INTER-LABORATORY PRECISION OF ACUTE TOXICITY

TESTS WITH AQUATIC ORGANISMS, USING REFERENCE

TOXICANTS1

REFERENCE TOXICANT

SILVER ENDOSULFAN

TEST ORGANISM N LC50 CV(%)2 N LC50 CV(%)

1. Pimephales promelas (96 h, 22·C)

96-h static test (Meas) 10 14.0 53 12 2.03 38

96-h flow-through test (Meas) 9 7.49 40 12 0.96 46

2. Oncorhyncus mykiss (96 h, 12·C)

96-h static test (Meas) 10 34.5 88 12 1.15 50

96-h flow-through test (Meas) 9 11.5 33 12 0.40 42

3. Daphnia magna (48 h, 20·C)

48-h static (Meas) 12 10.6 166 11 328 51

4. Mysidopsis bahia (96 h, 22·C)

96-h static test (Nom) 6 210 27 5 0.84 62

96-h flow-through test (Nom) 6 251 22 6 1.02 58

96-h flow-through test (Meas) 6 192 58 5 0.94 167

5. Cyprinodon variegatus (96 h, 22·C)

96-h static test (Nom) 4 1122 35 6 2.41 37

96-h flow-through test (Nom) 5 1573 50 6 1.69 46

96-h flow-through test (Meas) 5 1216 50 6 0.81 46

'Data for Pimephales promelas (fathead minnow), Oncorhyncus mykiss

(rainbow trout), and Daphnia magna were taken from USEPA, 1983b.

Data for, Mysidopsis bahia, and Cyprinodon variegatus (sheepshead

minnow) were taken from USEPA, 1981. Six laboratories participated

in each study. Test salinity was 2~_

LC50s expressed in ~g/L.

In the studies with the freshwater organisms, the water hardness for

five of the six laboratories ranged between 36 and 75 mg/L. However,

the water hardness for the sixth laboratory was 255 mg/L, resulting in

LC50 values for silver more than an order of magnitude larger than for

the other five. These values were rejected in calculating the CV%.

The mean weights of test fish were from 0.05-0.26 g for fathead minnows,

and 0.22-1.32 g for rainbow trout. Daphnia were 24-h old.

In studies with the marine organisms, only one LC50 (presumably the

combined LC50 from duplicate tests) was reported for each toxicity

test. LC50s for flow-through tests with Mysidopsis bahia and Cyprinodon

variegatus were calculated two different ways -- (1) on the basis of

the nominal toxicant concentrations (Nom), and (2) on the basis of

measured (Meas) toxicant concentrations. Test organism age was < 2 days

for Mysidopsis bahia, and 28 days for Cyprinodon variegatus. The

salinity of test solutions was 2~.

N, the total number of LC50 values used in calculating the CV(%) varied

with organism and toxicant because some data were rejected due to

water hardness, lack of concentration measurements, and/or because some of the LC50s were not calculable.

2CV% = Percent coefficient of variation = (standard deviation x 100)/mean.

15
TABLE 4. INTER-LABORATORY STUDY OF ACUTE TOXICITY TEST PRECISION, 1990:

SUMMARY OF RESPONSES USING KCL AS THE REFERENCE TOXICANT'

TEST PRECISION (CV%)2__

NO. LABS GRAPH] STAT'

SUBMITTING METHOD METHOD TOTALs__

TEST TYPE VALID DATA N LCSO CV% N LCSO CV% N LCSO CV%

Pimephales promelas (96 h, 22°C)6 17 6 944 28.8 13 832 27.8 17 864 29.6

Pimephales promelas (24 h, 25°C)' 6 6 832 11.5 6 832 11.5

Ceriodaphnia dubia (48 h, 25°C)' 11 11 256 53.1 11 264 48.5

Mysidopsis bahia (96 h, 22°C)8 14 7 292 32.9 11 250 36.0 14 268 37.3

Interlaboratory study of toxicity test precision conducted in 1990 by the Environmental

Monitoring Systems Laboratory-Cincinnati, U.S. Environmental Protection Agency, Cincinnati, Ohio

45268, in cooperation with the states of New Jersey and North Carolina, and the Office of Water

Enforcement and Permits, U.S. Environmental Protection Agency, Washington, DC.

CV% Percent coefficient of variation = (standard deviation X 100)/mean. Calculated for LCSO from

acute tests. LCSOs expressed as mg/L KCl added to the dilution water.

LCSO estimated by the Graphical Method.

LCSO estimated by Probit, Litchfield-Wilcoxon, or Trimmed Spearman-Karber method.

LCSO usually reported for only one method of analysis for each test. Where more than one LCSO was reported for a test, the lowest value was used to calculate the statistics for "Total."

Data from the New Jersey Department of Environmental Protection: static daily-renewal tests,

using moderately-hard synthetic freshwater.

Data from North Carolina certified laboratories: static non-renewal tests, using moderately-hard

reconstituted freshwater.

Data from the New Jersey Department of Environmental Protection: static daily-renewal tests,

using 25 ppt salinity, FORTY FATHOMS® synthetic seawater.

16
TABLE 5. NATIONAL INTER-LABORATORY STUDY OF ACUTE TOXICITY

TEST PRECISION, 199f: SUMMARY OF RESPONSES USING

REFERENCE TOXICANTS

TEST TYPE

Pimephales promelas (48 h, 25· C)3

Ceriodaphnia dubia (48 h, 25·C)3

Mysidopsis bahia (48 h, 25' C)6

Menidia beryllina (48 h, 25' C)5

NO. LABS

SUBMITTING

DATA LC50 CV%2

203 8964 28.6

171 4324 39.8

61 5324 30.1

39 1646 42.2

'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. LC50s were estimated by the graphical or Trimmed Spearman-Karber method.

2Percent coefficient of variation =(standard deviation X 100)/mean.

3Stat ic non-renewal tests, using moderately-hard synthetic freshwater

(total hardness =80-100 mg/L as CaC~).

4Expressed as mg KCI added per liter of dilution water.

5Stat ic non-renewal tests, using 30 ppt modified GP2 artificial seawater.

6Expressed as ~g Cu" added per liter of dilution water.

17
at the same concentrations, in the same dilution water, using the same data

analysis methods. Precision may vary with the test species, reference

toxicant, and type of test.

4.15.2 A control chart should be prepared for each combination of reference

toxicant, test species, test condition, and endpoint. Toxicity endpoints from

five or six tests are adequate for establishing the control charts. In this

technique, a running plot is maintained for the toxicity values (Xi) from

successive tests with a given reference toxicant (Figure 1), and endpoints

(LC50s) are examined to determine if they are within prescribed limits. The

types of control charts illustrated (see USEPA, 1979a) are used to evQluate

the cumulative trend of results from a series of samples. The mean (X) and

upper and lower control limits (± 2S) are recalculated with each successive

test result. After two years of data collection, or a minimum of 20 data

points, the control (cusum) chart should be maintained using only the 20 most

recent data points.

4.15.3 The outliers, which are values falling outside the upper and lower

control limits, and trends of increasing or decreasing sensitivity, are

readily identified. At the Po 05 probability level, one in 20 tests would be

expected to fall outside of the control limits by chance alone. If more than

one out of 20 reference toxicant tests fall outside the control limits, the

effluent toxicity tests conducted during the month in which the second

reference toxicant test failed are suspect, and should be considered as

provisional and subject to careful review.

4.15.4 If the toxicity value from a given test with the reference toxicant

falls well outside the expected range for the test organisms when using the

standard dilution water, the sensitivity of the organisms and the overall

credibility of the test system are suspect. In this case, the test procedure

should be examined for defects and should be repeated with a different batch

of test organisms.

4.15.5 Performance should improve with experience, and the control limits for

point estimates should gradually narrow. However, control limits of ± 2S, by

definition, will be exceeded 5% of the time, regardless of how well a

laboratory performs. Highly proficient laboratories which develop very narrow

control limits may be unfairly penalized if a test which falls just outside

the control limits is rejected de facto. For this reason, the width of the

control limits should be considered by the permitting authority in determining whether or not an outlier is to be rejected.

4.16 REFERENCE TOXICANTS

4.16.1 Reference toxicants such as sodium chloride (NaCl), potassium chloride

(KC1), cadmium chloride (Cdel ), copper sulfate (CuS04), sodium dodecyl

sulfate (SOS), and potassium aichromate (KZCrZ07), are suitable for use in the

NPOES and other Agency programs requiring aquatic toxicity tests. EMSLCincinnati hopes to release EPA-certified solutions of cadmium and copper,

with accompanying toxicity data for the recommended test species, for use as

reference toxicants through cooperative research and development agreements

with commercial suppliers, and will continue to develop additional reference

18
A UPPER CONTROL LIMIT (X + 28)

-----------------

CENTRAL TENDENCY gl------------

LOWER CONTROL LIMIT (X-2S)

7 2 4 I I 101214111120 22 24 21 21 30 32 34 3138 41142 44 41 48 !SO

3.0_~ I,· ·'1.':vJv..;;;·.. ···V., Y.";;1

2.0 V" . . g -..,r- "

o 5 10 15 20

TOXICITY TEST WITH REFERENCE TOXICANTS

B 12 .......---------------,

c

1.5l..1......;~"7--:-:':~-:':-::~:-=-~~-=-':!::'":t~-=-~=-' 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

CUMULATIVE TEST NUMBER ----- -----

Figure 1. Control (cusum) charts: A, General case; Band C, 48-h acute tests

with sodium chloride. (B) Fathead minnow (Pimephales promelas) , and

(C) Ceriodaphnia dubia, with the individual LC50s (Triangles), cumulative LC50 means (dotted 1ine), and upper and lower control

limits of two standard deviations (squares). (Provided by the

Environmental Services Division, U.S. Environmental Protection Agency, Kansas City, KS).

19
toxicants for future release. Interested parties can determine the

ava il abil ity of II EPA Certifi ed II reference toxi cants by checki ng the EPACincinnati electronic bulletin board, using a modem to access the following

telephone number: 513-569-7610. Standard reference materials also can be

obtained from commercial supply houses, or can be prepared inhouse using

reagent grade chemicals. The regulatory agency should be consulted before

reference toxicant(s) are selected and used.

4.17 RECORD KEEPING

4.17.1 Proper record keeping is important. A complete file should be

maintained for each individual toxicity test or group of tests on closely

related samples. This file should contain a record of the sample chain-ofcustody; 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.

4.17.2 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)].

20
SECTION 5

FACILITIES AND EQUIPMENT

5.1 GENERAL REQUIREMENTS

5.1.1 Effluent toxicity tests may be performed in a fixed or mobile

laboratory. Facilities should include equipment for rearing and/or holding organisms.

5.1.2 The facilities must be well ventilated and free of toxic fumes. Sample preparation, culturing, and toxicity testing areas should be separated to

avoid cross contamination of cultures or toxicity test solutions with toxic

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. 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.

5.1.3 Control of test solution temperature can best be achieved using circulating water baths, heat exchangers, or environmental chambers.

Photoperiod can be controlled using automatic timers in the laboratory or

environmental chambers.

5.1.4 Water used for rearing, holding, and testing organisms may be

reconstituted synthetic water, ground water, surface water, or dechlorinated

tap water. Dechlorination can be accomplished by carbon filtration,

laboratory water conditioning units, or the use of sodium thiosulfate. After

dechlorination, total residual chlorine should be non-detectable. Sodium

thiosulfate may be toxic to the test organisms, and if used for

dechlorination, paired controls with and without sodium thiosulfate should be

incorporated in effluent toxicity tests. Use of 3.6 mg (anhydrous) sodium

thiosulfate/L will reduce 1.0 mg chlorine/L. After dechlorination, total

residual chlorine should be non-detectable.

5.1.4.1 A deioniZing system providing 18 mega-ohm, laboratory grade water

should be provided with sufficient capacity for laboratory needs. 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

CULLIGEN®, CONTINENTAL®, or equivalent, mixed-bed water treatment system.

5.1.5 Air used for aeration must be free of oil and fumes. Oil-free air pumps should be used where possible. Particulates can be removed from the air using

BALSTON® Grade BX or equivalent filters (Balston, Inc., Lexington, MA), and

oil and other organic vapors can be removed using activated carbon filters

(BALSTON®, C-l filter, or equivalent).

21
5.1.6 During rearing, holding, and testing, test organisms should be shielded

from external disturbances such as rapidly changing light conditions

(especially salmonids) and pedestrian traffic.

5.1.7 Materials used for exposure chambers, tubing, etc., that 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, and may be

reused after cleaning. Containers made of plastics, such as polyethylene, polypropylene, polyvinyl chloride, TYGON®, etc., may be used to ship, store,

and transfer effluents and receiving waters, but they should not be reused

unless absolutely necessary, because they could carryover adsorbed toxicants

from one test to another. 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.

5.1.8 New plastic products should be tested for toxicity before general use

by exposing organisms to them under ordinary test conditions.

5.1.9 Equipment which cannot be discarded after each use because of cost,

must be decontaminated according to the cleaning procedures listed below.

Fiberglass, in addition to the previously mentioned materials, can be used for

holding and dilution water storage tanks, and in the water delivery system. All material should be flushed or rinsed thoroughly with dilution water before

using in the test.

5.1.10 Copper, galvanized material, rubber, brass, and lead must not come in

contact with holding or dilution water, or with effluent samples and test

solutions. Some materials, such as neoprene rubber (commonly used for

stoppers), may be toxic and should be tested before use.

5.1.11 Silicone adhesive used to construct glass test chambers absorbs some

organochlorine and organophosphorus pesticides, which are difficult to remove.

Therefore, as little of the adhesive as possible should be in contact with

water. Extra beads of adhesive inside the containers should be removed.

5.2 CLEANING TEST CHAMBERS AND LABORATORY APPARATUS

5.2.1 New plasticware used for effluent or dilution water collection or

organism test chambers does not require thorough cleaning before use. It is

sufficient to rinse new sample containers once with sample dilution water

before use. New glassware must be soaked overnight in 10% acid (see below)

and rinsed well in deionized water and dilution water.

5.2.2 All non-disposable sample containers, test vessels, tanks, and other

equipment that has come in contact with effluent must be washed after use in

the manner described below to remove surface contaminants as described below:

1. Soak 15 min in tap water, and scrub with detergent, or clean in an

automatic dishwasher.

22
2. Rinse twice with tap water.

3. Carefully rinse once with fresh, dilute (10%, V:V) hydrochloric 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.

5.2.3 All test chambers and equipment should be thoroughly rinsed with the

dilution water immediately prior to use in each test.

5.3 APPARATUS AND EQUIPMENT FOR CULTURING AND TOXICITY TESTS

5.3.1 Culture units -- see Appendix. It is preferable to obtain test

organisms from in-house culture units. If it is not feasible to maintain

cultures in-house, test organisms can be obtained from commercial sources, and

should be shipped to the laboratory in well oxygenated water in insulated

containers to minimize excursions in water temperature during shipment. The

temperature of the water in the shipping containers should be measured on

arrival, to determine if the organisms were subjected to obvious undue thermal

stress.

5.3.2 Samplers -- automatic samplers, preferably with sample cooling capability, that can collect a 24-h composite sample of 2 L or more.

5.3.3 Sample containers -- for sample shipment and storage (see Section 8,

Effluent and Receiving Water Sampling and Sample Handling).

5.3.4 Environmental chamber or equivalent facility with temperature control

(20°C or 25°C)

5.3.5 Water purification system -- MILLIPORE® MILLI-Q®, MILLIPORE® QPAKm

2 , or

equivalent. Depending on the quantity of high grade water needed, a firststage pre-conditioner deionizer, such as a CULLIGEN® or CONTINENTAL® System,

or equivalent, may be needed to provide feed water to the high-purity system.

5.3.6 Balance -- analytical, capable of accurately weighing to 0.0001 g.

5.3.7 Reference weights, Class S -- for documenting the performance of the

analytical balance(s). The balance(s) should be checked with reference

weights which are at the upper and lower ends of the range of the weighings

made when the balance is used. Abalance should be checked at the beginning of each series of weighings, periodically (such as every tenth weight) during

a long series of weighings, and after the last weight of a series is taken.

5.3.8 Test chambers -- borosilicate glass or non-toxic disposable plastic

test chambers are suitable. Test chamber volumes are indicated in the method

summaries. 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 (1/4 in) thick.

23
5.3.9 Volumetric flasks and graduated cylinders -- Class A, borosilicate

glass or non-toxic plastic labware, 10-1000 mL for making test solutions.

5.3.10 Volumetric pipets -- Class A, 1-100 mL.

5.3.11 Serological pipets -- 1-10 mL, graduated.

5.3.12 Pipet bulbs and fillers -- PROPIPET®, or equivalent.

5.3.13 Droppers, and glass tubing with fire polished edges, 4 mm ID -- for

transferring test organisms.

5.3.14 Wash bottles -- for rinsing small glassware and instrument electrodes

and probes.

5.3.15 Glass or electronic thermometers -- for measuring water temperature.

5.3.16 Bulb-thermograph or electronic-chart type thermometers -- for

continuously recording temperature.

5.3.17 National Bureau of Standards Certified thermometer (see USEPA Method

170.1; USEPA 1979b).

5.3.18 pH, DO, and specific conductivity meters -- for routine physical and

chemical measurements. Unless the test is being conducted to specifically

measure the effect of one of the above parameters, a portable, field-grade

instrument is acceptable.

5.3.19 Refractometer -- for measuring effluent, receiving, and test solution

salinity.

5.3.20 Amperometric titrator -- for measuring total residual chlorine.

5.4 REAGENTS AND CONSUMABLE MATERIALS

5.4.1 Reagent water -- defined as MILLIPORE® MILLI-Q®, MILLIPORE® QPAKm

2 or

equivalent water (see Subsection 5.3.5 above).

5.4.2 Effluent, dilution water, and receiving water -- see Section 7,

Dilution Water, and Section 8, Effluent and Receiving Water Sampling and

Sample Handling.

5.4.3 Reagents for hardness and alkalinity tests (see USEPA Methods 130.2 and

310.1; USEPA 1979b).

5.4.4 Standard pH buffers 4, 7, and 10 (or as per instructions of instrument

manufacturer) for instrument calibration (see USEPA Method 150.1; USEPA

1979b).

5.4.5 Specific conductivity and salinity standards (see USEPA Method 120.1;

USEPA 1979b).

24
5.4.6 laboratory quality control check samples and standards for the above

chemistry methods.

504,7 Reference toxicant solutions (see Section 4, Quality Assurance).

5.4.8 Membranes and filling solutions for dissolved oxygen probe (see USEPA

Method 360.1; USEPA 1979b), or reagents for modified Winkler analysis.

5.4.9 Sources of Food for Cultures and Toxicity Tests.

5.4.9.1 All food should be tested for nutritional sUitability, and chemically

analyzed for organic chlorine, PCBs, and toxic metals (see Section 4, Quality

Assurance).

5.4.9.2 Brine Shrimp (Artemia) -- see Appendix A.

1, Brine Shrimp (Artemia) Cysts.

There are many commercial sources of brine shrimp cysts. Sources

include: Aquarium Products, 180l Penrod Ct., Glen Burnie, MD 21061);

San Francisco Bay Brand, 8239 Enterprise Dr., Newark, CA 94560

(Phone: 415-792-7200); Argent Aquaculture, 8702 152nd Ave, N.E.,

Redmond, WA 98052 (206-885-3777)(Argentina brine shrimp eggs, Grade

1, Gold Label); and Jungle, Inc. Additional sources are listed in

the section on Artemia culture in Appendix A. The quality of the

cysts may vary from one batch to another, and the cysts in each new

batch (can or lot) should be evaluated for nutritional suitability

and chemical contamination. The nutritional suitability (see Leger

et al., 1985, 1986) of each new batch is checked against known

suitable reference cysts by performing a side-by-side growth and/or

reproduction tests using the "new" and "reference" cysts. If the

results of tests for nutritional suitability or chemical

contamination do not meet standards, the Artemia should not be used.

2. Frozen Adult Brine Shrimp

Frozen adult brine shrimp are available from San Francisco Bay Brand,

8239 Enterprise Dr., Newark, CA 94560 (415-792-7200).

5.4.9.3 Trout Chow

Starter or No.1 pellets, prepared according to current U.S. Fish and

Wildlife Service specifications, are available from: Zeigler Bros., Inc., P.O.

Box 95, Gardners, PA 17324 (717-780-9009); Glencoe Mills, 1011 Elliott,

Glencoe, MN 55336 (612-864-3181); and Murray Elevators, 118 West 4800 South,

Murray, UT 84107 (800-521-9092). (The flake food, TETRAMIN® or BIORIL®, can

be used regularly as a substitute for trout chow in preparing food for

daphnids, and can be used as a short-term substitute for trout chow in feeding fathead minnows.)

25
5.4.9.4 Dried, Powdered Leaves (CEROPHYLL®)

Dried, powdered, cereal leaves are available from Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178 (800-325-3010); or as CEROPHYLL®, from

Ward's Natural Science Establishment, Inc., P.O. Box 92912, Rochester, NY

14692-9012 (716-359-2502). Dried, powdered, alfalfa leaves obtained from

health food stores have been found to be a satisfactory substitute for cereal

leaves.

5,4.9.5 Yeast

Packaged dry yeast, such as FLEISCHMANN'S®, or equivalent, can be purchased

at the local grocery store or is available from Lake States Yeast, Rhineland,

WI.

5.4.9.6 Flake Fish Food

The flake foods, TETRAMIN® and BIORIL®, are available at most pet supply shops,

5.5 TEST ORGANISMS

5.5.1 Test organisms are obtained from inhouse cultures or commercial

suppliers (see Section 6, Test Organisms).

26
SECTION 6

TEST ORGANISMS

6.1 TEST SPECIES

6.1.1 The species used in characterizing the acute 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 are provided in this

manual for the following principal test organisms:

Freshwater Organisms:

1. Ceriodaphnia dubia (daphnid) (Table 11).

2. Daphnia pu7ex and D. magna (daphnids) (Table 12).

3. Pimepha7es prome7as (fathead minnow) (Table 13).

4. Oncorhynchus mykiss (rainbow trout) and Sa7ve7inus fontina7is (brook

trout) (Table 14).

Estuarine and Marine Organisms:

1. Mysidopsis bahia (mysid) (Table 15).

2. Cyprinodon variegatus (sheepshead minnow) (Table 16).

3. Menidia bery77ina (inland silverside), M. menidia (Atlantic

silverside), and M. peninsu7ae (tidewater silverside) (Table 17).

6.1.3 The test species listed in Subsection 6.1.2 are the recommended acute

toxicity test organisms. They are easily cultured in the laboratory, are

sensitive to a variety of pollutants, and are generally available throughout the year from commercial sources. Summaries of test conditions for these

species are provided in Tables 11-17. Guidelines for culturing and/or holding the organisms are provided in Appendix A.

6.1.4 Additional species may be suitable for toxicity tests in the NPDES

Program. A list of alternative acute toxicity test species and minimal

testing requirements (i.e., temperature, salinity, and life stage) for these

species are provided in Appendix B. It is important to note that these

species may not be as easily cultured or tested as the species on the list in

Subsection 6.1.2, and may not be available from commercial sources.

6.1.5 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 6.1.2. However, EPA allows the use of indigenous species only where state regulations require their use or prohibit importation

27
of the species in Subsection 6.1.2. Where state regulations prohibit importation or use of the recommended test species, permission must be

requested from the appropriate state agency prior to their use.

6.1.6 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. EPA 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.

6.1.7 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,

1991d).

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 INHOUSE CULTURES

6.2.1.1 Inhouse cultures should be established wherever it is cost effective.

If inhouse cultures cannot be maintained, test organisms should be purchased from experienced commercial suppliers (see Appendix for sources).

6.2.2 COMMERCIAL SUPPLIERS

6.2.2.1 All of the principal test organisms listed in Subsection 6.1.2 are

available from commercial suppliers.

6.2.3 FERAL (NATURAL OCCURRING, WILD CAUGHT) ORGANISMS

6.2.3.1 The use of test organisms taken from the receiving water has strong appeal, and would seem to be the logical approach. However, it is impractical 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 i$ usually more cost effective to culture the

28
organisms in the laboratory or obtain them from private, state, or

Federal sources. Fish such as fathead minnows, sheepshead minnows,

and silversides, and invertebrates such as daphnids and 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 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 assure 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.

6.2.3.2 Guidelines for collection of feral organisms are provided in USEPA,

1973; USEPA 1990a.

6.2.4 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 first instars of

daphnids and juvenile mysids and fish, is recommended for all tests. There

may be special cases, however, where the limited availability of organisms

will require some deviation from the recommended life stage. In a given test,

all organisms should be approximately the same age and should be taken from

the same source. 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 Appendix A.

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 30/00 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

29
small-mesh nylon netting, silk bolting cloth, plankton netting, or similar

material. Wide-bore, smooth glass tubes (4 to 8 mm inside diameter) with

rubber bulbs or pipettors (such as a PROPIPETTE® or other pipettor) should be

used for transferring smaller organisms such as daphnids, mysids, and larval

fish.

6.5.3 Holding tanks for fish are supplied with a good quality water (see

Section 5, Facilities and Equipment) with a flow-through rate of at least two

tank-volumes per day. Otherwise, use a recirculation system where the water

flows through an activated carbon or undergravel filter to remove dissolved

metabolites. Culture water can also be piped through high intensity ultraviolet light sources for disinfection, and to photodegrade dissolved

organics.

6.5.4 Crowding should be avoided. The DO must be maintained at a mlnlmum of

4.0 mg/L for marine and warm water, freshwater species, and 6.0 mg/L for coldwater, freshwater species. The solubility of oxygen depends on temperature, salinity, and altitude. Aerate if necessary.

6.5.5 Fish should be fed as much as they will eat at least once a day with

live or frozen brine shrimp or dry food (frozen food should be completely thawed before use). Brine shrimp can be supplemented with commercially prepared food such as TETRAMIN® 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.

6.5.6 Fish should be observed carefully each day for signs of disease,

stress, physical damage, and mortality. Dead and abnormal specimens should be

removed as soon as observed. It is not uncommon to have some fish (5-10%)

mortality during the first 48 h in a holding tank because of individuals that

refuse to feed on artificial food and die of starvation.

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 for marine and warm-water, freshwater species, and 6.0 mg/L for cold-water, freshwater species.

6.6.2 Upon arrival at the test site, organisms are transferred to receiving

water if receiving water ;s 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 min period with dilution water. If

receiving water is used as dilution water, caution must be exercised in

30
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 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 In static tests, marine organisms can be used at all concentrations of

effluent by adjusting the salinity of the effluent to a standard salinity

(such as 25~) 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. However, if this is impractical because of the large

volumes of water required, such as in flow-through tests, the highest effluent

concentration (lowest salinity) that could be tested would depend upon the

salinity of the receiving water and the tolerance of the test organisms. The

required salinities for toxicity tests with estuarine and marine species are

listed in Tables 15-17. However, the tolerances of other candidate test

species would have to be determined by the investigator in advance of the

test.

6.6.7 Because of the circumstances described above, when performing

flow-through tests of effluents discharged to saline waters, it is advisable

to acclimate groups of test organisms to each of three different salinities,

such as 10, 20, and 300/00, prior to transporting them to the test site. It may

also be advisable to maintain cultures of these test organisms at a series of

salinity levels, including at least 10, 20, and 30~, so that the change in

salinity upon acclimation at the desired test dilutions does not exceed 6~.

31
6.7 TEST ORGANISM DISPOSAL

6.7.1 When the toxicity test is concluded, all test organisms (including

controls) should be humanely destroyed and disposed of in an appropriate

manner.

32
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 acute

toxicity of the effluent, which is a primary objective of NPDES

permit-related toxicity testing, 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 acute toxicity of the

effluent in uncontaminated receiving water, the test may be conducted using

dilution water consisting of a single grab sample of receiving water (if

non-toxic), collected either upstream and outside the influence of the

outfall, or with other uncontaminated natural water (ground or surface water)

or standard dilution water having approximately the same characteristics

(hardness and/or salinity) as the receiving water. Seasonal variations in the

quality of surface waters may affect effluent toxicity. Therefore, the

hardness of fresh receiving water, and 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 immediately upstream or outside the influence of the outfall. A

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

7.2 STANDARD, SYNTHETIC DILUTION WATER

7.2.1 Standard, synthetic dilution water is prepared with deionized water and

reagent grade chemicals or mineral water (Tables 6 and 7) and commercial sea

salts (FORTY FATHOMS®, HW MARINEMIX®) (Table 8). The source water for the

deionizer can be groundwater 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® QPAK~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 MILLIPORE® System to extend the life of the MILLIPORE®

cartridges.

7.2.2.2

( i . e. , The recommended order of the cartridges in a four-cartridge deionizer

MILLI-Q® System or equivalent) is: (1) ion exchange, (2) ion exchange,

33
(3) carbon, and (4) organic cleanup (such as ORGANEX-Q®, or equivalent), followed by a final bacteria filter. The QPAKN

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 FRESHWATER

7.2.3.1 To prepare 20 L of standard, synthetic, moderately hard,

reconstituted water, use the reagent grade chemicals in Table 6 as follows:

1.

2.

3.

4.

5.

6.

7.

Place 19 L of MILLI-Q®, or equivalent, deionized water in a properly

cleaned plastic carboy. Add 1.20 g of MgS04, 1.92 g NaHC03 , and 0.080g KCl to the carboy.

Aerate overnight. Add 1.20 g of CaS04 • 2 H2

0 to 1 L of MILLI-Q® or equivalent

deionized water in a separate flask. Stir on magnetic stirrer until

calcium sulfate is dissolved, add to the 19 L above, and mix well.

For Ceriodaphnia culture and testing, add sufficient sodium selenate

(Na 2Se04 ) to provide 2 ~g selenium per liter of final dilution water.

Aerate the combined solution vigorously for an additional 24 h to

dissolve the added chemicals and stabilize the medium.

The measured pH, hardness, etc., should be as listed in Table 6.

7.2.3.2 To prepare 20 L of standard, synthetic, moderately hard,

reconstituted water, using 20% mineral water such as PERRIER® Water, or

equivalent (Table 7), follow the instructions below.

1. Place 16 L of MILLI-Q® or equivalent deionized water in a properly

cleaned plastic carboy.

2. Add 4 L of PERRIER® Water, or equivalent.

3. Aerate vigorously for 24 h to stabilize the medium.

4. The measured pH, hardness, and alkalinity of the aerated water will be

as indicated in Table 7.

5. This synthetic water is referred to as diluted mineral water (DMW) in

the toxicity test methods.

7.2.4 STANDARD, SYNTHETIC SEAWATER

7.2.4.1 To prepare 20 L of a standard, synthetic, reconstituted seawater

(modified GP2), with a salinity of 31~ (Table 8), follow the instructions

below. Other salinities can be prepared by making the appropriate dilutions.

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 8 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.

34
Larger or smaller volumes of modified GP2 can be prepared by using

proportionately larger or smaller amounts of salts and dilution water.

7.2.4.2 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 product per liter of deionized water. The

salinity of the resulting solutions should be checked with a refractometer.

7.3 USE OF RECEIVING WATER AS DILUTION WATER

7.3.1 If the objectives of the test require the use of uncontaminated surface

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

upstream 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'l 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 4°C during or immediately following collection,

and maintained at that temperature prior to use in the test.

7.3.3 In the case of freshwaters, the regulatory authority may require that

the hardness of the dilution water be comparable to the receiving water at the

discharge site. This requirement can be satisfied by collecting an

uncontaminated surface water with a suitable hardness, or adjusting the

hardness of an otherwise suitable surface water by addition of reagents as

indicated in Table 6.

7.3.4 In an estuarine environment, 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® or equivalent, a

natural seawater of higher salinity, or hypersaline brine. Instructions for

the preparation of hypersaline brine by concentrating natural seawater are

provided below.

7.3.5 Receiving water containing debris or indigenous organisms, that may be

confused with or attack the test organisms, should be filtered through a sieve

having 60 ~m mesh openings prior to use.

35
TABLE 6. PREPARATION OF SYNTHETIC FRESHWATER USING REAGENT GRADE CHEMICALS'

Reagent Added (mg/Ll 2 Final Water Quality

Water

Hardness4 Al kaType NaHC03 CaS04·2H2O MgS04 KCl pH3 1initl

Very soft 12.0 7.5 7.5 0.5 6.4-6.8 10-13 10-13

Soft 48.0 30.0 30.0 2.0 7.2-7.6 40-48 30-35

Moderately Hard 96.0 60.0 60.0 4.0 7.4-7.8 80-100 60-70

Hard 192.0 120.0 120.0 8.0 7.6-8.0 160-180 110-120

Very hard 384.0 240.0 240.0 16.0 8.0-8.4 280-320 225-245

'Taken in part from Marking and Dawson (1973).

2Add reagent grade chemicals to deionized water.

~~~~~~~~~~t~se~~i~~~~~~. pH after 24 h of aeration.

TABLE 7. PREPARATION OF SYNTHETIC FRESHWATER USING MINERAL WATER'

Final Water Qual ity

Volume of Proportion

Water Mineral Wate~ of Mineral AlkaType Added (mL/L) Water (%) pH3 Hardness4 linitl

•

Very soft 50 2.5 7.2-8.1 10-13 10-13

Soft 100 10.0 7.9-8.3 40-48 30-35

Moderately Hard 200 20.0 7.9-8.3 80-100 60-70

Hard 400 40.0 7.9-8.3 160-180 110-120

Very hards

~From Mount et al., 1987; data provided by Philip Lewis, EMSL-Cincinnati.

Add mineral water to MILLI-Q® water or equivalent to prepare DMW (Diluted

3 Mineral Water).

4Approximate equilibrium pH after 24 h of aeration.

sExpressed as mg CaCOviL.

Dilutions of PERRIE~® Water form a precipitate when concentrations

equivalent to "very hard water" are aerated.

36
TABLE 8. PREPARATION OF SYNTHETIC SEAWATER USING REAGENT GRADE CHEMICALS1

,2,3

NaCl 21.03

Na 2S04 3.52

KCl 0.61

KBr 0.088

Na 2B407 . 10 H2O 0.034

MgC1 2 • 6 H2O 9.50

CaC1 2 • 2 H2O 1.32

SrC1 2 • 6 H2O 0.02

NaHC03 0.17

Compound

1Modified GP2.

Concentration

(giL)

Amount (g) Required for

20 L

420.6

70.4

12.2

1. 76

0.68

190.0

26.4

0.400

3.40

2The constituent salts and concentrations were taken from

USEPA, I990b. The salinity is 30.89 giL.

3GP2 can be diluted with deionized (01) water to the desired test salinity.

7.3.6 When receiving water is used as dilution water in flow-through tests,

it is preferable to pump the dilution water continuously to the acclimation

chamber andlor dilutor. However, where it is not feasible to pump the

dilution water continuously, grab samples of the dilution water are

transported to the test site in tanks, and continuously pumped from the tanks

to the acclimation chamber andlor dilutor.

7.3.7 HYPERSALINE BRINE

7.3.7.1 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.

37
7.3.7.2 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.

7.3,7.3 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 ~m before placing into the brine generator. Water should be collected on

an incoming tide to minimize the possibility of contamination.

7,3.7.4 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.

7.3.7.5 After the required salinity is attained, the HSB should be filtered a

second time through a l-~m 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.

7.3.7.6 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.

7.3.7.7 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).

7.3.7.8 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 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,

38
p. 4-36). Following dechlorination, total residual chlorine should not exceed

0.01 mg/L. Because of the possible toxicity of thiosulfate to test organisms,

a control lacking thiosulfate should be included in toxicity tests utilizing

thiosulfate-dechlorinated water.

7.4.2 To be adequate for general laboratory use following 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.

39
SECTION 8

EFFLUENT AND RECEIVING WATER SAMPLING AND SAMPLE HANDLING

8,1 EFFLUENT SAMPLING

8.1.1 The effluent sampling point is ordinarily the same as that specified in

the NPDES discharge permit (USEPA, 1979c). Conditions for exception would be:

(1) better access to a sampling point between the final treatment and the

discharge outfall; (2) if the effluent is chlorinated prior to discharge to

the receiving waters, it may also be desirable to take samples prior to

contact with the chlorine to determine toxicity of the unchlorinated effluent;

or (3) in the event there is a desire to evaluate the toxicity of the influent

to publicly owned treatment works or separate process waters in industrial

facilities prior to their being combined with other process waters 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 requirements of the NPDES permit, 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 within the treatment facility are short, grab

samples may seem preferable because of the ease of collection and the

potential of observing peaks (spikes) in toxicity. However, the sampling

duration of a grab sample is so short that full characterization of an

effluent over a 24-h period would require a prohibitive number of separate

samples and tests. Collection of a 24-h composite sample, however, may dilute

toxicity spikes, and average the quality of the effluent over the sampling

period. Sampling recommendations are provided below.

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.

40
Disadvantages:

1. Samples are collected over a

relatively infrequent basis.

toxicity would depend on the

of missing spikes is high.

very short period of time and on a

The chances of detecting a spike in

frequency of sampling, and the probability

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, samples are collected once, or

daily, and used for test initiation and renewal.

8.3.3 Sufficient sample must be collected to perform the required toxicity

and chemical tests. A 4-L (I-gal) CUBITAINER® will provide sufficient sample

volume for most tests (see Tables 11-17).

8.3.4 The following effluent sampling methods are recommended:

8.3.4.1 Continuous Discharges

1. If the facility discharge is continuous, but the calculated retention

time of the continuously discharged effluent is less than 14 days and

the variability of the effluent toxicity is unknown, at a minimum, four

grab samples or four composite samples are collected over a 24-h

period. For example, a grab sample is taken every 6 h (total of four

samples) and each sample is used for a separate toxicity test, or four

successive 6-h composite samples are taken and each is used in a

separate test.

2. If the calculated retention time of a continuously discharged effluent

is greater than 14 days, or if it can be demonstrated that the

wastewater does not vary more than 10% in toxicity over a 24-h period,

regardless of retention time, a single grab sample is collected for a

single toxicity test.

41
3. The retention time of the effluent in the wastewater treatment facility

may be estimated from calculations based on the volume of the retention

basin and rate of wastewater inflow. However, the calculated retention

time may be much greater than the actual time because of

short-circuiting in the holding basin. Where short-circuiting is

suspected, or sedimentation may have reduced holding basin capacity, a

more accurate estimate of the retention time can be obtained by

carrying out a dye study.

8.3.4.2 Intermittent Discharges

8.3.4.2.1 If the facility discharge is intermittent, a grab sample is

collected midway during each discharge period. Examples of intermittent

discharges are:

1. When the effluent is continuously discharged during a single 8-h work

shift (one sample is collected), or two successive 8-h work shifts (two

samples are collected).

2. When the facility retains the wastewater during an 8-h work shift, and

then treats and releases the wastewater as a batch discharge (one

sample is collected).

3. When the facility discharges wastewater to an estuary only during an

outgoing tide, usually during the 4 h following slack high tide (one

sample is collected).

8.3.4.3 At the end of a shift, clean up activities may result in the

discharge of a slug of toxic waste, which may require sampling and testing.

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, it is common practice to collect a single grab

sample and use it throughout the test.

8.4.2 The sampling point is determined by the objectives of the test. In

rivers, grab samples should be collected at mid-stream and mid-depth, if

accessible. At estuarine and marine sites, samples are collected at middepth.

8.4.3 To determine the extent of the zone of toxicity in the receiving water

downstream from the outfall, receiving water samples are collected at several

distances downstream from the discharge. The time required for the effluentreceiving-water mixture to travel to sampling points downstream from the

outfall, and the rate and degree of mixing, may be difficult to ascertain.

Therefore, it may not be possible to correlate downstream toxicity with

effluent toxicity at the discharge point unless a dye study is performed. The

toxicity of receiving water samples from five stations downstream from the

discharge point can be evaluated using the same number of test vessels and

42
test organisms as used in one effluent toxicity test with five effluent

dilutions.

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, it is recommended that they be held at 4°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 the sample in test

initiation should not exceed 36 h. EPA believes that 36 h is adequate time to

deliver the samples to the laboratories performing the tests 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 EPA Regional

Administrator under 40 CFR 136.3(e) must include supportive data which show

that the toxicity of the effluent sample is not reduced (e.g., because of

volatilization and/or sorption of toxics on the sample container surfaces) by

extending the holding time beyond more than 36 h. However in no case should

more than 72 h elapse between collection and first use of the sample. In

static-renewal tests, the original sample may also be used to prepare test

solution for renewal at 24 h, 48 h, and/or 72 h after test initiation, if

stored at 4·C, with minimum head space, as described in Subsection 8.5.

Guidance for determining the persistence of the sample is provided in

Subsection 8.7.

8.5.5 To minimize the loss of toxicity due to volatilization of toxic

constituents, all sample containers should be "completely" filled, leaving no

air space between the contents and the lid.

8.5.6 SAMPLES USED IN ON-SITE TESTS

8.5.6.1 Samples collected for on-site tests should be used within 24 h.

8.5.7 SAMPLES SHIPPED TO OFF-SITE FACILITIES

8.5.7.1 Samples collected for off-site toxicity testing are to be chilled to

4°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

43
the laboratory and is unpacked. Insulating material must not be placed

between the ice and the sample in the shipping container.

8.5.7.2 Samples may be shipped in one or more 4-L (I-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.

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 4°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 before first use unless a variance has been granted by the

NPDES permitting authority.

8.7 PERSISTENCE OF EFFLUENT TOXICITY DURING SAMPLE SHIPMENT AND HOLDING

8.7.1 The persistence of the toxicity of an effluent prior to its use in a

toxicity test is of interest in assessing the validity of toxicity test data,

and in determining the possible effects of allowing an extension of the

holding time. Where a variance in holding time (>36 h, but ~72 h) is

requested by a permittee (see Subsection 8.5.4 above), information on the

effects of the extension in holding time on the toxicity of the samples must

be obtained by comparing the results of multi-concentration acute 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.

44
SECTION 9

ACUTE TOXICITY TEST PROCEDURES

9.1 PREPARATION OF EFFLUENT AND RECEIVING WATER SAMPLES FOR TOXICITY TESTS

9.1.1 When aliquots are removed from the sample container, the head space

above the remaining sample should be held to a minimum. Air which enters a

container upon removal of sample should be expelled by compressing the

container before reclosing, if possible (i.e., where a CUBITAINER® used), or

by using an appropriate discharge valve (spigot).

9.1.2 It may be necessary to first coarse-filter samples through a 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 must be

filtered through a sieve with 60 ~m mesh openings. Caution: filtration may

remove some toxicity.

9.1.3 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.

9.1.4 It is recommended that total alkalinity and total hardness also be

measured in the undiluted test water (effluent or receiving water) and the

dilution water.

9.1.5 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 9, under the appropriate pH and temperature, and

multiplying it by the concentration (mg/L) of total ammonia in the sample.

9.1.6 Effluents and receiving waters can be dechlorinated using 6.7 mg/L anhydrous sodium thiosulfate to reduce 1 mg/L chlorine (Standard Methods, 18th

Edition, APHA, 1992, p. 9-32; note that the amount of thiosulfate required to

dechlorinate effluents is greater than the amount needed to dechlorinate tap 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.

9.1.7 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.

9.1.8 If the samples must be warmed to bring them to the prescribed test

temperature, supersaturation of the dissolved oxygen and nitrogen may become a

problem. To avoid this problem, the effluent and dilution water are checked

45
TABLE 9. PERCENT UNIONIZED NH3 IN AQUEOUS AMMONIA SOLUTIONS: TEMPERATURES

IS-26°C AND PH's 6.0-8.9 1

pH TEMPERATURE COC)

i5 i6 i7 i8 i9 20 2i 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 0.193

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

'Table provided by Teresa Norberg-King, Environmental Research Laboratory, Duluth, Minnesota.

Also see Emerson, et. al., 1975, Thurston, et. al, 1974, and USEPA, 1985.

46
with a DO probe after reaching test temperature and, if the DO is greater than

100% saturation or lower than 4.0 mg/L, the solutions are aerated moderately

(approximately 500 mL/min) for a few minutes, using an airstone, until the DO

is within the prescribed range (~4.0 mg/L when using warm water species, or ~6.0 mg/L when using cold water species). Caution: avoid excessive aeration.

9.1.9 Mortality 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 IN NaOH or IN HCl dropwise,

as required, being careful to avoid overadjustment.

9.2 PRELIMINARY TOXICITY RANGE-FINDING TESTS

9.2.1 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, acute, definitive toxicity test. However, when preparing to perform a

static test with an sample of completely unknown quality, or before initiating

a flow-through test, it is advisable to conduct a preliminary toxicity rangefinding test.

9.2.2 A toxicity range-finding test ordinarily consists of a down-scaled,

abbreviated static acute test in which groups of five organisms are exposed to

several widely-spaced sample dilutions in a logarithmic series, such as 100%,

10.0%, 1.00%, and 0.100%, and a control, for 8-24 h. Caution: if the sample

must also be used for the full-scale definitive test, the 36-h limit on

holding time (Section 8, Effluent and Receiving Water Sampling and Sample

Handling, Subsection 8.5.4) must not be exceeded before the definitive test is

initiated.

9.2.3 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 definitive test because: (1) the definitive test is usually

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.

9.3 MULTI-CONCENTRATION (DEFINITIVE) EFFLUENT TOXICITY TESTS

9.3.1 The tests recommended for use in determining discharge permit

compliance in the NPDES program are multi-concentration, or definitive, tests

which provide (1) a point estimate of effluent toxicity in terms of a LC50, or

(2) a No-Observed-Adverse-Effect Concentration (NOAEC) defined in terms of

mortality, and obtained by hypothesis testing. The tests may be static nonrenewal, static renewal, or flow-through.

9.3.2 The tests consist of a control and a minimum of five effluent

concentrations commonly selected to approximate a geometric series, such as

100%, 50%, 25%, 12.5%, and 6.25%, by using a dilution factor of 0.5.

47
9.3.3 These tests are also to be used in determining compliance with permit limits on the mortality of the "instream" or receiving water concentration

(RWC) of effluents by bracketing the RWC with effluent concentrations in the

following manner: (1) 100% effluent, (2) [RWC + 100]/2, (3) RWC, (4) RWC/2,

and (5) RWC/4. For example, where the RWC = 50%, the effluent concentrations

used in the test would be 100%, 75%, 50%, 25%, and 12.5%.

9.3.4 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., temperature, water

hardness, salinity, etc.

9.4 RECEIVING WATER TESTS

9.4.1 Receiving water toxicity tests generally consist of 100% receiving

water and a control. The total hardness or salinity of the control should be

comparable to the receiving water.

9.4.2 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. A minimum of four replicates and 10 organisms

per replicate are required for each treatment (see Tables 11-17).

9.4.3 In cases where the objective of the test is to estimate the degree of

toxicity of the receiving water, a definitive, multi-concentration test is

performed by preparing dilutions of the receiving water, using a ~0.5 dilution

series, with a suitable control water.

9.5 STATIC TESTS

9.5.1 Static tests may be non-renewal or renewal.

9.5.2 An excess volume of each dilution is prepared to provide sufficient

material for toxicity testing and routine chemical analyses. The solutions

are well mixed with a glass rod, TEFLON® stir bar, or other means. Aliquots of each sample concentration are delivered to the test chambers, and the

chambers are arranged in random order. The test solutions are brought to the

required temperature, and the test organisms are added. The remaining volumes

of each sample concentration are used, as necessary, for the chemical

analyses.

9.5.3 Saline dilution water can be prepared by adding dry salts (FORTY

FATHOMS® or equivalent, or modified GP2) or hypersaline brine to de-ionized

water, or a suitable surface freshwater, to adjust the salinity of the entire

dilution series. If saline receiving water is used as the diluent, a salinity control must be prepared using deionized water and dried sea salts to

determine if the addition of sea salts alone has an adverse effect on the test

organisms. It may be desirable to conduct static toxicity tests at several

salinities.

9.5.4 If the effluent has low salinity, but the test is to be conducted with

a salt water organism, the test solutions may be prepared by adding dry ocean

48
salts or hypersaline brine to a sufficient quantity of 100% effluent to raise

the salinity to the required level, which will depend on the objectives of the

test and the policy of the regulatory agency. After the addition of the dried

salts, stir gently for 30 to 60 min, preferably with a magnetic stirrer, to

ensure that the salts are in solution. It is important to check the final

salinity with a refractometer.

9.5.5 Addition of dry salts to effluents and dilution water may change the pH

and affect the toxicity of the waste. If the objective of the test is to

determine the toxicity of the effluent at the original pH, the pH of the

salinity-adjusted solutions can be brought to the required level by dropwise

addition of IN Hel or IN NaOH. It is recommended that a concurrent test be

conducted with salinity-adjusted effluent in which the pH has not been altered

after adding the salt.

9.5.6 The volume of the effluent used must be sufficient to prepare all

percent concentrations of the effluent needed for the toxicity test and for

routine chemical analysis. For example, to conduct tests with Menidia, the use

of 200 mL of test solution in each of duplicate exposure vessels and five

concentrations of effluent (10 exposure vessels), would require a total of 1 L

of 100% effluent. However, to provide sufficient volumes of test solutions

for routine chemical analysis and for toxicity testing, additional effluent

would be required (1.5-2.0 L).

9.5.7 A standard control lacking thiosulfate should be included in tests

where the dilution water was prepared by dechlorinating tap water with

thiosulfate.

9.5.8 If, within 1 h of the start of the test, 100% mortality has occurred in

the higher effluent concentrations (such as 100% and 50%), additional

concentrations of effluents, such as 3.1%, 1.6%, and 0.8%, are added to the

test at the lower end of the concentration series.

9.5.9 Increases in pH may occur in test solutions during acute, static nonrenewal toxicity of pollutants such as ammonia. This problem can be avoided

by conducting a test in a static renewal or flow through mode rather than a

static non-renewal mode.

9.6 FLOW-THROUGH TESTS

9.6.1 Flow-through tests are usually performed with the same effluent

concentrations that are used for static tests, except that where the receiving

water is saline and the effluent is not, 100% effluent cannot be tested with a

marine organism. Examples of flow-through test systems are provided in the

Appendix. Small organisms, such as mysids and daphnids, are confined in

screened enclosures placed in the flow-through chambers. More than one

species may be used in the same test chamber in a given test, if segregated.

9.6.2 The dilutor system should be operated long enough prior to adding the

test organisms to calibrate the dilutor and make the necessary adjustments in

the temperature, flow rate through the test chambers, and aeration. The flow

rate through the proportional dilutor must provide for a minimum of five 90%

49
replacements of water volume in each test chamber every 24 h (see Figure 2).

This replacement rate should provide sufficient flow to maintain an adequate concentration of dissolved oxygen. The dilutor should also be capable of

maintaining the test concentration at each dilution within 5% of the starting concentration for the duration of the test. The calibration of the dilutor

should be checked carefully before the test begins to determine the volume

of effluent and dilution water used in each portion of the effluent delivery

system and the flow rate through each test chamber. The general operation of

the dilutor should be checked at least at the beginning and end of each day during the test.

9.6.3 The control consists of the same dilution water, test conditions,

procedures, and organisms used in testing the effluent. In the event a test

is to be conducted with salt water organisms, where each effluent dilution has

a different salinity, a static control is prepared for the lowest (or highest, in the case of high salinity, e.g. brine wastes) salinity level used in the

flow-through test to determine if salinity alone has any adverse effects on

the test organisms.

9.7 NUMBER OF TEST ORGANISMS

9.7.1 A minimum of 20 organisms of a given species are exposed to each

effluent concentration (Jensen, 1972). Small fish and invertebrates are

captured with 4- to 8-mm inside diameter pipettes. Organisms larger than 10-

mm can be captured by dip net. In a typical toxicity test involving five

effluent concentrations and a control (six concentrations X 20 organisms per concentration), fish and other large test organisms are captured from a common

pool and distributed sequentially to the test chambers until the required

number of organisms are placed in each. The test chambers are then positioned randomly. To avoid carryover of excess culture water in transferring small

organisms to the test chambers, it may be advantageous to distribute small

organisms, such as daphnids, mysids, and larval fish, first to small holding

vessels, such as weighing boats, petri dishes, or small beakers. The water in

the intermediary holding vessels is then drawn down to a small volume and the

entire lot is transferred to a test chamber. In the case of daphnids, both

excessive handling and carryover of culture water can be avoided by placing the tip of the transfer pipettes below the surface of the water in the test

chambers and allowing the organisms to swim out of the pipettes without

discharging the contents.

9.8 REPLICATE TEST CHAMBERS

9.8.1 Two or more test chambers are provided for each effluent concentration

and the control. Although the data from duplicate chambers are usually

combined to determine the LC50 and confidence interval, the practice of

dividing the test population for each effluent concentration between two or

more replicate chambers has several advantages and is considered good laboratory practice because it: (1) permits easier viewina and countina of

test organisms; (2) more easily avoids possible violations of loading limits,

which might occur if all of the test organisms are placed in a single test

vessel; and (3) ensures against the invalidation of the test which might

50
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t:: 6

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0.4 0.6 1 2 4 10 20

Volume of Water in Tank

Flow of Water Per Hour

40 60 100

Figure 2. Approximate times required to replace water in test chambers in

flow-through tests. For example: for a chamber containing 4 L,

with a flow of 2 L/h, the above graph indicates that 90% of the

water would be replaced every 4.8 h. The same time period (such as

hours) must be used on both axes, and the same unit of volume (such

as liters) must be used for both volume and flow (From: Sprague, 1969).

51
result from accidental loss of a test vessel, where all of the test organisms

for a given treatment are in a single chamber.

9.9 LOADING OF TEST ORGANISMS

9.9.1 A limit is placed on the loading (weight) of organisms per liter of

test solution to minimize the depletion of dissolved oxygen, the accumulation ,,~ VI ;"';""';0"" III,""UI I U~ '""Vll'-'-I r",.,ra,.,t",,,t;,,,.,,, ...... """'VII,J "oF VI III""" mat::lhnl .... ""...,"" ••

if'""" W::lc::to •• _..,""_ 1"'1"" nY'nrlllf'ts'" , ;lnrl/nr

_ •• _, ................ c::.trpc::.c::. --- innll('pn ••• - ..... ---

by crowding, any of which could significantly affect the test results.

However, the probability of exceeding loading limits is greatly reduced with

the use of very young test organisms.

9.9.2 For both renewal and non-renewal static tests, loading in the test

solutions must not exceed the following live weights: 1.1 giL at 15°C,

0.65 giL at 20°C, or 0.40 giL at 25°C.

9.9.3 For flow-through tests, the live weight of test organisms in the test

chambers must not exceed 7.0 giL of test solution at 15°C, or 2.5 giL at 25°C.

9.10 ILLUMINATION

9.10.1 Light of the quality and intensity n9rmally obtained in the laboratory

during working hours is adequate (10-20 ~E/m Is or 50-100 ft-c). A uniform

photoperiod of 16 h light and 8 h darkness can be achieved in the laboratory

or environmental chamber, using automatic timers.

9.11 FEEDING

9.11.1 Where indicated in the test summary tables (Tables 11-17), food is

made available to test organisms while holding before they are placed in the

test chambers. The organisms are fed at test renewal, 48 h after the test is

initiated, if Regional or State policy requires a 96-h test duration.

9.11.2 Where Artemia nauplii are fed, the nauplii are first concentrated on a

NITEX® screen and then are resuspended in fresh or salt water, depending on

the salinity of the test solutions, using just enough water to form a slurry

that can be transferred by pipette. It should be noted that Artemia nauplii

placed in freshwater usually die in 4 h, generally are not eaten after death,

and decay rapidly, whereas those placed in saline water remain viable and can

serve as food for the duration of the test.

9.11.3 Problems caused by feeding, such as the possible alteration of the

toxicant concentration, the build-up of food and metabolic wastes and

resulting oxygen demand, are common in static test systems. Where feeding is

necessary, excess food should be removed daily by aspirating with a pipette.

9.11.4 Feeding does not cause the above problems in flow-through systems.

U"..,n\l~V' ;+;C" ~rh,;C"~h'n +n 'V'l"\mn\l" nvro"C"l" +1'\1'\,.1 +I"\I""~' m':\+nV'l;~' ~nrl :an\! Ilvn't;¥'l;;I, I'.. I~ QUYI~UUI'f;;; \"u 1t;;IIIUY'l;;;; c;A.~'I;;")") IVVU, I~'-QI IIIU ..... oc;IIUI, \,lIlY \4IIJ

particulate matter that settles from the effluent, from the bottom of the test

vessels daily by aspirating with a pipette.

52
9.12 TEST TEMPERATURE

9.12.1 Test temperature will depend on the test species and objectives of the

test (see Tables 11-17). Where acute and short-term chronic toxicity tests

are performed simultaneously with the same species to determine acute:chronic

ratios, both tests must be performed at the chronic test temperature. The

average daily temperature of the test solutions must be maintained within ±

1°C of the selected test temperature, for the duration of the test. This can

be accomplished for static tests by use of a water bath or environmental

chamber, and in flow-through tests by passing the effluent and/or dilution

water through separate coils immersed in a heating or cooling water bath prior

to entering the dilutor system. Coils should be made from materials

recommended in Section 5, Facilities and Equipment.

9.13 STRESS

9.13.1 Minimize stress on test organisms by avoiding unnecessary

disturbances.

9.14 DISSOLVED OXYGEN CONCENTRATION

9.14.1 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 its toxicity by altering the pH. However, the

DO in the test solution must not be permitted to fall below 4.0 mg/L for warm

water species and 6.0 mg/L for cold water species. Oxygen saturation values

in fresh and saline waters can be determined from Figure 3 and Table 10,

respectively.

9.14.2 In static tests, low DOs 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 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.

9.14.3 In most flow-through tests, DO depletion is not a problem in the test

chambers because aeration occurs as the liquids pass through the dilutor

system. If the DO decreases to a level that would be a source of additional

stress, the turnover rate of the solutions in the test chambers must be

increased sufficiently to maintain acceptable DO levels. If the increased

turnover rate does not maintain adequate DO levels, aerate the dilution water

prior to the addition of the effluent, and aerate all test solutions. To

reduce the potential for driving off volatile compounds in the wastewater,

aeration may be accomplished by bubbling air through a 1-mL pipet at a rate of

no more than 100 bubbles/min, using an air valve to control the flow.

9.14.4 Caution must be exercised to avoid excessive aeration. Turbulence

caused by aeration should not result in a physical stress to the test

53
TABLE 10. OXYGEN SOLUBILITY (MG/L) IN WATER AT EQUILIBRIUM WITH AIR AT 760

MM HG (AFTER RICHARDS AND CORWIN, 1956)

TEMP SALINITY (%0)

°C " .. ,,, 15 20 ')1: 30 35 40 JI-' V ;) lV Co;) "T ....

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 1l.8 11.4 11.0 10.6 10.3

2 13.4 13.0 12.6 12.2 11. 9 1l.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 10.2 9.8 9.5 9.3

6 12.1 11.7 11.4 11.0 10.7 10.3 10.0 9.6 9.3 9.1

8 1l.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

54
CORRECTION FACTORS FOR OXYGEN

SATURATION AT VARIOUS ALTITUDES

ALTllUDE PRESSURE

-f!- _M_ MM FACTOR i I iii' , 'I"""i1'I""I""1

0 0 760 --:uiO 0 5 10 15 20 25 30

330 100 750 1.01 Waler temperalures ·C

665 200 741 1.03

980 300 732 1.04

1310 400 723 1.05

1640 500 714 1.06

1970 600 705 1.08 \\0(\

2300 700 696 1.09 -;,3'1.-.}18

2630 600 687 1.11 .,. \,,0 2950 900 679 1.12 '1.').0

3280 1000 671 1.13

3610 1100 663 1.15

3940 1200 655 1.16

4270 1300 647 1.17

4600 1400 639 1.19

4930 1500 631 1.20

5250 1600 623 1.22

5580 1700 615 1.24

5910 1600 608 1.25

6240 1900 601 1.26 0 6560 2000 594 1.28

6900 2100 587 1.30

7220 2200 580 1.31 0 2 3 4 5 6 7 8 9 10 11 12

7550 2300 573 1.33 Oxygen cc. per filer 7880 2400 568 1.34

8200 2500 560 1.36

Figure 3. Rawson's nomograph for obtaining oxygen saturation values in

freshwater at different temperatures at sea level. When a

straightedge is used to connect the water temperature on the upper scale and the concentration on the lower scale, the percent saturation can be read from the point of intersection on the

diagonal scale. To determine the percent saturation at locations

above sea level, factors are provided to convert oxygen concentrations measured at various altitudes to sea level values

in the table at the upper left. For example, an oxygen concentration of 6.4 mg/L measured in a body of water at an

altitude of 1000 mand a temperature of 15°C would be eqUivalent

to a concentration of 6.4 x 1.13, or 7.2 mg/L, at sea level.

To determine the percent saturation, a straightedge is used to

connect the point at 15°C on the temperature scale with the

point, 7.2 mg/L on the concentration scale, and the percent saturation is read at the point of intersection (68%) on the

diagonal scale. (From Welch, 1948).

55
organisms. When aeration is used, the methodology must be detailed in the

report. For safety reasons, pure oxygen should not be used to aerate test

solutions.

9.15 TEST DURATION

9.15.1 Test duration may vary from 24 to 96 h depending on the objectives of

the test and the requirements of the regulatory authority. For specific

information on test duration, see the tables summarizing the test conditions

below.

9.16 ACCEPTABILITY OF TEST RESULTS

9.16.1 For the test results to be acceptable, survival in controls must be at

least 90%. Tests in which the control survival is less than 90% are invalid,

and must be repeated. In tests with specific chemicals, the concentration of

the test material must not vary more than 20% at any treatment level during the exposure period.

9.16.2 Upon subsequent completion of a valid test, the results of all tests,

valid and invalid, are reported to the regulatory authority with an

explanation of the tests performed and results.

9.17 SUMMARY OF TEST CONDITIONS FOR THE PRINCIPAL TEST ORGANISMS

9.17.1 Summaries of the test conditions for the daphnids, Ceriodaphnia dubia,

Daphnia pulex, and D. magna, fathead minnows, Pimephales promelas, rainbow

trout, Oncorhynchus mykiss, brook trout, Salvelinus fontinalis, the mysid, Mysidopsis bahia, sheepshead minnows, Cyprinodon variegatus, and silversides,

Menidia beryllina, M. menidia, and M. peninsulae, are provided in Tables 11-

17.

56
TABLE 11. SUMMAR~ OF TEST CONDITIONS AND TEST ACCEPTABILIT~ CRITERIA FOR

CERIODAPHNIA DUBIA ACUTE TOXICITY TESTS WITH EFFLUENTS AND

RECEIVING WATERS

I. Test type:

2, Test duration:

3, Temperature: '

4. Light quality:

5. light i ntensity:

6, Photoperiod:

7. Test chamber size:

8. Test solution volume:

9. Renewal of test

solutions:

10. Age of test organisms:

II. No. organisms per

test chamber:

12. No. replicate chambers

per concentration:

13. No. organisms per concentration:

Static non-renewal, static-renewal, or flowthrough

24, 48, or 96 h

Ambient laboratory illumination

10-20 ~E/m2/s (50-100 ft-c)

(ambient laboratory levels)

16 h light, 8 h darkness

30 mL (minimum)

15 mL (minimum)

Minimum, after 48 h

Less than 24-h old

Minimum,S for effluent and receiving water

tests

Minimum, 4 for effluent and receiving water

tests

Minimum, 20 for effluent and receiving water

tests

'Acute and chronic toxicity tests performed simultaneously to obtain

acute/chronic ratios must use the same temperature and water hardness.

57
TABLE 11. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

CERIODAPHNIA DUBIA ACUTE TOXICITY TESTS WITH EFFLUENTS AND

RECEIVING WATERS (CONTINUED)

14. Feeding regime:

15. Test chamber cleaning:

16. Test chamber aeration:

17. Dilution water:

18. Test concentrations:

19. Dilution series:

20. Endpoint:

21. Sampling and sample

holding requirements:

22. Sample volume required:

23. Test acceptability

criterion:

Feed YCT and Se7enastrum while holding prior

to the test; newly-released young should

have food available a minimum of 2 h prior

to use in a test; add 0.1 mL each of YCT and

Se7enastrum 2h prior to test solution

renewal at 48 h

Cleaning not required

None

Moderately hard synthetic water prepared using MILLIPORE MILLI-Q® or equivalent deionized water and reagent grade chemicals

or 20% DMW (see Section 7 Dilution Water),

receiving water,ground water, or synthetic

water, modified to reflect receiving water

hardness.

Effluents: Minimum of five effluent

concentrations and a control

Receiving Waters: 100% receiving water and a

control

Effluents: 0.5 dilution series

Receiving Waters: None, or 0.5 dilution

series

Effluents: Mortality (LC50 or NOAEC)

Receiving Waters: Mortality (Significant difference from control)

Effluents and Receiving Waters: Grab or

composite samples are used within 36 h of

completion of the sampling period.

1 L

90% or greater survival in controls

58
TABLE 12. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

DAPHNIA PULEX AND D. MAGNA ACUTE TOXICITY TESTS WITH EFFLUENTS

AND RECEIVING WATERS

1. Test type:

2. Test duration:

3. Temperature: 1

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. organisms per

test chamber:

12. No. replicate chambers

per concentration:

13. No. organisms per

concentration:

14. Feeding regime:

Static non-renewal, static-renewal, or flowthrough

24, 48, or 96 h

20°C ± laC; or 25°C ± laC

Ambient laboratory illumination

10-20 ~E/m2/s (50-lOa ft-c)

(ambient laboratory levels)

16 h light, 8 h darkness

30 mL (minimum)

25 mL (minimum)

Minimum, after 48 h

Less than 24-h old

Minimum,S for effluent and receiving water

tests

Minimum, 4 for effluent and receiving water

tests

Minimum, 20 for effluent and receiving water

tests

Feed YCT and Se7enastrum while holding prior

to the test; newly-released young should

have food available a minimum of 2 h prior

to use in a test; add 0.1 mL each of YCT and

Se7enastrum 2 h prior to test solution

renewal at 48 h

'Acute and chronic toxicity tests performed simultaneously to obtain

acute/chronic ratios must use the same temperature and water hardness.

59
TABLE 12. SUMMARY OF CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

DAPHNIA PULEX AND D. MAGNA ACUTE TOXICITY TESTS WITH EFFLUENTS

AND RECEIVING WATERS (CONTINUED)

15. Test chamber cleaning:

16. Test chamber aeration:

17. Dilution water:

18. Test concentrations:

19. Dilution series:

20. Endpoint:

21. Sampling and sample

holding requirements:

22. Sample volume required:

23. Test acceptability criterion:

Cleaning not required

None

Moderately hard synthetic water prepared using MILLIPORE MILLI-Q® or equivalent deionized water and reagent grade chemicals

or 20% DMW (see Section 7, Dilution Water),

receiving water, ground water, or synthetic

water, modified to reflect receiving water

hardness.

Effluents: Minimum of five effluent

concentrations and a control

Receiving Waters: 100% receiving water and a

control

Effluents: ~0.5 dilution series

Receiving Waters: None, or ~0.5 dilution

series

Effluents: Mortality (LC50 or NOAEC)

Receiving Waters: Mortality (Significant difference from control)

Effluents and Receiving Waters: Grab or

composite samples are used within 36 h of

completion of the sampling period.

1 L

90% or greater survival in controls

60
TABLE 13. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

FATHEAD MINNOW, PIMEPHALES PROMELAS, ACUTE TOXICITY TESTS WITH

EFFLUENTS AND RECEIVING WATERS'

L Test type:

2. Test duration:

3. Temperature: 2

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:

II. No. organisms per

test chamber:

12. No. replicate chambers

per concentration:

13. No. organisms

per concentration:

14. Feeding regime:

Static non-renewal, static-renewal, or flowthrough

24, 48, or 96 h

Ambient laboratory illumination

10-20 ~E/m2/s (50-100 ft-c)

(ambient laboratory levels)

16 h light, 8 h darkness

250 mL (minimum)

200 mL (minimum)

Minimum, after 48 h

1-14 days; 24-h range in age

Minimum, 10 for effluent and receiving water

tests

Minimum, 2 for effluent tests

Minimum, 4 for receiving water tests

Minimum, 20 for effluent tests

Minimum, 40 for receiVing water tests

Artemia nauplii are made available while

holding prior to the test; add 0.2 mL

Artemia nauplii concentrate 2 h prior to

test solution renewal at 48 h

2

Cyprinella leedsi (Bannerfin shiner, formerly Notropis leedsi) can be

used with the test conditions in thi3 table, where it is the required

test organism in discharge permits.

Acute and chronic toxicity tests perfcrmed simultaneously to obtain

acute/chronic ratios must use the same temperature and water hardness.

61
TABLE 13. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

FATHEAD MINNOW, PIMEPHALES PROMELAS, ACUTE TOXICITY TESTS WITH

EFFLUENTS AND RECEIVING WATERS (CONTINUED)

15. Test chamber cleaning: Cleaning not required

16. Test solution aeration: None, unless DO concentration falls below

4.0 mg/L; rate should not exceed 100

bubbles/min

17. Dilution water: Moderately hard synthetic water prepared using MILLIPORE MILLI-Q® or equivalent deionized water and reagent grade chemicals

or 20% DMW (see Section 7, Dilution Water),

receiving water, ground water, or synthetic

water, modified to reflect receiving water

hardness.

18. Test concentrations: Effluents: Minimum of five effluent

concentrations and a control

Receiving Waters: 100% receiving water and a

control

19. Dilution series:

20. Endpoint:

21. Sampling and sample

holding requirements:

22. Sample volume required:

23. Test acceptability criterion:

Effluents: ~0.5 dilution series

Receiving Waters: None, or ~0.5 dilution

series

Effluents: Mortality (LC50 or NOAEC)

Receiving Waters: Mortality (Significant

difference from control)

Effluents and Receiving Waters: Grab or

composite samples are used within 36 h of

completion of the sampling period.

2 L for effluents and receiving waters

90% or greater survival in controls

62
TABLE 14. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

RAINBOW TROUT, ONCORHYNCHUS MYKlSS, AND BROOK TROUT,

SALVELlNUS FONTlNALlS, ACUTE TOXICITY TESTS WITH EFFLUENTS AND

RECEIVING WATERS

1. Test type:

2. Test duration:

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. organisms per

test chamber:

12. No. replicate chambers

per concentration:

13. No. organisms per concentration:

14. Feeding regime:

15. Test chamber cleaning:

Static non-renewal, static-renewal, or flowthrough

24, 48, or 96 h

Ambient laboratory illumination

10-20 ~E/m2/s (50-100 ft-c)

(ambient laboratory levels)

16 h light, 8 h darkness. Light intensity

should be raised gradually over a 15 min

period at the beginning of the photoperiod, and lowered gradually at the end of the

photoperiod, using a dimmer switch or other

suitable device.

5 L (minimum) (test chambers should be

covered to prevent fish from jumping out)

4 L (minimum)

Minimum, after 48 h

Rainbow Trout: 15-30 days (after yolk sac

absorption to 30 days)

Brook Trout: 30-60 days

Minimum, 10 for effluent and receiving water

tests

Minimum, 2 for effluent tests

Minimum, 4 for receiving water tests

Minimum, 20 for effluent tests

Minimum, 40 for receiving water tests

Feeding not required

Cleaning not required

63
TABLE 14. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

RAINBOW TROUT, ONCORHYNCHUS MYKISS, AND BROOK TROUT,

SALVELINUS FONTINALIS, ACUTE TOXICITY TESTS WITH EFFLUENTS AND

RECEIVING WATERS (CONTINUED)

16. Test solution aeration: None, unless DO concentration falls below

6.0 mg/L; rate should not exceed iOO

bubbles/min

17. Dilution water: Moderately hard synthetic water prepared using MILLIPORE MILLI-Q® or equivalent deionized water and reagent grade chemicals

or 20% DMW (see Section 7, Dilution Water),

receiving water, ground water, or synthetic

water, modified to reflect receiving water

hardness.

18. Test concentrations: Effluents: Minimum of five effluent

concentrations and a control

Receiving Waters: 100% receiVing water and a

control

19. Dilution series:

20. Endpoint:

21. Sampling and sample holding requirements:

22. Sample volume reqUired:

23. Test acceptability criterion:

Effluents: ~0.5 dilution series

Receiving Waters: None, or ~0.5 dilution

series

Effluents: Mortality (LCSO or NOAEC)

ReceiVing Waters: Mortality (Significant difference from control)

Effluents and ReceiVing Waters: Grab or

composite samples are used within 36 h of

completion of the sampling period.

20 L for effluents

40 L for receiving waters

90% or greater survival in controls

64
TABLE 15. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

FOR MYSID, MYSIDOPSIS BAHIA, A~UTE TOXICITY TESTS WITH

EFFLUENTS AND RECEIVING WATERS

1. Test type:

2. Test duration:

3. Temperature: 2

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. organisms per

test chamber:

12. No. replicate chambers

per concentration:

13. No. organisms per concentration:

14. Feeding regime:

Static non-renewal, static-renewal, or flowthrough

24, 48, or 96 h

20°C ± 1°C; or 25°C ± 1°C

Ambient laboratory illumination

10-20 ~E/m2/s (50-100 ft-c)

(ambient laboratory levels)

16 h light, 8 h darkness

250 mL (minimum)

200 mL (minimum)

Minimum, after 48 h

1-5 days; 24-h range in age

Minimum, 10 for effluent and receiving water

tests

Minimum, 2 for effluent tests

Minimum, 4 for receiving water tests

Minimum, 20 for effluent tests

Minimum, 40 for receiving water tests

Artemja nauplii are made available while

holding prior to the test; feed 0.2 mL of

concentrated suspension of Artemja nauplii ~24-h old, daily (approximately 100 nauplii

per mysid)

'Homesjmysjs costata (mysid) can be used with the test conditions in this

table (except at a temperature of 12°C or 25°C, and a salinity of 32-34%,

where it is the required test organism in discharge permits.

2Acute and chronic toxicity tests performed simultaneously to obtain

acute/chronic ratios must use the same temperature and salinity.

65
TABLE 15. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

MYSID, MYSIDOPSIS BAHIA, ACUTE TOXICITY TESTS WITH EFFLUENTS

AND RECEIVING WATERS (CONTINUED)

15. Test chamber cleaning: Cleaning not required

16~ Test solution aeration: None, unless DO concentration falls below

4.0 mg/L; rate should not exceed 100

bubbles/min

17. Dilution water: 5-30~ ± 10%; Uncontaminated source of

seawater, deionized water mixed with

hypersaline brine or artificial sea salts

(HW MARINEMIX®, FORTY FATHOMS®, modified

GP2, or eqUivalent) prepared with MILLI-Q®,

or equivalent, deionized water (see Section

7, Dilution Water); or receiving water

18. Test concentrations: Effluents: Minimum of five effluent

concentrations and a control

Receiving Waters: 100% receiving water and a

control

19. Dilution series:

20. Endpoint:

21. Sampling and sample holding requirements:

22. Sample volume required:

23. Test acceptability criterion:

Effluents: ~0.5 dilution series

Receiving Waters: None, or ~0.5 dilution

series

Effluents: Mortality (LC50 or NOAEC)

Receiving Waters: Mortality (Significant

difference from control)

Effluents and Receiving Waters: Grab or

composite samples are used within 36 h of

completion of the sampling period.

1 L for effluents

2 L for receiving waters

90% or greater survival in controls

66
TABLE 16. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, ACUTE TOXICITY TESTS

WITH EFFLUENTS AND RECEIVING WATERS

1. Test type:

2. Test duration:

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:

II. No. organisms per

test chamber:

12. No. replicate chambers

per concentration:

13. No. organisms per concentration:

14. Feeding regime:

15. Test chamber cleaning:

Static non-renewal, static-renewal, or flowthrough

24, 48, 96 h

20°C ± 1°C; or 25°C ± 1°C

Ambient laboratory illumination

10-20 ~E/m2/s (50-100 ft-c)

(ambient laboratory levels)

16 h light, 8 h darkness

250 mL (minimum)

200 mL (minimum)

Minimum, after 48 h

1-14 days; 24-h range in age

Minimum, 10 for effluent and receiving water

tests

Minimum, 2 for effluent tests

Minimum, 4 for receiving water tests

Minimum, 20 for effluent tests

Minimum, 40 for receiving water tests

Artemia nauplii are made available while

holding prior to the test; add 0.2 mL

Artemia nauplii concentrate 2 h prior to

test solution renewal at 48 h

Cleaning not required

'Acute and chronic toxicity tests performed simultaneously to obtain

acute/chronic ratios must use the same temperature and salinity.

67
TABLE 16. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS, ACUTE TOXICITY TESTS

WITH EFFLUENTS AND RECEIVING WATERS (CONTINUED)

16. Test solution aeration: None, unless DO concentration falls below

4.0 mgjL; rate should nct exceed 100

bubbles/min

17. Dilution water: 5-32~ ± 10%; Uncontaminated source of

seawater, deionized water mixed with

hypersaline brine or artificial sea salts

(HW MARINEMIX®, FORTY FATHOMS®, modified

GP2, or equivalent) prepared with MILLI-Q®

or equivalent deionized water (see Section

7, Dilution Water); or receiving water

18. Test concentrations: Effluents: Minimum of five effluent

concentrations and a control

Receiving Waters: 100% receiving water and a

control

19. Dilution series:

20. Endpoint:

21. Sampling and sample holding requirements:

22. Sample volume required:

23. Test acceptability criterion:

Effluents: ~0.5 dilution series

Receiving Waters: None, or ~0.5 dilution

series

Effluents: Mortality (LC50 or NOAEC)

Receiving Waters: Mortality (Significant

difference from control)

Effluents and Receiving Waters: Grab or

composite samples are used within 36 h of

completion of the sampling period.

1 L for effluents

2 L for receiving waters

90% or greater survival in controls

68
TABLE 17. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR

SILVERSIDE, MENIDIA BERYLLINA, M. MENIDIA, AND M. PENINSULAE,

ACUTE TOXICITY TESTS WITH EFFLUENTS AND RECEIVING WATERS

1. Test type:

2. Test duration:

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. organisms per

test chamber:

12. No. replicate chambers

per concentration:

13. No. organisms per concentration:

14. Feeding regime:

15. Test chamber cleaning:

Static non-renewal, static-renewal, or flowthrough

24, 48, or 96 h

Ambient laboratory illumination

10-20 pE/m2/s (50-100 ft-c)

(ambient laboratory levels)

16 h light, 8 h darkness

250 mL (minimum)

200 mL (minimum)

Minimum, after 48 h

9-14 days; 24-h range in age

Minimum, 10 for effluent and receiving water

tests

Minimum, 2 for effluent tests

Minimum, 4 for receiving water tests

Minimum, 20 for effluent tests

Minimum, 40 for receiving water tests

Artemia nauplii are made available while

holding prior to the test; add 0.2 mL

Artemia nauplii concentrate 2 h prior to

test solution renewal at 48 h

Cleaning not required

'Acute and chronic toxicity tests performed simultaneously to obtain

acute/chronic ratios must use the same temperature and salinity.

69
TABLE 17. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

FOR SILVERSIDE, MENIDIA BERYLLINA, M. MENIDIA, AND M.

PENINSULAE, ACUTE TOXICITY TESTS WITH EFFLUENTS AND RECEIVING

WATERS (CONTINUED)

16. Test solution aeration: None, unless DO concentration falls below

4.0 mg/L; rate should not exceed 100

bubbles/min

17. Dilution water: Uncontaminated source of seawater, deionized

water mixed with hypersaline brine or

artificial sea salts (HW MARINEMIX®, FORTY

FATHOMS®, modified GP2, or equivalent) prepared with MILLI-Q® or equivalent deionized water (see Section 7, Dilution

Water): or receiving water: 1-32~ ± 10% for M. beryllina; 15-32~ ± 10% for M. menidia; and

M. peninsulae

18. Test concentrations: Effluents: Minimum of five effluent

concentrations and a control

Receiving Waters: 100% receiving water and a

control

19. Dilution series:

20. Endpoint:

21. Sampling and sample holding requirements:

22. Sample volume required:

23. Test acceptability criterion:

Effluents: ~0.5 dilution series

Receiving Waters: None, or ~0.5 dilution

series

Effluents: Mortality (LC50 or NOAEC)

Receiving Waters: Mortality (Significant difference from control)

Effluents and Receiving Waters: Grab or

composite samples are used within 36 h of

completion of the sampling period.

1 L for effluents

2 L for receiving waters

90% or greater survival in controls

70
SECTION 10

TEST DATA

10.1 BIOLOGICAL DATA

10.1.1 Death is the "effect" used for determining toxicity to aquatic

organisms in acute toxicity tests.

10.1.2 Death is not easily determined for some organisms. The criteria

usually employed in establishing death are: (1) no movement of gills or

appendages; and (2) no reaction to gentle prodding.

10.1.3 The death of some organisms, such as mysids and larval fish, is easily

detected because of a change in appearance from transparent or translucent to

opaque. General observations of appearance and behavior, such as erratic

swimming, loss of reflex, discoloration, excessive mucus production,

hyperventilation, opaque eyes, curved spine, hemorrhaging, molting, and

cannibalism, should also be noted in the daily record.

10.1.4 The test chambers should be checked for early mortality during the

first few hours of the test. The number of surviving organisms in each test

chamber is recorded at the end of each 24-h period (Figure 4). When

recognizable, dead organisms should be removed during each observation period.

10.1.5 The species, source, and age of the test organisms should be recorded.

10.2 CHEMICAL AND PHYSICAL DATA

10.2.1 In static tests, at a minimum pH, salinity or conductivity, and total

residual chlorine are measured in the highest concentration of test solution

and in the dilution water at the beginning of the test, at test solution

renewal, and at test termination. DO, pH, and temperature are measured in the

control and all test concentrations at the beginning of the test, daily

thereafter, and at test termination.

10.2.1.1 It is recommended that total alkalinity and total hardness also be

measured in the control and highest effluent concentration at the beginning of

the test and at test solution renewal.

10.2.1.2 Total ammonia is measured in samples where toxicity may be

contributed by unionized ammonia (where total ammonia might be ~5 mg/L).

10.2.1.3 The DO should be monitored closely (every 2 h) for the first 4 to

8 h, to guard against rapid DO depletion, and is measured daily thereafter in

all effluent concentrations in which there are surviving organisms, and at

test termination. It is recommended that test solution DO be recorded

continuously in the test chamber at the highest test solution concentration or

in a surrogate vessel at a comparable test solution concentration and

containing the standard complement of test organisms.

71
I I (DATE)

FROM: -- AM/PM; __I__I__(DATE)

INDUSTRY/TOXICANT:

_

ADDIRESS:

_

CONITACT:

EFFLUENT"""'S"'EO";;R"IA..L'--'ON""O-:---------------

NPDES PERMIT NO.:

SAMPLE COLLECTOR:---------------

GRAB SAMPLE: COLLECTED (1) AM/PM; / I (DATE)

(2) AM/PM; --I--I--(DATE)

(3) AM/PM; ==/==/==(DATE)

(4) AM/PM; __I__I__(DATE)

COMPOSITE SAMPLE: COLLECTED (1) FROM: AM/PM; / / (DATE)

______AM/PM; I I (DATE) -- -- --(2) FROM: AM/PM; I / (DATE

-- TO: AM/PM; I I (DATE) (3) FROM: 

-- TO: AM/PM; / / (DATE) 

-- 1'0:-- AM/PM; __I __I 

""-l

N

PERSON CONDUCTING TEST:

TEST PER 100:

BEGINNING: DATE TIME

_

ENDING: DATE TIME

_

TEST ORGANISM:

SPECIES:

_

AGE:

SOURCE:

DILUTION WA1ER-USEl):

01 SSOLVED OXYGEN (mg/"L')----,T...O...T..AL......R""E... S"'ID"'U"A'L.....A"'M""""MON I A : _

o IiRS FREE AMMONIA:

_

24 II

48 II

72 II

96 II

Cone. Test Number of Oxygen pH Total Alkalinity Total Hardness

or Container Li ve Organisms (mg/l) (mg/ l as (mg/l as Number 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 7

Figure 4. Example of data sheet for effluent toxicity tests.
Test solution surface area: cm2

1. EXPOSURE CHAMBER

Total capacity:

Test solution volume:

Water depth (constant):

(cyclic):

3. AERATION

None:

mL ---

mL ---

___ cm

--to cm ---

2. FEEDING SCHEDULE

Not Fed:---

Fed daily:

Fed irregularly:

(describe): _

Food used:-----

4. SCREENED ANIMAL

ENCLOSURES

Not used:

Slow: (Bubbles or mL/min) Used:

(cm) Diameter

Moderate:

Vigorous:

II

II

II

II

From: AM/PM; -1-1_ (DATE)

To: AM/PM; -1-.-1_ (DATE)

5. Condition/appearance of surviving organisms at end of test: (i.e., alive but

immobile; loss of orientation; erratic movement; etc.) _

6. Comment s : _

NPDES NO: Inspection Date: Outfall number:

Facil ity N'-am-e-:-- Test Date: Macro Test: --------

City Name: Inspection Code: Type Macro:

County Name: Type Inspection: Expo Time:

Receiving Water: Date Info to WSD: Resul-;-t-s-:------ Permit Issued: Date Info to State: Fish Test:

Permit Expires: Date of WMD Action: Type Fish: ---- SIC Code: Data of Static Action: Expo Time: ---------- Present Treatment: Type of Act ion: Results:

Remarks: Annua1 Status Update: Remarks: -----

Figure 5. Check list on back of effluent toxicity data sheet.

73
10.2.1.4 At a mlnlmum, test solution temperature is measured at the beginning of the test, and daily thereafter. Temperature measurements are made by placing thermometers or other temperature sensing devices directly in test

solutions or in a comparable volumes of water in chambers positioned in

several locations among the test vessels to determine test solution

temperatures. It is recommended that test solution temperature be recorded

continuously in at least one test chamber or in a comparable volume of water

in a surrogate vessel which is comparable to the test chambers.

10.2.2 In flow-through tests, at a minimum pH, salinity or conductivity, total alkalinity, total hardness, and total residual chlorine are measured

daily in the highest effluent concentration. 00 and temperature are measured

at the beginning of the test, daily thereafter in the control and all test

concentrations, and at test termination.

10.2.3 The measurement of specific conductance is recommended because it is a

very useful parameter in detecting transient fluctuations in the chemical

characteristics of effluents, and will indicate errors in test dilutions.

10.2.4 Where acute toxicity test methods are utilized to determine permit limits for toxic chemicals, at a minimum, the concentration of the test

material must be measured in each test concentration at test initiation, daily thereafter, and at test termination.

10.2.5 Methods used for chemical analysis should be those specified for

Section 304(h) of the CWA (USEPA, 1993c). For salinity measurements, a

refractometer may be used if calibrated with a sample of known salinity.

74
SECTION 11

ACUTE TOXICITY DATA ANALYSIS

11.1 INTRODUCTION

11.1.1 The objective of acute toxicity tests with effluents and recelvlng

waters is to identify discharges of toxic effluents in acutely toxic amounts.

Data are derived from tests designed to determine the adverse effects of

effluents and receiving waters on the survival of the test organisms. The

recommended effluent toxicity test consists of a control and five or more

concentrations of effluent (i.e., multi-effluent-concentration, or definitive

tests), in which the endpoint is (1) an estimate of the effluent concentration

which is lethal to 50% of the test organisms in the time period prescribed by the test, expressed as the LC50, or (2) the highest effluent concentration at

which survival is not significantly different from the control (No-ObservedAdverse-Effect Concentration, or NOAEC). Receiving water tests may be single

concentration or multi-concentration tests. The LC50 is determined by the

Graphical, Spearman-Karber, Trimmed Spearman-Karber, or Probit Method. The

NOAEC is determined by hypothesis testing.

11.1.2 Some states require tests consisting of a control and a single

concentration of effluent with a pass/fail endpoint. Control survival must be

90% or greater for an acceptable test. The test "passes" if survival in the

control and effluent concentration equals or exceeds 90%. The test "fails" if

survival in the effluent is less than 90%, and is significantly different from

control survival (which must be 90% or greater), as determined by hypothesis

testing.

11.1.3 The toxicity of receiving (surface) water can be determined with (1) a

paired test consisting of four replicates each of a suitable control and 100%

surface water, or (2) a multi-concentration test. The results of the first

type of test (100% receiving water and a control) are analyzed by hypothesis

testing. The results of the second type of test may be analyzed by hypothesis

testing or used to determine an LC50.

11.1.4 The data analysis methods recommended in this manual have been chosen

primarily because they are (1) well-tested and well-documented, (2) applicable

to most types of test data sets for which they are recommended, but still

powerful, and (3) most easily understood by non-statisticians. Many other

methods were considered in the selection process, and it is recognized that

the methods selected are not the only possible methods of analysis of acute

toxicity data.

11.1.5 ROLE OF THE STATISTICIAN

11.1.5.1 The use of the statistical methods described in this manual for

routine data analysis does not require the assistance of a statistician.

However, if the data appear unusual in any way, or fail to meet the necessary

assumptions, a statistician should be consulted. The choice of a statistical

method to analyze toxicity test data and the interpretation of the results of

75
the analysis of the data can become problematic if there are anomalies in the

data. Analysts who are not proficient in statistics are strongly advised to

seek the assistance of a statistician before selecting alternative methods of

analysis and using the results.

11.1.6 INDEPENDENCE, RANDOMIZATION, AND OUTLIERS

11.1.6.1 A critical assumption in the statistical analysis of toxicity data

is statistical independence among observations. Statistical independence

means that given knowledge of the true mean for a given concentration or

control, knowledge of the error in anyone actual observation would provide no

information about the error in any other observation. One of the best ways to

insure independence is to properly follow randomization procedures. The

purpose of randomization is to avoid situations where test organisms are

placed serially, by level of concentration, 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.

11.1.6.2 Another area for potential bias of results is the presence of

outliers. 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

statistical analysis should be performed both with and without the outlier,

and the results of both analyses should be reported. For a discussion of

techniques for evaluating outliers, see Draper and John (1981).

11.2 DETERMINATION OF THE LC50 FROM DEFINITIVE, MULTI-EFFLUENT-CONCENTRATION

ACUTE TOXICITY TESTS

11.2.1 The method used to estimate the LC50 from multi-concentration acute

toxicity tests depends on the shape of the tolerance distribution, and how

well the effluent concentrations chosen characterize the cumulative

distribution function for the tolerance distribution (i.e., the number of

partial mortalities). A review of effluent acute toxicity data from the last

248 tests performed by the Ecological Support Branch, Environmental Services

Division, EPA Region 4, indicated the following pattern in the number of

partial mortalities: (1) no partial mortalities (all or nothing response) - 28%; (2) one partial mortality - 54%; (3) two or more partial mortalities - 16%; (4) LC50 occurring at one of the test concentrations - 2%.

11.2.1.1 Four methods for estimating the LC50 are presented below: the

Graphical Method, the Spearman-Karber Method, the Trimmed Spearman-Karber

Method, and the Probit Method. The analysis scheme is shown in Figure 6.

Included in the presentation of each method is a description of the method,

the requirements for the method, a description of the calculations involved in

the method or a description of the computer program, and an example of the

calculations.

76
DETERMINATION OF THE lC50

FROM A MULTI-EFFLUENT-CONCENTRATION

ACUTE TOXICITY TEST

MORTALITY DATA

# DEAD

"

TWO OR MORE

PARTIAL MORTALITIES?

YES

"

IS PROBIT MODEL

APPROPRIATE?

(SIGNIFICANT X2 TEST)

YES

"

NO

NO

" ONE OR MORE NO GRAPHICAL METHOD

PARTIAL MORTALITIES?'" LC50

"YES

I PROBIT METHOD I

ZERO MORTALITY IN THE

LOWEST EFFLUENT CONC.

AND 100% MORTALITY IN THE

HIGHEST EFFLUENT CONC.?

YES

"

SPEARMAN-KARBER

METHOD

"

NO

l'

TRIMMED SPEARMAN

KARBER METHOD

.. LC50AND95%

CONFIDENCE

INTERVAL

....

-

Figure 6. Flowchart for determination of the LC50 for multi-effluentconcentration acute toxicity tests.

77
11.2.2 THE GRAPHICAL METHOD

11.2.2.1 Description

1. The Graphical Method is a mathematical procedure for calculating

the LC50.

2. The procedure estimates the LC50 by linearly interpolating between points of a plot of observed percent mortality versus

the base 10 logarithm (log,o) of percent effluent concentration.

3. It does not provide a confidence interval for the LC50 estimate.

4. Use of the Graphical Method is only recommended when there are

no partial mortalities.

11.2.2.2 Requirements

1. The only requirement for the Graphical Method is that the

observed percent mortalities bracket the 50%.

11.2.2.3 General Procedure

1. Let Pp' P" ... , Pk denote the observed proport ion mortal iti es

for the control and the k effluent concentrations. The first

step is to smooth the Pi if they do not satisfy Po ... Pk.

The smoothing replaces any adjacent Pi'S that do not conform to

Pp ... Pk' with their average. For example, if Pi is less

than Pi-" then:

where: p~ = the smoothed observed proportion mortality for effluent

concentration i.

2. 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:

p!=(p;-p:)/(l-p:)

where: p~ = the smoothed observed proportion mortality for the control.

3. 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.

4. Locate the two points on the graph which bracket 50% mortality and connect them with a straight line.

78
5. 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.

11.2.2.4 Example Calculation

1. All-or-nothing data (Graphical Method) in Table 18 are used in the

calculations. Note that in this case, the data must be smoothed and

adjusted for mortality in the controls.

2. To smooth the data, the observed proportion mortality for the

control and the lower three effluent concentrations must be

averaged. The smoothed observed proportion mortalities are as

follows: 0.0125, 0.0125, 0.0125, 0.0125, 1.0, and 1.0.

3. The smoothed responses are adjusted for control mortality (see

11.2.2.3), where the smoothed response for the control (p~) = 0.0125.

The smoothed, adjusted response proportions for the effluent

concentrations are as follows: 0.0, 0.0, 0.0, 1.0, and 1.0.

4. A plot of the smoothed, adjusted data is shown in Figure 7.

5. 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.

6. The point at which the plotted line intersects the 50% mortality

line is the estimated LC50. The estimated LC50 = 35% effluent.

TABLE 18. MORTALITY DATA (NUMBER OF DEAD ORGANISMS) FROM ACUTE TOXICITY

TESTS USED IN EXAMPLES OF LC50 DETERMINATIONS (20 ORGANISMS IN

THE CONTROL AND ALL TEST CONCENTRATIONS)

METHOD OF ANALYSIS

EFFLUENT GRAPHICAL SPEARMAN- TRIMMED PROBIT

CONe KARBER SPEARMAN-

(%) KARBER

CONTROL 1 1 1 a

6.25% a 1 a a

12.5% a a 2 3

25.0% 0 0 0 9

50.0% 20 13 0 20

100.0% 20 20 16 20

79
100

90

80

70

60

50

W

40

:E 30 ::>

.....J

20

I- 15 Z

W

::>

.....J 10

u.. 9

u.. 8

W 7

I- 6

Z 5

W

(,) 4

([

W 3 a..

2

f...--

l-- r - -- .... - 4r4~

.~

1

o 10 20 30 40 50 60 70 80 90 100

PERCENT MORTALITY

Figure 7. Plotted data and fitted line for graphical method, using all-ornothing data.

80
11.2.3 THE SPEARMAN-KARBER METHOD

11.2.3.1 Description

L

2.

3.

4.

5.

The Spearman-Karber Method is a nonparametric statistical procedure for

estimating the LC50 and the associated 95% confidence interval (Finney,

1978).

This procedure estimates the mean of the distribution of the 109, of

the tolerance. If the log tolerance distribution is symmetric, t~is estimate of the mean is equivalent to an estimate of the median of the

log tolerance distribution.

If the response proportions are not monotonically non-decreasing with

increasing concentration (constant or steadily increasing with

concentration), the data are smoothed.

Abbott's procedure is used to "adjust" the test results for mortality

occurring in the control.

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.

11.2.3.2 Requirements

1. To calculate the LC50 estimate, the following must be true:

a. The smoothed adjusted proportion mortality for the lowest

effluent concentration (not including the control) must be zero.

b. The smoothed adjusted proportion mortality for the highest effluent concentration must be one.

2. 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.

11.2.3.3 General Procedure

1. The first step in the estimation of the LC50 by the Spearman-Karber Method is to smooth the observed response proportions, Pi if they do

not satisfy Po ... Pk (see 11.2.2.3, Step 1). '

2. Adjust the smoothed observed proportion mortality in each effluent

concentration for mortality in the control group using Abbott's formula

(see 11.2.2.3, Step 2).

3. 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.

81
4. Calculate the log,o of the estimated LC50, m, as follows:

m=1!

i=l

(pt+l -pt) (Xi+Xi +1)

2

where p~ = the smoothed adjusted proportion mortality at concentration i

X. the log,o of concentration i

k

1

= the number of effluent concentrations tested, not including

the control.

5. Calculate the estimated variance of m as follows:

V(m) = Pia (l-pt) (Xi+1-Xi -1) 2

i=2 4 (ni-l)

where Xi = the log,o of concentration i

n· = the number of organisms tested at effluent concentration i

pt = the smoothed adjusted observed proportion mortality at

effluent concentration i

k = the number of effluent concentrations tested, not including the control.

6. Calculate the 95% confidence interval for m: m ± 2.0

7. The estimated LC50 and a 95% confidence interval for the

estimated LC50 can be found by taking base,o antilogs of the

above values.

8. With the exclusion of the plot in item 3, the above calculations

can be carried out using the Trimmed Spearman-Karber computer

program mentioned in 11.2.4.3 and 11.2.4.4.

11.2.3.4 Example Calculation

1. Mortality data from a definitive, multi-concentration, acute

toxicity test are given in Table 18. Note that the data must be

smoothed and adjusted for mortality in the controls.

2. To smooth the data, the observed proportion mortality for the

control, and the observed proportion mortality for the 6.25%,

12.5%, and 25% effluent concentrations must be averaged. The

smoothed observed proportion mortalities are as follows: 0.025,

0.025, 0.025, 0.025, 0.65, and 1.00.

82
3. To adjust the smoothed, observed proportion mortality in each

effluent concentration for mortality in the control group,

Abbott's formula must be used. After smoothing and adjusting, the

proportion mortalities for the effluent concentrations are as

follows: 0.000, 0.000, 0.000; 0.641, and 1.000.

4. The data will not be plotted for this example. For an example of

the plotting procedures, see 11.2.2.4.

5. The log,o of the estimated Le50, m, is calculated as follows:

m = [(0.0000 - 0.0000)(0.7959 + 1.0969)]/2 +

[(0.0000 - 0.0000)(1.0969 + 1.3979)]/2 +

[(0.6410 - 0.0000)(1.3979 + 1.6990)]/2 +

[(1.0000 - 0.6410)(1.6990 + 2.0000)]/2 = 1.656527

6. The estimated variance of m, V(m), is calculated as follows:

V(m) = (0.0000)(1.0000)(1.3979 - 0.7959)~4(19) +

(0.0000)(1.0000)(1.6990 - 1.0969)~4(19) +

(0.6410)(0.3590)(2.0000 - 1.3979) /4(19)

0.0010977

7. The 95% confidence interval for m is calculated as follows:

1.656527 ± 2 VO.0010977 = (1.5902639,1.7227901)

8. The estimated LC50 is as follows: antilog(1.656527) = 45.3%.

9. The upper limit of the 95% confidence interval for the estimated LC50 is

as follows:

antilog(1.7227901) = 52.8%

10. The lower limit of the 95% confidence interval for the estimated LC50 is

as follows:

antilog(1.5902639) = 38.9%

11.2.4 THE TRIMMED SPEARMAN-KARBER METHOD

11.2.4.1 Description

1. The Trimmed Spearman-Karber Method is a modification of the SpearmanKarber nonparametric statistical procedure for estimating the LC50 and

the associated 95% confidence interval (Hamilton, et al, 1977).

2. This procedure estimates the trimmed mean of the distribution of

the 109,0 of the tolerance. If the 109 tolerance distribution is

symmetrlc, this estimate of the trimmed mean is equivalent to an

estimate of the median of the log tolerance distribution.

83
3. Use of the Trimmed Spearman-Karber Method is only appropriate when

the requirements for the Probit Method and the Spearman-Karber

Method are not met.

11.2.4.2 Requirements

1. To calculate the LC50 estimate with the Trimmed Spearman-Karber Method, the smoothed, adjusted, observed proportion mortalities must

bracket 0.5.

2. To calculate a confidence interval for the LC50 estimate, one or

more of the smoothed, adjusted, observed proportion mortalities

must be between zero and one.

11,2.4.3 General Procedure

1. Smooth the observed proportion mortalities as described in

11.2.2.3, Step 1.

2. Adjust the smoothed observed proportion mortality in each effluent

concentration for mortality in the control group using Abbott's

formula (see 11.2.2.3, Step 2).

3. Plot the smoothed, adjusted data as described in 11.2.2.3, Step 3.

4. Calculate the amount of trim to use in the estimation of the LC50

as follows:

Trim = max(p~, 1 - p:)

where: p~ = the smoothed, adjusted proportion mortality for the lowest

effluent concentration, exclusive of the control.

the smoothed, adjusted proportion mortality for the highest

effluent concentration.

the number of effluent concentrations, exclusive of the

control.

5. Due to the intensive nature of the calculation for the estimated LC50

and the calculation for the associated 95% confidence interval using the Trimmed Spearman-Karber Method, it is recommended that the data

be analyzed by computer.

6. A machine-readable, compiled, version of a computer program which

estimates the LC50 and associated 95% confidence interval using the Trimmed Spearman-Karber Method can be obtained from

EMSL-Cincinnati by sending a diskette with a written request to

the Ecological Monitoring Research Division, Environmental

Monitoring Systems Laboratory, at 26 W. Martin Luther King Drive, ~~--~ __ ~+~ nu AC~CO

l. 111l. 1IIIIa l.. I, VII 't,JLVO.

84
7. The modified program automatically performs the following functions:

a. Smoothi ng. b. Adjustment for mortality in the control.

c. Calculation of the trim.

d. Calculation of the LC50.

e. Calculation of the associated 95% confidence interval.

11.2.4.4 Example Calculation Using the Computer Program

1. Data from Table 18 are used to illustrate the analysis using the

Trimmed Spearman-Karber program.

2. The program requests the following input (see Figure 8):

a. Output destination (D = disk file or P = printer).

b. Title for output.

c. Control data.

d. Data for each toxicant concentration.

3. The program output includes the following (see Figure 9):

a. A table of the concentrations tested, number of organisms exposed,

and mortal ities.

b. The amount of trim used in the calculation.

c. The estimated LC50 and the associated 95% confidence interval.

4. The analysis results for this example are as follows:

a. The observed proportion mortalities smoothed and adjusted for

mortality in the control.

b. The amount of trim used to calculate the estimate:

trim = max {O.OO, 0.204) = 0.204.

c. The estimate of the LC50 is 77.3%. The 95% confidence interval could

not be calculated for the data set.

11.2.5 THE PROBIT METHOD

11.2.5.1 Description

1. The Probit Method is a parametric statistical procedure for estimating

the LC50 and the associated 95% confidence interval (Finney, 1978).

2. The analysis consists of transforming the observed proportion mortalities

with a probit transformation, and transforming the effluent

concentrations to log,o'

3. Given the assumption of normality for the log,o of the tolerances, the

relationship between the transformed variables mentioned above is

approximately linear.

4. This relationship allows estimation of linear regression parameters, using an iterative approach.

85
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5

ENTER DATE OF TEST:

08/19/93

ENTER TEST NUMBER:

1

~HAT IS TO BE ESTIMATED?

(ENTER "L" FOR LC50 AND "E" FOR EC50)

L

ENTER TEST SPECIES NAME:

Fathead minnow

ENTER TOXICANT NAME:

Effluent

ENTER UNITS FOR EXPOSURE CONCENTRATION OF TOXICANT:

%

ENTER THE NUMBER OF INDIVIDUALS IN THE CONTROL:

20

ENTER THE NUMBER OF MORTALITIES IN THE CONTROL:

1

ENTER THE NUMBER OF CONCENTRATIONS

(NOT INCLUDING THE CONTROL; MAX = 10):

5

ENTER THE 5 EXPOSURE CONCENTRATIONS (IN INCREASING ORDER):

6.25 12.5 25 50 100

ARE THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION EQUAL(Y/N)?

Y

ENTER THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION:

20

ENTER UNITS FOR DURATION OF EXPERIMENT

(ENTER "H" FOR HOURS, "D" FOR DAYS, ETC.):

H

ENTER DURATION OF TEST:

96

ENTER THE NUMBER OF MORTALITIES AT EACH EXPOSURE CONCENTRATION:

o 2 0 0 16

~OULD YOU LIKE THE AUTOMATIC TRIM CALCULATION(Y/N)?

Y

Figure 8. Example of input for computer program for Trimmed Spearman-Karber Method.

86
TRIMMED SPEARMAN-KARBER METHOD, VERSION 1.5

DATE: 08/18/93 TEST NUMBER: 1 DURATION: 96 H

TOXICANT: Effluent

SPECIES: Fathead minnow

RAIJ DATA: Concentration Number Morta lities --. --.. (%) Exposed

.00 20 1

6.25 20 0

12.50 20 2

25.00 20 0

50.00 20 0

100.00 20 16

SPEARMAN-KARBER TRIM: 20.51%

SPEARMAN-KARBER ESTIMATES: Le50: 77.11

95% Lower Confidence: 69.74

95% Upper Confidence: 85.26

NOTE: MORTALITY PROPORTIONS IJERE NOT MONOTONICALLY INCREASING.

ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION.

WOULD YOU LIKE TO HAVE A COPY SENT TO THE PRINTER(Y/N)?

Figure 9. Example of output from computer program for Trimmed

Spearman-Karber Method.

87
5. The estimated LC50 and associated confidence interval are calculated from

the estimated linear regression parameters.

11.2.5.2 Requirements

1. To obtain a reasonably precise estimate of the LC50 with the Probit

Method, the observed proportion mortalities must bracket 0.5.

2. The log,o of the tolerance is assumed to be normally distributed.

3. To calculate the LC50 estimate and associated 95% confidence interval,

two or more of the observed proportion mortalities must be between zero

and one.

11.2.5.3 General Procedure

1. Due to the intensive nature of the calculations for the estimated LC50

and associated 95% confidence interval using the Probit Method, it is

recommended that the data be analyzed by a computer program.

2. A machine-readable, compiled, version of a computer program to estimate

the LCI and LC50 and associated 95% confidence intervals using the Probit

Method can be obtained from EMSL-Cincinnati by sending a diskette with a

written request to the Ecological Monitoring Research Division,

Environmental Monitoring Systems Laboratory, 26 W. Martin Luther King

Drive, Cincinnati, OH 45268.

11.2.5.4 Example Using the Computer Program

1. Data from Table 18 are used to illustrate the operation of the

Probit program for calculating the LC50 and the associated 95%

confidence interval.

2. The program begins with a request for the following initial input (see

Figure 10):

a. Desired output of abbreviated (A) or full (F) output?

b. Output designation (P = printer, D = disk file).

c. Title for the output.

d. Control data.

e. The number of exposure concentrations

f. Data for each toxicant concentration.

3. The program output for the abbreviated option includes the

following (see Figure 11):

a. A table of the observed proportion responding and the proportion

responding adjusted for controls.

b. The calculated chi-squared 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.

88
EPA PROB1T ANALYS1S PROGRAM

USED FOR CALCULATING LC/EC VALUES

Version 1.5

Do you wish abbreviated (A) of fulL (F) output? A

Output to printer or disk file (P / D)? P

Title? PROBIT EXAMPLE

Number of responders in the control group = ? 0

Number of exposure concentrations, exclusive of controls? 5

Input data starting with the lowest exposure concentration

Concentration =? 6.25

Number responding =? 0

Number exposed = ? 20

Concentration = ? 12.5

Number responding =? 3

Number exposed =? 20

Concentration = ? 25

Number responding =? 9

Number exposed =? 20

Concentration =? 50

Number responding =? 20

Number exposed = ? 20

Concentration =? 100

Number responding =? 20

Number exposed =? 20

Number Number

Number Cone. Resp. Exposed

--1- 6.2500 --0- -zo

2 12.5000 3 20

3 25.0000 9 20

4 50.0000 20 20

5 100.0000 20 20

Do you wish to modify your data? n The control response rate = 0

Do you wish to modify it? n

Figure 10. Example of input for computer program for Probit

Method.

89
EPA PROBIT ANALYSIS PROGRAM

USED FOR CALCULATING LC/EC VALUES

Version 1.5

PROBIT EXAMPLE

Proportion

Observed Responding

Number Number Proportion Adjusted for

Conc. Exposed Resp. Responding Controls

6.2500 20 0 0.000 0.000

12.5000 20 3 0.1500 0.1500

25.0000 20 9 0.4500 0.4500

50.0000 20 20 1.0000 1.0000

100.0000 20 20 1.0000 1.0000

Chi Square for Heterogeneity (calculated)

Chi Square for Heterogeneity

(tabular value at 0.05 level)

PROB IT EXAMPLE

=

=

3.076

7.815

Estimated lC/EC Values and Confidence Limits

Point Exposure

Conc.

Lower Upper

95% Confidence limits

LC/EC 1.00

LC/EC 50.00

7.924

22.872

4.147

18.787

10.959

27.846

Figure 11. Example of output for computer program for Probit

Method.

90
c. The estimated LCI and LC50 and 95% confidence limits.

d. A plot of the fitted regression line with observed data

overlaid on the plot.

4. The results of the data analysis for this example are as follows:

a. The observed proportion responding were not adjusted for mortality in

the control.

b. The test for heterogeneity was not significant (the calculated Chisquare was less than the tabular value), thus the Probit Method

appears to be appropriate for this data.

c. The estimate of the LC50 is 22.9% with a 95% confidence interval of

(18.8%, 27.8%).

11.3 DETERMINATION OF NO-OBSERVED-ADVERSE-EFFECT CONCENTRATION (NOAEC) FROM

MULTI-CONCENTRATION TESTS, AND DETERMINATION OF PASS OR FAIL (PASS/FAIL)

FOR SINGLE-CONCENTRATION (PAIRED) TESTS

11.3.1 Determination of the No-Observed-Adverse-Effect Concentration (NOAEC),

for multi-concentration toxicity tests, and pass or fail (Pass/Fail) for single- concentration toxicity tests is accomplished using hypothesis testing. The

NOAEC is the highest concentration at which survival is not significantly different from the control. In Pass/Fail tests, the objective is to determine

if the survival in the single treatment (effluent or receiving water) is

significantly different from the control survival.

11.3.2 The first step in these analyses is to transform the responses,

expressed as the proportion surviving, by the arc sine square root

transformation (Figures 12 and 13). The arc sine square root transformation is

commonly used on proportional data to stabilize the variance and satisfy the

normality and homogeneity of variance requirements. Shapiro-Wilk's test may be

used to test the normality assumption.

11.3.3 If the data do not meet the assumption of normality and there are four

or more replicates per group, then the nonparametric Wilcoxon Rank Sum Test can

be used to analyze the data.

11.3.4 If the data meet the assumption of normality, the F test for equality of

variances is used to test the homogeneity of variance assumption. Failure of

the homogeneity of variance assumption leads to the use of a modified t test,

where the pooled variance estimate is adjusted for unequal variance, and the

degrees of freedom for the test are adjusted.

11.3.5 GENERAL PROCEDURE

11.3.5.1 Arc Sine Square Root Transformation

11.3.5.1.1 The arc sine square root transformation consists of determining the

angle (in radians) represented by a sine value. In this transformation, the

proportion surviving is taken as the sine value, the square root of the sine

value is calculated, and the angle (in radians) for the square root of the sine

value is determined. Whenever the proportion surviving is 0 or 1, a special

91
modification of the transformation must be used (Bartlett, 1937). Illustrations

of the arc sine square root transformation and modification are provided below.

1. Calculate the response proportion (RP) for each replicate within a group,

where:

RP = (number of surviving organisms)/(number exposed)

2. Transform each RP, as follows.

a. For RPs greater than zero or 1ess than one:

Angle (in radians) = arc sinelTRPf

b. Modification of the arc sine when RP = O.

Angle (in radians) =arc sine~ 1

4n

where n = number animals/treatment replicate.

c. Modification of the arc sine when RP = 1.0.

Angle = 1.5708 radians- (radians for RP=O)

11.3.5.2 Shapiro-Wilk's Test

11.2.5.2.1 After the data have been transformed, test the assumption of

normality using Shapiro-Wilk's test. The test statistic, W, is obtained by dividing the square of an appropriate linear combination of the sample order

statistics by the usual symmetric estimate of variance (D). 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, and there must be more than two

replicates per concentration for the test to be valid.

1. To calculate W, first center the observations by subtracting the mean of

all the observations within a concentration from each observation in that

concentration.

92
DETERMINATION OF PASS OR FAIL

FROM A SINGLE -EFFLUENT-CONCENTRATION

ACUTE TOXICTY TEST

SURVIVAL DATA

PROPORTION SURVIVING

" ARCSINE

TRANSFORMATION

l'

NORMALITY?

(SHAPIRO-WILK'S TEST)

YES

"

NO ... WILCOXON RANK 1-------,

- SUM TEST

HOMOGENEITY OF VARIANCE NO "'_1 MODIFIED l

(F-TEST) I-----'--'---~. T-TEST r---I~~

YES

"

T-TEST

SIGNIFICANT DIFF.

IN SURVIVAL? ,...-----------'

NO I YES

t

1 PASS I

t

FAIL

Figure 12. Flowchart for analysis of single-effluent-concentration test data.

93
DETERMINATION OF THE NOAEC

FROM A MULTI-EFFLUENT-CONCENTRATION

ACUTE TOXICITY TEST

SURVIVAL DATA

PROPORTION SURVIVING

ARCSINE

•

TRANSFORMATION

" NORMALITY? NO

(SHAPIRO-WILK'S TEST)

" YES

YES HOMOGENEITY OF VARIANCE NO

(BARTLETT'S TEST) -...

"

NOl'EQUAL NUMBER OF

REPLICATES?

YES YES

EQUAL NUMBER OF

REPLICATES?

NO

f-

"

T-TESTWITH

BONFERRONI

ADJUSTMENT

DUNNETI'S

TEST

I

STEEL'S MANY-ONE

RANK TEST

I

ENDPOINT ESTIMATES

NOAEC

WILCOXON RANK SUM

TEST WITH

BONFERRONIADJUSTMENT

I

Figure 13. Flowchart for analysis of multi-effluent-concentration test data.

94
2. Calculate the denominator, 0, of the test statistic:

n

D=L (Xi-X) 2

i=l

where: Xi = the ith centered observation

X = the overall mean of the centered observations.

3. Order the centered observations from smallest to largest.

X(1) s X(2 ) S ••• s XCi)

where: X(i) denotes the ith ordered observation.

4. From Table 19, for the number of observations, n, obtain the

coefficients a" az' ... , ak, where k is n/2 if n is even, and (n - 1)/2

if n is odd.

5. Compute the test statistic, W, as follows:

w = [t a. (X (n-i+1) _ X (1)) ] 2

D i=l

11.3.5.2.2 The decision rule for the test is to compare the critical value from

Table 20 to the computed W. If the computed value is less than the critical

value, conclude that the data are not normally distributed.

11.3.5.3 F Test

11.3.5.3.1 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.

11.3.5.3.2 To make the two-tailed F test at the 0.01 level of significance, put

the larger of the two sample variances in the numerator of F.

11.3.5.3.3 Compare the calculated F with the 0.005 level of a tabulated F value

with n, - 1 and nz - 1 degrees of freedom, where n1 and nz are the number of

replicates for each of the two groups (Snedecor and Cochran, 1980). If the

calculated F value is less than or equal to the tabulated F, conclude that the

variances of the two groups are equal.

11.3.5.4 T Test

11.3.5.4.1 If the variances for the two groups are found to be statistically

equivalent, then the equal variance t test is the appropriate test.

95
TABLE 19. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST1 (CONOVER, 1980)

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

i\n 11 12 13 14 15 16 17 18 19 20

, 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 0.3290 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

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 0.0823 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

96
TABLE 19. COEFFrC IENTS FOR THE SHAPIRO-WILK'S TEST (CONTINUED)

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

;\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

97
TABLE 20. 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

98
11.3.5.4.2 Calculate the following test statistic:

where: X,

X2

Mean for the control

Mean for the effluent concentration

f(nl -l)S;+(n2 -1)S; S = ...!.V_-=-- ----=. _

p n l +n2 -2

S2 Estimate of the variance for the control 1

S2 Estimate of the variance for the effl uent concentration 2

n, = Number of replicates for the control

n2 Number of replicates for the effl uent concentration

11.3.5.4.3 Since we are concerned with a decrease in survival from the control,

a one-tailed test is appropriate. Thus, compare the calculated t with a

critical t, where the critical t is at the 5% level of significance with n, + n2

- 2 degrees of freedom. If the calculated t exceeds the critical t, the mean

responses are declared different.

11.3.5.5 Modified T Test

11.3.5.5.1 If the F test for equality of variance fails, the t test is still a

valid test. However, the denominator and the degrees of freedom for the test

are modified.

11.3.5.5.2 The t statistic, with the modification for the denominator, is

calculated as follows:

t = Xl - X2

S2 S2

_1 +_2

n l n2

where: X, Mean for the control

X2 = Mean for the effl uent concentration

S2

, -- Estimate of the variance for the control

99
S~ = Estimate of the variance for the effluent concentration

n, = Number of replicates for the control

n2 Number of replicates for the effluent concentration

11.3.5.5.3 Additionally, the degrees of freedom for the test are adjusted using

the following formula:

dfl = (n1 -1) (nz-l)

( n z - 1 ) c z + (1 - c ) z (n1 - 1 )

11.3.5.5.4 The modified degrees of freedom is usually not an integer. Common

practice is to round down to the nearest integer.

11.3.5.5.5 The modified t test is then performed in the same way as the equal

variance t test. The calculated t is compared to the critical t at the 0.05

significance level with modified degrees of freedom. If the calculated t

exceeds the critical t, the mean responses are found to be statistically

different.

11.3.5.6 Wilcoxon Rank Sum Test

11.3.5.6.1 If the data fail the test for normality and there are four or more

replicates per group, the nonparametric Wilcoxon Rank Sum Test may be used to

analyze the data. If less than four replicates were used, a nonparametric alternative is not available.

11.3.5.6.2 The Wilcoxon Rank Sum Test consists of jointly ranking the data and

calculating the rank sum for the effluent concentration. The rank sum is then

compared to a critical value to determine acceptance or rejection of the null

hypothesis.

11.3.5.6.3 To carry out the test, combine the data for the control and the

effluent concentration 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. Sum the ranks for the effluent concentration.

11.3.5.6.4 If the survival in the effluent concentration is significantly less

than that of the control, the rank sum for the effluent concentration would be

lower than the rank sum of the control. Thus, we are only concerned with

comparing the rank sum for the effluent concentration with some "minimum" or

100
critical rank sum, at or below which the effluent concentration survival would

be considered to be significantly lower than the survival in the control. For a

test at the 5% level of significance, the critical rank sum can be found in

Table 21.

11.3.6 SINGLE CONCENTRATION TEST

11.3.6.1 Data from an acute effluent toxicity test with Ceriodaphnia are

provided in Table 22. The proportion surviving in each replicate is transformed

by the arc sine square root transformation prior to statistical analysis of the

data (Figure 12).

11.3.6.2 After the data have been transformed, test the assumption of normality

with the Shapiro-Wilk's test.

11.3.6.2.1 The first step in 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 23.

11.3.6.2.2 Calculate the denominator, D, of the test statistic:

8

D= L (Xi -X)2

i=l

For this set of data, X = 0 and D = 0.060.

101
TABLE 22. DATA FROM AN ACUTE SINGLE-CONCENTRATION TOXICITY TEST WITH

CERIODAPHNIA

PROPORTION SURVIVING

100% EFFLUENT

REPLICATE CONTROL CONCENTRATION

A 1.00 0.40

RAW B 1.00 0.30

DATA C 0.90 0.40

D 0.90 0.20

ARC SINE A 1.412 0.685

TRANSFORMED B 1. 412 0.580

DATA C 1.249 0.685

D 1.249 0.464

1.330

0.0088

0.604

0.0111

TABLE 23. EXAMPLE OF SHAPIRO-WILK'S TEST: CENTERED OBSERVATIONS

TREATMENT

A B

REPLICATE

C D

Control

100% Effluent

0.082

0.081

0.082

-0.024

-0.081

0.081

-0.081

-0.140

11.3.6.2.3 Order the centered observations from smallest to largest. The

ordered observations are listed in Table 24.

11.3.6.2.4 From Table 1, for n = 8 and k = n/2 = 4, obtain the coefficients

a" a2 , ... , ak • The aj values are listed in Table 25.

102
TABLE 24. EXAMPLE OF SHAPIRO-WILK'S TEST: ORDERED OBSERVATIONS

1

2

3

4

5

6

7

8

-0.140

-0.081

-0.081

-0.024

0.081

0.081

0.082

0.082

11.3.6.2.5 Compute the test statistic, W, as follows:

w= 1 '(0.2200)2=0.0807 0.060

The differences, X(n-i+1) - X(i), are listed in Table 25.

11.3.6.2.6 From Table 20, the critical Wvalue for n = 8 and a significance

level of 0.01, is 0.749. Since the calculated W, 0.807, is not less than the

critical value the conclusion of the test is that the data are normally

distributed.

11.3.6.3 The F test for equality of variances is used to test the homogeneity

of variance assumption.

103
TABLE 25. EXAMPLE OF SHAPIRO-WILK'S TEST: TABLE OF COEFFICIENTS AND

DIFFERENCES

ai

Xcn- i+l) _ XCi)

1 0.6052 0.222 X(8) _ X(' )

2 0.3164 0.163 X(7) _ X(2)

3 0.1743 0.162 X(6) _ X(3 )

4 0.0561 0.105 X(5) _ X(4)

11.3.6.3.1 From Table 22, obtain the sample variances for the control and the

100% effluent. Since the var~ability of the 100% effluent is greater than the

variability of the control, S for the 100% effluent concentration is placed in

the numerator of the F statistic and S2 for the control is placed in the

denominator.

F= 0.0111 =1.2614

0.0088

11.3.6.3.2 There are four replicates for the control and four replicates for

the 100% effluent concentration. Thus there are three degrees of freedom for

the numerator and the denominator. For a two-tailed test at the 0.01 level of

significance, the critical F value is 47.467. The calculated F, 1.2614, is less

than the critical F, 47.467, thus the conclusion is that the variances of the

control and 100% effluent are equal.

11.3.6.4 The assumptions of normality and homogeneity of variance have been met

for this data set. An equal variance t test will be used to compare the mean

responses of the control and 100% effluent.

11.3.6.4.1

Table 22.

- - 2 2 To perform the t test, obtain the values for X" X2, S" and S2 from

Calculate the t statistic as follows:

1.330-0.604 t=-------

O. 0997~ 1 + 1 4 4

where:

s =/(4-1)0.0088+(4-1) (0.0111)

p 4 +4-2

104
11.3.6.4.2 For a one-tailed test at the 0.05 level of significance with 6

degrees of freedom, the critical t value is 1.9432. Since the calculated t,

10.298, is greater than the critical t, the conclusion is that the survival in

the 100% effluent concentration is significantly less than the survival in the

control.

11.3.6.5 If the data had failed the normality assumption, the appropriate analysis would have been the Wilcoxon Rank Sum Test. To provide an example of

this test, the survival data from the t test example will be reanalyzed with the

nonparametric procedure.

11.3.6.5.1 The first step in the Wilcoxon Rank Sum Test is to combine the data

from the control and the 100% effluent concentration and arrange the values in

order of size, from smallest to largest.

11.3.6.5.2 Assign ranks to the ordered observations, a rank of 1 to the

smallest, 2 to the next smallest, etc. The combined data with ranks assigned is

presented in Table 26.

TABLE 26. EXAMPLE OF WILCOXON'S RANK SUM TEST: ASSIGNING RANKS

TO THE CONTROL AND 100% EFFLUENT CONCENTRATIONS

PROPORTION CONTROL

RANK SURVIVING OR 100%

EFFLUENT

1 0.20 100% EFFLUENT

2 0.30 100% EFFLUENT

3.5 0.40 100% EFFLUENT

3.5 0.40 100% EFFLUENT

5.5 0.90 CONTROL

5.5 0.90 CONTROL

7.5 1.00 CONTROL

7.5 1.00 CONTROL

11.3.6.5.3 Sum the ranks for the 100% effluent concentration.

11.3.6.5.4 For this set of data, the test is for a significant reduction in

survival in the 100% effluent concentration as compared to the control. The

critical value, from Table 21, for four replicates in each group and a

significance level of 0.05 is 11. The rank sum for the 100% effluent

concentration is 10 which is less than the critical value of 11. Thus the

conclusion is that survival in the effluent concentration is significantly less

than the control survival.

105
11.3.7 MULTI-CONCENTRATION TEST

11.3.7.1 Formal statistical analysis of the survival data is outlined in

Figure 13. The response used in the analysis is the proportion of animals

surviving in each test or control chamber. Concentrations at which there is

no survival in any of the test chambers are excluded from statistical

determination of the NOAEC.

11.3.7.2 For the case of equal numbers of replicates across all

concentrations and the control, the determination of the NOAEC endpoint is

made via a parametric test, Dunnett's Procedure, or a nonparametric test,

Steel's Many-one Rank Test, on the arc sine 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 fail, the nonparametric test, Steel's Many-one Rank Test, is used

to determine the NOAEC endpoints. If the assumptions of Dunnett's Procedure

are met, the endpoints are estimated by the parametric procedure.

11.3.7.3 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 a Bonferroni adjustment. The

Wilcoxon Rank Sum Test with the Bonferroni adjustment is the nonparametric alternative.

11.3.7.4 Example of Analysis of Survival Data

11.3.7.4.1 This example uses survival data from a fathead minnow test. The

proportion surviving in each replicate must first be transformed by the arc

sine square root transformation procedure. The raw and transformed data,

means and standard deviations of the transformed observations at each toxicant

concentration and control are listed in Table 27. A plot of the survival

proportions is provided in Figure 14.

11.3.7.4.2 Test for Normality

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 28.

106
CONNECTS THE MEAN VALUE FOR EACH CONCENTRATION

REPRESENTS THE CRrnCAlVALUE FOR DUNNETT'S TEST

(ANY PROPORTION BELOW THIS VALUE WOULD BE

SIGNIFICANTLY DIFFERENT FROM THE CONTROL)

1.0,* * * * *

0.6

0.5

0.4

*

*

*

*

*

*

0.7

0.9-.1*

0.3

z 0.8

o

i=

a:

o

a..

o

a:

a..

...J

>a:

:::>

en

.......

o

........

64 128 256

TOXICANT CONCENTRATION (1l9/L)

o 32

I I

*

Figure 14. Plot of mean survival proportion data in Table 27.
TABLE 27. FATHEAD MINNOW SURVIVAL DATA

TOXICANT CONCENTRATION CUG/U

REPLICATE CONTROL 32 64 128 256 512

A 1.0 0.8 0.9 0.9 0.7 0.4

RAW B 1.0 0.8 1.0 0.9 0.9 0.3

C 0.9 1.0 1.0 0.8 1.0 0.4

D 0.9 0.8 1.0 1.0 0.5 0.2

ARC SINE A 1. 412 1.107 1.249 1.249 0.991 0.685

TRANS- B 1.412 1.107 1.412 1.249 1.249 0.580

FORMED C 1.249 1. 412 1.412 1.107 1. 412 0.685

D 1.249 1.107 1.412 1. 412 0.785 0.464

MEfN(Y) 1.330 1.183 1.371 1.254 1.109 0.604

~i 0.0088 0.0232 0.0066 0.0155 0.0768 0.0111

1 1 2 3 4 5 6

TABLE 28. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

REPLICATE

A

B

C

D

CONTROL

0.082

0.082

-0.081

-0.081

TOXICANT CONCENTRATION (UG/L)

32 64 128 256 512

-0.076 -0.122 -0.005 -0.118 0.081

-0.076 0.041 -0.005 0.140 -0.024

0.229 0.041 -0.147 0.303 0.081

-0.076 0.041 0.158 -0.324 -0.140

2. Calculate the denominator, D, of the statistic:

n

D=:E (Xi-X) 2

i=l

where: Xi = the ith centered observation

X = the overall mean of the centered observations

n = the total number of centered observations

108
3. For this set of data: n = 24 (number of observations)

x= __1__ (0.000) = 0.000

24

D= 0.4265

4. Order the centered observations from smallest to largest

X(1) :5 X(2) :5 ••• :5 X,n)

where: X(i) denotes the ith ordered observation.

The ordered observations for this example are listed in Table 29.

TABLE 29. ORDERED CENTERED OBSERVATIONS FOR THE SHAPIRO-WILK'S EXAMPLE

1

2

3

4

5

6

7

8

9

10

11

12

-0.324

-0.147

-0.140

-0.122

-0.118

-0.081

-0.081

-0.076

-0.076

-0.076

-0.024

-0.005

13

14

15

16

17

18

19

20

21

22

23

24

-0.005

0.041

0.041

0.041

0.081

0.081

0.082

0.082

0.140

0.158

0.229

0.303

5. From Table 17, for the number of observations, n, obtain the

coefficients a1, a~, ... ak , where k is approximately n/2 if n is even;

(n-l)/2 if n is odd. For the data in this example, n = 24 and k = 12.

The ai values are listed in Table 30.

109
6. Compute the test statistic, W, as follows:

W = .! [t a. (X (n-i+l) - X (i)) ] 2

D i=l

The differences X(n-i+1) - XCi) are 1isted in Table 30. For the data in

this example,

1

W= (0.6444)2 = 0.974

0.4265

TABLE 30. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE

ai X(n-i+1) _ XCi)

1 0.4493 0.627 X(24) _ X( 1)

2 0.3098 0.376 X(23) _ X(2)

3 0.2554 0.298 X(22) _ X(3)

4 0.2145 0.262 X(21 ) _ X(4)

5 0.1807 0.200 X(20) _ Xes)

6 0.1512 0.163 X(19) _ X(6)

7 0.1245 0.162 X(18) _ X(7)

8 0.0997 0.157 X(17) _ X(8)

9 0.0764 0.117 X( 16) _ X(9)

10 0.0539 0.117 X(1S) _ X(10)

11 0.0321 0.065 X(14) _ X(11)

12 0.0107 0.0 X(13) _ X(12)

7. The decision rule for this test is to compare Was calculated in #6 to a

critical value found in Table 21. If the computed Wis less than the

critical value, conclude that the data are not normally distributed.

For the data in this example, the critical value at a significance level

of 0.01 and n = 24 observations is 0.884. Since W= 0.974 is greater

than the critical value, conclude that the data are normally distributed.

110
B =

where: Vi

11.3.7.4.3 Test for Homogeneity of Variance

1. The test used to examine whether the variation in mean proportion

surviving is the same across all toxicant concentrations including the

control, is Bartlett's Test (Snedecor and Cochran, 1980). The test

statistic is as follows:

[(t VJ in EP - tv. in 51]

i=l i=l

C

degrees of freedom for each toxicant concentration and

control, Vi = (n i - 1)

ni = the number of replicates for concentration i.

In = loge

i 1, 2, ... , P where p is the number of concentrations

including the control

(t ViS})

If = i=l

tv.

i=l

C=l + [3 (p-l)] -1 [t l/V. - (t V.) -1]

i=l i=l

2. For the data in this example, (See Table 27) all toxicant concentrations

including the control have the same number of replicates (n i = 4 for all

i) . Thus, Vi = 3 for all i.

3. Bartlett's statistic is therefore:

B = [(18)ln(O.0236) - 3tin(sj)]/1.1296

i=l

[18(-3.7465) - 3(-24.7516)]/1.1296

6.8178/1.1296

6.036

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 15.086. Since B = 6.036 is less

than the critical value of 15.086, conclude that the variances are not

different.

III
11.3.7.4.4 Dunnett's Procedure

1. To obtain an estimate of the pooled variance for the Dunnett's

Procedure, construct an ANOVA table (Table 31).

TABLE 31. ANOVA TABLE

SOURCE

BETWEEN

WITHIN

Total

DF

P - 1

N - P

N - 1

SUM OF SQUARES

(SS)

SSB

SSW

SST

MEAN SQUARE(MS)

(SS/DF)

Sa2 = SSB/(P-1)

S~ = SSW/(N-P)

where: p = number toxicant concentrations including the control

N = total number of observations n1 + n ... + np

nj = number of observations in concentration i

SSE= f T}/ni -G2 /N Between Sum of Squares

i=l

SST=f EY}j-G2 /N Total Sum of Squares

i=lj=l

SSW = SST - SSB Within Sum of Squares

G = the grand total of all sample observations, G=fT.

i=l

y.. = 1 J

the total of the replicate measurements for concentration Hi"

the jth observation for concentration Hi" (represents

the proportion surviving for toxicant concentration i in test

chamber j)

112
2. For the data in this example:

N = 24

T1 = Y11 + Y12 + YB + Y14 = 5.322

T2 = Y21 + Y22 + Y23 + Y24 = 4.733

T3 = Y31 + Y32 + Y33 + Y34 = 5.485

T4 = Y41 + Y42 + Y43 + Y44 = 5. 017

Ts = YS1 + YS2 + YS3 + YS4 = 4.437

T6 = Y61 + Y62 + Y63 + Y64 = 2.414

G = T1 + T2 + T3 + T4 + TS + T6 = 27.408

SSB = t Tf/ni -G2 /N

i=l

= __1__ (131.495) - (27.408)2 = 1.574

4 24

SST = t Ey? -G2

. . ~J /N ~=lJ=l

= 33.300 - (27.408)2 2.000

24

SS~ = SST - SSB = 2.000 - 1.574 = 0.4260

SB = SSB/(p - 1) = 1.574/(6 - 1) = 0.3150

Sw2 = SSW/(N - p) = 0.426/(24 - 6) = 0.024

113
3. Summarize these calculations in the ANOVA table (Table 32).

TABLE 32. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE

Source

BETWEEN

WITHIN

Total

OF

5

18

23

SUM OF SQUARES

(5S)

1. 574

0.426

2.002

MEAN SQUARE(MS)

(SS/DF)

0.315

0.024

4. To perform the individual comparisons, calculate the t statistic for

each concentration and control combination as follows:

(Y1 - YJ

t i = -::s::-v.;--;::;:(;::l!::;:n::::l~)=+:::;:;(l;:!:;::n:;:i~)

= mean proportion surviving for concentration = mean proportion survlvlng for the control

square root of the within mean square = number of replicates for the control = number of replicates for concentration i.

5. Table 33 includes the calculated t values for each concentration and

control combination. In this example, comparing the 32 ~g/L

concentration with the control, the calculation is as follows:

t = (1.330-1.183) =1.341

2 [0.155v'(1!4)+(1!4)]

6. Since the purpose of this test is to detect a significant reduction in

proportion surviving, a one-sided test is appropriate. The critical

value for this one-sided test is found in Table 34. For an overall

alpha level of 0.05, 18 degrees of freedom for error and five

concentrations (excluding the control) the critical value is 2.41. The

mean proportion surviving for concentration Iii" is considered

significantly less than the mean proportion surviving for the control if

t j is greater than the critical value. Since t 6 is greater than 2.41,

the 512 ~g/L concentration has significantly lower survival than the

control. Hence the NOAEC for survival is 256 ~g/L.

114
TABLE 33. CALCULATED T VALUES

TOXICANT CONCENTRATION (~G/L)

32

64

128

256

512

2

3

4

5

6

1.341

-0.374

0.693

2.016

6.624

7. To quantify the sensitivity of the test, the minimum significant difference (MSD) that can be detected statistically may be calculated.

MSD=d Sw!(1/n1 } + (l/n)

Where: d = the critical value for the Dunnett's procedure

Sw = the square root of the within mean square

n = the common number of replicates at each concentration

(this assumes equal replication at each concentration)

nj = the number of replicates in the control.

8. In this example:

MSD = 2 . 41 (0 . 155) ..; (1/4) + ( 1/4 )

2.41 (0.155)(0.707)

0.264

9. The MSD (0.264) is in transformed units. To determine the MSD in terms

of percent survival, carry out the following conversion.

(1) Subtract the MSD from the transformed control mean.

1.330 - 0.264 = 1.066

(2) Obtain the untransformed values for the control mean and the

difference calculated in 1.

[Sine (1.330) ]2 = 0.943

[Sine (1.066) ]2 = 0.766

115
(3) The untransformed MSD (MSD~) is determined by subtracting the

untransformed values from l.

MSDu = 0.943 - 0.766 = 0.177

10. Therefore, for this set of data, the minimum difference in mean

proportion surviving between the control and any toxicant

concentration that can be detected as statistically significant is

0.177.

11. This represents a decrease in survival of 19% from the control.

116
......

......

"'"-J

TABLE 34. DUNNETT'S "T" VALUES (MILLER, 1981)

(One' tai ted)cJak

a = .05

1 2 3 4 5 6 7 8 

5 2.02 2.44 2.58 2.85 2.98 3.08 3.16 

6 1.94 2.34 2.56 2.71 2.83 2.92 3.00 

7 1.89 2.27 2.48 2.62 2.73 2.82 2.89 

8 1.86 2.22 2.42 2.55 2.66 2.74 2.81 

9 1.83 2.18 2.37 2.50 2.60 2.68 2.75 

10 1.81 2.15 2.34 2.47 2.56 2.64 2.70 

11 1.80 2.13 2.31 2.44 2.53 2.60 2.67 

12 1.78 2.11 2.29 2.41 2.50 2.58 2.64 

13 1.77 2.09 2.27 2.39 2.48 2.55 2.61 14 1.76 2.08 2.25 2.37 2.46 2.53 2.59 15 1.75 2.07 2.24 2.36 2.44 2.51 2.57 

16 1. 75 2.06 2.23 2.34 2.43 2.50 2.56 

17 1.74 2.05 2.22 2.33 2.42 2.49 2.54 

18 1.73 2.04 2.21 2.32 2.41 2.48 2.53 

19 1.73 2.03 2.20 2.31 2.40 2.47 2.52 

20 1. 72 2.03 2.19 2.30 2.38 2.46 2.51 

24 1. 71 2.01 2.17 2.28 2.36 2.43 2.48 

30 1. 70 1.99 2.15 2.25 2.33 2.40 2.45 

40 1.68 1.97 2.13 2.23 2.31 2.37 2.42 

60 1.67 1.95 2.10 2.21 2.28 2.35 2.39 

120 1.86 1.93 2.08 2.18 2.26 2.32 2.37 

a 1.64 1.92 2.06 2.16 2.23 2.29 2.34 
......

......

co

TABLE 34. DUNNETT'S "T" VALUES (CONTINUED) (MILLER, 1981)

(One-tailed)c:P

(l = 0.1

1 2 3 4 5 6 7 8 

5 3.37 3.90 4.21 4.43 4.50 4.73 4.85 

6 3.14 3.61 4.88 4.07 4.21 4.33 4.43 

7 3.00 3.42 3.56 3.83 3.96 4.07 4.15 

8 2.90 3.20 3.51 3.67 3.79 3.88 3.96 

9 2.82 3.19 3.40 3.55 3.64 3.75 3.82 

10 2.76 3.11 3.31 3.45 3.56 3.64 3.71 

11 2.72 3.06 3.25 3.38 3.46 3.56 3.63 

12 2.68 3.01 3.19 3.32 3.42 3.50 3.56 13 2.65 2.97 3.15 3.27 3.37 3.44 3.91 14 2.62 2.94 3.11 3.23 3.32 3.40 3.46 

15 2.60 2.91 3.08 3.20 3.29 3.36 3.42 

16 2.58 2.38 3.05 3.17 3.28 3.33 3.39 

17 2.57 2.86 3.03 3.14 3.23 3.30 3.36 

18 2.55 2.84 3.01 3.12 3.21 3.27 3.33 

19 2.54 2.83 2.99 3.10 3.18 3.25 3.31 

20 2.53 2.81 2.97 3.08 3.17 3.23 3.29 

2.40 2.77 2.92 3.03 3.11 3.17 3.22 

30 2.46 2.72 2.87 2.97 3.05 3.11 3.16 

40 2.42 2.68 2.82 2.92 2.99 3.06 3.10 

60 2.39 2.64 2.78 2.87 2.94 3.08 3.04 

120 2.36 2.60 2.73 2.82 2.90 2.94 2.90 

(l 2.33 2.56 2.68 2.77 2.34 2.90 2.83 
SECTION 12

REPORT PREPARATION

The following general format and content are recommended for the report:

12.1 INTRODUCTION

1. Permit number

2. Toxicity testing requirements of permit

3. Plant location

4. Name of receiving water body

5. Contractor (if contracted)

a. Name of firm

b. Phone number

c. Address

12.2 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 discharge (MGO, CFS, GPM)

8. Design flow of treatment facility at time of sampling

12.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION WATER

1. Effluent Samples

a. Sampling point

b. Sample collection method

c. Collection dates and times

d. Mean daily discharge on sample collection date

e. Lapsed time from sample collection to delivery

f. Sample temperature when received at the laboratory

g. Physical and chemical data

2. Receiving Water Samples

a. Sampling point

b. Sample collection method

c. Collection dates and times

d. Streamflow at time of sampling and 7Q10

e. Lapsed time from sample collection to delivery

f. Sample temperature when received at the laboratory

g. Physical and chemical data

119
3. Dilution Water Samples

a. Source

b. Collection date(s) and time(s) (where applicable)

c. Pretreatment

d. Physical and chemical characteristics (pH, hardness, salinity, etc.)

12.4 TEST CONDITIONS

1. Toxicity test method used (title, number, source)

2. Endpoint(s) of test

3. Deviations from reference method, if any, and reason(s)

4. Date and time test started

5. Date and time test terminated

6. Type and volume of test chambers

7. Volume of solution used per chamber

8. Number of organisms per test chamber

9. Number of replicate test chambers per treatment

10. Feeding frequency, and amount and type of food

11. Acclimation temperature of test organisms (mean and range)

12. Test temperature (mean and range)

12.5 TEST ORGANISMS

l. Scientific name

2. Age

3. Life stage

4. Mean length and weight (where applicable)

5. Source

6. Diseases and treatment (where applicable)

12.6 QUALITY ASSURANCE

1. Reference toxicant used routinely; source; date received; lot no.

2. Date and time of most recent reference toxicant test; test results and

current cusum chart

3. Dilution water used in reference toxicant test

4. Physical and chemical methods used

12.7 RESULTS

1. Provide raw toxicity data in tabular form, including daily records of

affected organisms in each concentration (including controls)

2. Provide table of endpoints: LC50, NOAEC, Pass/Fail.

3. Indicate statistical methods used to calculate endpoints

4. Provide summary table of physical and chemical data

5. Tabulate QA data

12.8 CONCLUSIONS AND RECOMMENDATIONS

1. Relationship between test endpoints and permit limits.

2. Action to be taken.

120
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methods of analysis. 15th edition. Association of Official Analytical

Chemists, Arlington, VA.

APHA. 1992. Standard methods for the examination of water and wastewater.

18th edition, Part 8010E. American Public Health Association, Washington,

DoC.

Bartlett, M.S. 1937. Some examples of statistical methods of research in

agriculture and applied biology. J. Royal Statist. Soc. Supple 4:137-183.

Bidwell, J.P., and S. Spotte. 1985. Artificial Seawaters: formulas and

methods. Jones and Barlett, Publ., Boston, MA. 349 pp.

Conover, W.J. 1980. Practical nonparametric statistics. Second edition.

John Wiley and Sons, New York, NY.

Cowgill, U.M. 1987. Critical analysis of factors affecting the sensitivity of zooplankton and the reproducibility of toxicity test results. Wat. Res.

21(12):1453-1462.

Cowgill, U.M., D.P. Milazzo, and B.D. Landenberger. 1990. The

reproducibility of the three brood Ceriodaphnia test using the reference

toxicant sodium lauryl sulfate. Arch. Environ. Contam. Toxicol. 19:513-517.

DeGraeve, G.M., W.H. Clement, and M.F. Arthur. 1989. A method for conducting laboratory toxicity degradation evaluations of complex effluents. Battelle

Columbus Division, Columbus, OH. 22 pp.

DeWoskin, R.S. 1984. Good laboratory practice regulations: A comparison. Research Triangle Institute, Research Triangle Park, NC. 63 pp.

Dorn, P.B., and J.H. Rogers. 1989. Variability associated with

identification of toxics in National Pollutant Discharge Elimination System

(NPDES) effluent toxicity tests. Environ. Toxicol. Chern. 8:893-902.

Draper, N.R., and J.A. John. 1981. Influential observations and outliers in

regression. Technometrics 23:21-26.

Emerson, K., R.C. Russo, R.E. Lund, and R.V. Thurston. 1975. Aqueous ammonia

equilibrium calculations; effect of pH and temperature. J. Fish. Res. Bd.

Can. 32(12):2379-2383.

FDA. 1978. Good laboratory practices for non-clinical laboratory studies.

Part 58. Fed. Reg. 43(247):60013-60020. December 22, 1978.

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125

v
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132
APPENDICES

A. Distribution, Life Cycle, Taxonomy, and Culture Methods .. 134

A.l. Ceriodaphnia dubia--Prepared by Philip A. Lewis and James

M. Lazorchak. . . . . . . . . . . . . . . . . . . . . 134

A.2. Daphnia (D. magna and D. pu7ex)--Prepared by Philip A.

Lewis and James M. Lazorchak 151

A.3. Mysids (Nysidopsis bahia)--Prepared by Stephen H.

Ward 172

A.4. Brine Shrimp (Artemia sa7ina)--Prepared by Philip A.

Lewis and David A. Bengtson . . . . . . . . . . .. 191

A.5. Fathead Minnow (Pimepha7es prome7as)--Prepared by

Donald J. Klemm, Quentin H. Pickering, and Mark E.

Smith . . . . . . . . . . . . . . . . . . . . . .. 200

A.6. Rainbow Trout (Oncorhynchus mykiss) and Brook Trout

(Sa7ve7inus fontina7is)--Prepared by Donald

J. Klemm 219

A.7. Sheepshead Minnow (Cyprinodon variegatus)--Prepared by

Donald J. Klemm. . . . . . . . . . . . . . . . .. 229

A.8. Silversides: Inland Silverside (Menidia bery77ina),

Atlantic Silverside, (N. menidia) , and Tidewater

Silverside (N. peninsu7ae)--Prepared by Douglas P.

Middaugh and Donald J. Klemm 247

B. Supplemental List of Acute Toxicity Test Species--Prepared by Margarete A. Heber . . . . . . . . . . . . . . . 264

C. Dilutor Systems--Prepared by William H. Peltier. . 267

D. Plans for Mobile Toxicity Test Laboratory--Prepared by

William H. Peltier . . . . . . . . . . . . . . . . 280

D.l Tandem-Axle Trailer. . . . . . . . . . . . . 280

D.2. Fifth Wheel Trailer. . . . . . . . . . . . . 283

E. Check Lists and Information Sheets--Prepared by William

H. Peltier. . . . . . . . . . . . . . . . . . . . . 284

£.1. Toxicity Test Field Equipment List . . . . . 284

E.2. Information Check List for On-Site Industrial or

Municipal Toxicity Test . 286

E.3. Daily Event Log . . . . . 291

E.4. Dilutor Calibration Form 292

E.5. Daily Dilutor Calibration Check 293

133
APPENDIX A

DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS

A.I. CfRIODAPHNIA DUBIA

1. SYSTEMATICS

1.1 MORPHOLOGY AND TAXONOMY

1.1.1 Ceriodaphnia are closely related and morphologically similar to

Daphnia, but are smaller and have a shorter generation time (USEPA, 1986).

They are generally more rotund, lack the prominent rostral projection typical

of Daphnia, and do not develop the dorsal helmets and long posterior spines

often observed in Daphnia.

1.1.2 With Ceriodaphnia dubia, the female has a heavy, setulated pecten on

the postabdominal claw (Figure lA), and the male was long antennules (Figure

lC), in contrast to the closely related C. reticu7ata, where the female has

heavy, triangular denticles in the pecten of the postabdominal claw (Figure

20), and the male has very short antennules (Figure 2C). Some clones having intermediate characters may be hybrids or phenotypic variants of C. dubia

(USEPA, 1986). Detailed descriptions of the males and females of both species

and the variant were given by USEPA (1986).

1.1.3 Although males are very similar to females, they can be recognized by

their rapid, erratic swimming habit, smaller size, denser coloration,

extended antennules and claspers, and rostrum morphology.

2. ECOLOGY AND LIFE HISTORY

2.1 DISTRIBUTION

2.1.1 C. dubia, has been reported from littoral areas of lakes, ponds, and

marshes throughout most of the world, but it is difficult to ascertain its

true distribution because it has been reported in the literature under several

other names (C. affinis, C. quadrangu]a, and C. reticu7ata. It has also been

suggested that reports of C. dubia in New Zealand and parts of Asia may be yet

another unnamed species (Berner, personal communication).

2.2 ECOLOGY

2.2.1 Ceriodaphnia ecology and life history are very similar to those of

other daphnids. Specific information on the ecology and life history of

Ceriodaphnia dubia is either not available or is widely scattered throughout

the literature. However, it is known to be a pond and lake dwelling species

that is usually common among the vegetation in littoral areas (Fairchild,

1981). In the Lake of Velence, Hungary, C. dubia was most common in regions

where "grey" and "dark brown" waters merged (Pal, 1980). In Par Pond

(Savannah River Plant, Aiken, SC) the Ceriodaphnia were much more abundant in

the heated water (effluent from the nuclear reactor) than in the ambient area

134
A

B

1

c

:..;.. 2

Figure 1. Ceriodaphnia dubia. A. (1) parthenogenetic female, (2) postabdomen,

and (3) claw; B. ephippial female; C. male. (From USEPA, 1986).

135
A

B

Figure 2.

\

Ceriodaphnia reticu7ata. A.

postabdomen, (3) and claw;

(From USEPA, 1986).

136

c

(1) parthenogenetic female, (2)

B. ephippial female; C. male.
(Vigerstad and Tilly, 1977), and in a reservoir in Russia, animals from the

heated water were larger and heavier than those living under normal water

temperatures (Kititsyna and Sergeeva, 1976). In Iran they are common in

warmer, montane, oligotrophic lakes (Smagowicz, 1976).

2.2.2 In Lake Kinneret, Israel, Ceriodaphnia reticu7ata are abundant only

between March and June, with a peak in May when the temperature ranges between

20 and 22°C. When summer temperatures reached 27-28°C, the Ceriodaphnia were

reduced in size and egg production became significantly less, leading to a

progressive decline of the population (Gophen, 1976). In Lake Parvin, France,

the period of development was from June to September (Devaux, 1980).

2.2.3 Ceriodaphnia typically swim with an erratic, jerking motion for a

period of time, and hang motionless in the water between swimming bouts. This

swimming behavior results in a mean speed of 1.5-2.5 mm/s. When approached by

a predator, however, it flees by swimming away qUickly along a straight path

(Wong, 1981).

2.2.4 During most of the year, populations of Ceriodaphnia consist almost

entirely of females; the males appearing principally in autumn. Production of

males appears to be induced primarily by low water temperatures, high

population densities, and/or a decrease in available food. As far as is

presently known, C. dubia reproduce only by cyclic parthenogenesis in which

the males contribute to the genetic makeup of the young during the sexual

stage of reproduction.

2.2.5 The females tend to aggregate during sexual reproductive activity, when

ephippia are produced (Brandl and Fernando, 1971). Ephippia are embryos

encased in a tough covering, and are resistent to drying. They can be stored

for long periods and shipped through the mail in envelopes, like seeds. When

placed in water at the proper temperature, ephippia hatch in a few days producing a new parthenogenetic population.

2.2.6 Ceriodaphnia have many predators, including fish, the mysid Mysis

re7icta, Chaoborus larvae, and copepods. As with Daphnia, it also reacts to

intense predation with defensive strategies. Ceriodaphnia reticu7ata

(possibly C.dubia) in a Minnesota lake, reacted to the copepod, Cyc70ps

verna7is, by producing large offspring and growing to a large size at the

expense of early reproduction (Lynch, 1979). They reacted to fish predators

by producing smaller offspring in larger numbers.

2.3 FOOD AND FEEDING

2.3.1 C1adocera are polyphagous feeders and find their food in the seston.

Daphnids, including the Ceriodaphnia, are classified as fine mesh filter

feeders by Geller and Mueller (1981). These fine mesh filter feeders are most

abundant in eutrophic lakes during summer phytoplankton blooms when suspended

bacteria are available as food only for filter-feeding species with fine mesh.

2.3.2 Lynch (1978) examined the gut contents of Ceriodaphnia reticu7ata

(possibly C. dubia) from a Minnesota pond and found bacteria, detritus and

partially digested algae. In this pond, Ceriodaphnia and Daphnia pu7ex shared

137
the same resource base and had very similar d elS vJ odaphnia fed

more intensively on diatoms. The Ceriodaphnia were considered to be less

sensitive to low food levels than Daphnia, because of their high rate of

population growth during periods of low food levels in late summer.

2.4 LIFE CYCLE

2.4.1 Four distinct periods may be recognized in the life cycle of

Ceriodaphnia: (1) egg, (2) juvenile, (3) adolescent, and (4) adult. The life

span of Ceriodaphnia, from the release of the egg into the brood chamber until

the death of the adult, is highly variable depending on the temperature and

other environmental conditions. Generally the life span increases as

temperature decreases, due to lowered metabolic activity. For example, the

average life span of Ceriodaphnia dubia is about 30 days at 25°C, and 50 days

at 20°C. One female was reported to have lived 125 days and produced 29

broods at 20°C (Cowgill et al., 1985).

2.4.2 Typically, a clutch of 4 to 10 eggs is released into the brood chamber,

but clutches with as many as 20 eggs are common. The eggs hatch in the brood

chamber and the juveniles, which are already similar in form to the adults,

are released in approximately 38 h, when the female molts (casts off her

exoskeleton or carapace). The total number of young produced per female

varies with temperature and other environmental conditions. The most young

are produced in the range of 18-25°C (124 young per female in a 28-day life

span at 24°C) (113 young per female in a 77-day life span at 18°C) but

production falls off sharply below 18°C (13 young per female in a 24-day life

span at 12°C) (McNaught and Mount, 1985).

2.4.3 The time required to reach maturity (produce their first offspring) in

C.dubia varies from three to five days and appears to be dependent on body

size and environmental conditions. A study of the growth and development of

parthenogenetic eggs by Shuba and Costa (1972) revealed that at 24°C the

embryos matured to free-swimming juveniles in approximately 38 h. The eggs

that did not develop fully usually were aborted after 12 hours.

2.4.4 The growth rate of the organism is greatest during its juvenile stages

(early instars), and the body size may double during each of these stages.

Each instar stage is terminated by a molt. Growth occurs immediately after

each molt while the new carapace is still elastic.

2.4.5 Following the juvenile stages, the adolescent period is very short, and

consists of a single instar. It is during the adolescent instar that the

first clutch of eggs reaches full development in the ovary. Generally, eggs

are deposited in the brood chamber within minutes after molting, and the young

which develop are released just before the next molt.

2.4.6 In general, the duration of instars increases with age, but also

depends on environmental conditions. Agiven instar usually lasts

approximately 24 h under favorable conditions. However, when conditions are

unfavorable, it may last as long as a week. Four events take place in a

matter of a few minutes at the end of each adult instar: (1) release of young from the brood chamber to the outside, (2) molting, (3) increase in size, and

138
(4) release of a new clutch of eggs into the brood chamber. The number of

young per brood is highly variable, depending primarily on food availability and environmental conditions. C. dubia may produce as many as 25 young in a

single brood, but more commonly the number is six to ten. The number of young

released during the adult instars reaches a maximum at about the fourth

instar, after which there is a gradual decrease.

3. CULTURING METHODS

3.1 Ceriodaphnia are available from commercial biological supply houses.

Guidance on the source of culture animals to be used by a permittee for selfmonitoring effluent toxicity tests should be obtained from the permitting

authority. Only a small number of organisms (20-30) are needed to start a

culture. Before test organisms are taken from a culture, the culture should

be maintained for at least two generations using the same food, water, and

temperature as will be used in the toxicity tests.

3.2 Cultures of test organisms should be started at least three weeks before

the brood animals are needed, to ensure an adequate supply of neonates for the

test. Only a few individuals are needed to start a culture because of their

prolific reproduction.

3.3 Starter animals may be obtained from an outside source by shipping in

polyethylene bottles. Approximately 20-30 animals and 3 mL of food (see

below) are placed in a l-L bottle filled full with culture water. Animals

received from an outside source should be transferred to new culture media

gradually over a period of 1-2 days to avoid mass mortality.

3.4 It is best to start the cultures with one animal, which is sacrificed

after producing young, embedded, and retained as a permanent microscope slide

mount to facilitate identification and permit future reference. The species

identification of the stock culture should be verified by a taxonomic

authority. The following procedure is recommended for making slide mounts of

Ceriodaphnia (Beckett and Lewis, 1982):

1. Pipet the animal onto a watch glass.

2. Reduce the water volume by withdrawing excess water with the pipet.

3. Add a few drops of carbonated water (club soda or seltzer water) or

70% ethanol to relax the specimen so that the post-abdomen is

extended. (Optional: with practice, extension of the postabdomen

may be accomplished by putting pressure on the cover slip).

4. Place a small amount (one to three drops) of mounting medium on a

glass microscope slide. The recommended mounting medium is

CMCP-9/9AF Medium, prepared by mixing two parts of CMCP-9 with one

part of CMCP-9AF. For more viscosity and faster drying, CMC-10

stained with acid fuchsin may be used. CMCP-9 and 9AF are available

from Polysciences, Inc., Paul Valley Industrial Park, Warrington, PA

18976 (215-343-6484).

5. Using a forceps or a pipet, transfer the animal to the drop of

mounting medium on the microscope slide.

139
6. Cover with a cover slip and exert minimum pressure to remove any air

bubbles trapped under the cover slip. Slightly more pressure will

extend the postabdomen. 7. Allow mounting medium to dry. 8. Make slide permanent by placing CMC-I0 around the edges of the

coverslip. 9. Identify to species (see Pennak, 1989, and USEPA, 1986).

10. Label with waterproof ink or diamond pencil.

11. Store for permanent record.

3.5 CULTURE MEDIA

3.5.1 Although Ceriodaphnia stock cultures can be successfully maintained in

some tap waters, well waters, and surface waters, use of synthetic water as

the culture medium is recommended because (1) it is easily prepared, (2) it is

of known quality, (3) it yields reproducible results, and (4) allows adequate growth and reproduction. Culturing may be successfully done in hard,

moderately hard or soft reconstituted water, depending on the hardness of the

water in which the test will be conducted. The quality of the dilution water

is extremely important in Ceriodaphnia culture. The use of MILLIPORE MILLI-Q®

or SUPER-Q®, or equivalent, to prepare reconstituted water is highly recommended. The use of diluted mineral water (DMW) for culturing and testing is widespread due to the ease of preparation.

3.5.2 The chemicals used and instructions for preparation of reconstituted

water are given in Section 7, Dilution Water. The compounds are dissolved in

distilled or deionized water and the media are vigorously aerated for several

hours before using. The initial pH of the media is between 7.0 and 8.0, but

it will rise as much as 0.5 unit after the test is underway.

3.6 MASS CULTURE

3.6.1 Mass cultures are used only as a "backup" reservoir of organisms. Neonates from mass cultures are not to be used directly in toxicity tests.

3.6.2 One-liter or 2L glass beakers, crystallization dishes, "battery jars,"

or aquaria may be used as culture vessels. Vessels are commonly filled to

three-fourths capacity. Cultures are fed daily. Four or more cultures are

maintained in separate vessels and with overlapping ages to serve as back-up in case one culture is lost due to accident or other unanticipated problems, such as low DO concentrations or poor quality of food or laboratory water.

3.6.3 Mass cultures which will serve as a source of brood organisms for

individual culture should be maintained in good condition by frequent renewal

of the medium and brood organisms. Cultures are started by adding 40-50

neonates per liter of medium. The stocked organisms should be transferred to

new culture medium at least twice a week for two weeks. After two weeks, the

culture is discarded and re-started with neonates in fresh medium. Using this

schedule, 1-L cultures will produce 500 to 1000 neonate Ceriodaphnia each

week.

140
3.6.6 Reserve cultures also may be maintained in large (80-L) aquaria or

other large tanks.

3.7 INDIVIDUAL CULTURE

3.7.1 Individual cultures are used as the immediate source of neonates for

toxicity tests.

3.7.2 Individual organisms are cultured in

(1 oz) plastic cups or 30-mL glass beakers.

cup. It is convenient to place the cups in

toxicity tests (see Figure 1).

15 mL of culture medium in 30-mL

One neonate is placed in each

the same type of board used for

3.7.3 Organisms are fed daily and are transferred to fresh medium a minimum

of three times a week, typically on Monday, Wednesday, and Friday. On the

transfer days, food is added to the new medium immediately before or after the

organisms are transferred.

3.7.4 To provide cultures of overlapping ages, new boards are started weekly,

using neonates from adults which produce at least eight young in their third

or fourth brood. These adults can be used as sources of neonates until

14 days of age. A minimum of two boards are maintained concurrently to

provide backup supplies of organisms in case of problems.

3.7.5 Cultures which are properly maintained should produce at least 20 young

per adult in three broods (seven days or less at 25°C). Typically, 60 adult

females (one board) will produce more than the minimum number of neonates

(120) required for two tests.

3.7.6 Records should be maintained on the survival of brood organisms and

number of offspring at each renewal. Greater than 20% mortality of adults or

less than an average of 20 young per adult on a board at 25°C during a

one-week period would indicate problems, such as poor quality of culture media

or food. Organisms on that board should not be used as a source of test

organisms.

3.8 CULTURE MEDIUM

3.8.1 Moderately hard synthetic water prepared using MILLIPORE MILLI-Q® or

equivalent deionized water and reagent grade chemicals or 20% DMW is recommended

as a standard culture medium (see Section 7, Dilution Water).

3.9 CULTURE CONDITIONS

3.9.1 Ceriodaphnia should be cultured at the temperature at which they will be

used in the toxicity tests (20°C or 25°C ± 2°C).

3.9.2 Day/night cycles prevailing in most laboratories will provide adequate

illumination for normal growth and reproduction. A 16-h/8-h day/night cycle is

recommended.

141
3.9.3 Clear, double-strength safety glass or 6 mm plastic panels are placed on

the culture vessels to exclude dust and dirt, and reduce evaporation.

3.9.4 The organisms are delicate and should be handled as carefully and as

little as possible so that they are not unnecessarily stressed. They are

transferred with a pipet of approximately 2-mm bore, taking care to release the

animals under the surface of the water. Any organism that is injured during

handling should be discarded.

3010 FOOD PREPARATION AND FEEDING

3.10.1 Feeding the proper amount of the right food is extremely important in

Ceriodaphnia culturing. The key is to provide sufficient nutrition to support

normal reproduction without adding excess food which may reduce the toxicity of

the test solutions, clog the animal's filtering apparatus, or greatly decrease

the DO concentration and increase mortality. A combination of Yeast,

CEROPHYLL®, and Trout chow (YCT) or flake food, along with the unicellular green

alga, Se7enastrum capricornutum, will provide suitable nutrition if fed daily.

3.10.2 The YCT and algae are prepared as follows:

3.10.2.1 Digested trout chow (or flake food):

1. Preparation of trout chow requires one week. Use starter or No.1 pellets

prepared according to current U.S. Fish and Wildlife Service

specifications, or flake food. Suppliers of trout chow include Zeigler

Bros., Inc., P.O. Box 95, Gardners, PA 17324 (717-780-9009); Glencoe

Mills, 1011 Elliott, Glencoe, MN 55336 (612-864-3181); and Murray

Elevators, 118 West 4800 South, Murray, UT 84107 (800-521-9092).

2. Add 5.0 g of trout chow pellets or flake food to 1 L of MILLI-Q® water.

Mix well in a blender and pour into a 2-L separatory funnel. Digest prior

to use by aerating continuously from the bottom of the vessel for one week

at ambient laboratory temperature. Water lost due to evaporation is

replaced during digestion. Because of the offensive odor usually produced

during digestion, the vessel should be placed in a fume hood or other

isolated, ventilated area.

3. At the end of digestion period, place in a refrigerator and allow to

settle for a minimum of 1 h. Filter the supernatant through a fine mesh

screen (i.e., NITEX® 110 mesh). Combine with equal volumes of supernatant

from CEROPHYLL® and yeast preparations (below). The supernatant can be

used fresh, or frozen until use. Discard the sediment.

3.10.2.2 Yeast:

1. Add 5.0 g of dry yeast, such as FLEISCHMANN'S® to 1 L of MILLI-Q® water.

2. Stir with a magnetic stirrer, shake vigorously by hand, or mix with a

blender at low speed, until the yeast is well dispersed.

3. Combine the yeast suspension immediately (do not allow to settle) with

equal volumes of supernatant from the trout chow (above) and CEROPHYLL®

preparations (below). Discard excess material.

142
3.10.2.3 CEROPHYLL® (Dried, Powdered, Cereal Leaves):

1. Place 5.0 g of dried, powdered, cereal leaves in a blender. (Available as

"CEREAL LEAVES,11 from Sigma Chemical Company, P.O. Box 14508, St. Louis,

MO 63178 (800-325-3010); or as CEROPHYLL®, from Ward's Natural Science

Establishment, Inc., P.O. Box 92912, Rochester, NY, 14692-9012

(716-359-2502). Dried, powdered, alfalfa leaves obtained from health food

stores have been found to be a satisfactory substitute for cereal leaves.

2. Add 1 L of MILLI-Q® water.

3. Mix in a blender at high speed for 5 min, or stir overnight at medium

speed on a magnetic stir plate.

4. If a blender is used to suspend the material, place in a refrigerator

overnight to settle. If a magnetic stirrer is used, allow to settle for

1 h. Decant the supernatant and combine with equal volumes of supernatant

from trout chow and yeast preparations (above). Discard excess material.

3.10.2.4 Combined YCT Food:

1. Mix equal (approximately 300 mL) volumes of the three foods as described

above.

2. Place aliquots of the mixture in small (50-mL to 100-mL) screw-cap plastic

bottles and freeze until needed.

3. Freshly prepared food can be used immediately, or it can be frozen until

needed. Thawed food is stored in the refrigerator between feedings, and

is used for a maximum of two weeks.

4. It is advisable to measure the dry weight of solids (dry 24 h at 105°C) in

each batch of YCT before use. The food should contain 1.7-1.9 g solids/L.

Cultures or test solutions should contain 12-13 mg solids/L.

3.10.3 Algal (Se7enastrum) Food

3.10.3.1 Algal Culture Medium

1. Prepare (five) stock nutrient solutions using reagent grade chemicals as

described in Table 1.

2. Add 1 mL of each stock solution, in the order listed in Table 1, to

approximately 900 mL of MILLI-Q® water. Mix well after the addition of

each solution. Dilute to 1 L and mix well. The final concentration of

macronutrients and micronutrients in the culture medium is given in

Table 2.

3. Immediately filter the medium through a 0.45 ~m pore diameter membrane at

a vacuum of not more than 380 mm (15 in.) mercury, or at a pressure of not

more than one-half atmosphere (8 psi). Wash the filter with 500 mL

deionized water prior to use.

4. If the filtration is carried out with sterile apparatus, filtered medium

can be used immediately, and no further sterilization steps are required

before the inoculation of the medium. The medium can also be sterilized

by autoclaving after it is placed in the culture vessels.

5. Unused sterile medium should not be stored more than one week prior to

use, because there may be substantial loss of water by evaporation.

143
TABLE 1. NUTRIENT STOCK SOLUTIONS FOR MAINTAINING ALGAL STOCK CULTURES

AND TEST CONTROL CULTURES

STOCK

SOLUTION

COMPOUND AMOUNT DISSOLVED IN

500 ML MILLI-Q® WATER

MgC1 2·6H2O 6.08 9

CaC1 2·2H2O 2.20 9

NaN03 12.75 9

MgS04·7H2O 7.35 9

K2HP04 0.522 9

NaHC03 7.50 9

B.

C.

D.

2. MICRONUTRIENTS

1. MACRONUTRIENTS

A.

92.8 mg

208.0 mg

1.64 mg a

79.9 mg

0.714 mg b

~:~~6 ~~~ 150.0 mg

1.196 mg e

aZnC12 - Weigh out 164 mg and dilute to 100 mL. Add 1 mL of this

solution to Stock #1.

bCoC12.6H~O - Weigh out 71.4 mg and dilute to 100 mL. Add 1 mL of

this sOlution to stock #1.

CN~Mo04.2H20 - Weigh out 36.6 mg and dilute to 10 mL. Add 1 mL

ot this solution to stock #1.

dCuC12.2H~O - Weigh out 60.0 mg and dilute to 1000 mL. Take 1 mL of

this sOlution and dilute to 10 mL. Take 1 mL of the second dilution and

add to Stock #1.

eNa2Se04 - Weigh out 119.6 mg and dilute to 100 mL. Add 1 mL of this solution

to Stock #1.

144
TABLE 2. FINAL CONCENTRATION OF MACRONUTRIENTS AND MICRONUTRIENTS

IN THE CULTURE MEDIUM

Concentration Element Concentration

Macronutrient (mg/L) (mg/L)

NaN03 25.5 N 4.20

MgC1 2 • 6H2O 12.2 Mg 2.90

CaC1 2 ·2H2O 4.41 Ca 1.20

MgS04 ·7H2O 14.7 S 1. 91

K2HP04 1.04 P 0.186

NaHC03 15.0 Na 11.0

K 0.469

C 2.14

Concentration Element Concentration

Micronutrient (Jlg/L) (Jlg/L)

H3803 185 8 32.5

MnC1 2 • 4H2O 416 Mn 115

ZnC1 2 3.27 Zn 1. 57

CoC1 2 ·6H2O 1.43 Co 0.354

CuC1 2 ·2H2O 0.012 Cu 0.004

Na 2Mo04 ·2H2O 7.26 Mo 2.88

FeC1 3 ·6H2O 160 Fe 33.1

Na 2EDTA.2H2O 300

Na 2Se04 2.39 Se 1.00

145
3.10.3.2 Algal Cultures

3.10.3.2.1 Two types of algal cultures are maintained: (1) stock cultures,

and, (2) "food" cultures.

3.10.3.2.2 Establishing and Maintaining Stock Cultures of Algae

1. Upon receipt of the "starter" culture (usually about 10 mL), a stock

culture is initiated by aseptically transferring one milliliter to each of

several 250-mL culture flasks containing 100 mL algal culture medium

(prepared as described above). The remainder of the starter culture can

be held in reserve for up to six months in a refrigerator (in the dark) at

4°C.

2. The stock cultures are used as a source of algae to initiate "food"

cultures for Ceriodaphnia toxicity tests. The volume of stock culture

maintained at anyone time will depend on the amount of algal food

required for the Ceriodaphnia cultures and tests. Stock culture volume

may be rapidly "scaled up" to several liters, if necessary, using 4-L

serum bottles or similar vessels, each containing 3 L of growth medium.

3. Culture temperature is not critical. Stock cultures may be maintained in

environmental chambers with cultures of other organisms if the

illumination is adequate (c2ntinuous "cool-white " fluorescent lighting of

approximately 86 ± 8.6 ~E/m Is, or 400 ft-c).

4. Cultures are mixed twice daily by hand or stirred continuously.

5. Stock cultures can be held in the refrigerator until used to start "food"

cultures, or can be transferred to new medium weekly. One-to-three

milljliters of 7-day old algal stock culture, containing approximately 1.5

X 10 cells/mL, are transferred to each 100 mL of fresh culture medium.

The inoculum should provide an initial cell density of approximately

10,000-30,000 cells/mL in the new stock cultures. Aseptic techniques

should be used in maintaining the stock algal cultures, and care should be

exercised to avoid contamination by other microorganisms.

6. Stock cultures should be examined microscopically weekly, at transfer, for

microbial contamination. Reserve quantities of culture organisms can be

maintained for 6-12 months if stored in the dark at 4°C. It is advisable

to prepare new stock cultures from "starter" cultures obtained from

established outside sources of organisms every four to six months.

3.10.3.2.3 Establishing and Maintaining "Food" Cultures of Algae

1. "Food" cultures are started seven days prior to use for Ceriodaphnia

cultures and tests. Approximately 20 mL of 7-day-old algal stock culture

(described in the previous paragraph), containing 1.5 X 10 cells/mL, are

added to each liter of fresh algal culture medium (i.e., 3 L of medium in

a 4-L bottle, or 18 L in a 20-L bottle). The inoculum should provide an

initial cell density of approximately 30,000 cells/mL. Aseptic techniques

should be used in preparing and maintaining the cultures, and care should

be exercised to avoid contamination by other microorganisms. However,

sterility of food cultures is not as critical as in stock cultures because

the food cultures are terminated in 7-10 days. A one-month supply of

algal food can be grown at one time, and the excess stored in the

refrigerator.

146
2. Food cultures may be maintained at 25°C in environmental chambers with the

algal stock cultures or cultures of other organisms if the illumination is

adequate (conlinuous "cool-white" fluorescent lighting of approximately

86 ± 8.6 ~E/m Is, or 400 ft-c).

3. Cultures are mixed continuously on a magnetic stir plate (with a medium

size stir bar) or in a moderately aerated separatory funnel, or are mixed

twice daily by hand. If the cultures are placed on a magnetic stir plate,

heat generated by the stirrer might elevate the culture temperature

several degrees. Caution should be exercised to prevent the culture

temperature from rising more than 2-3°C.

3.10.3.3 Preparing Algal Concentrate for Use as Ceriodaphnia Food

1. An algal concentrate containing 3.0 to 3.5 X 107 cells/mL is prepared from

food cultures by centrifuging the algae with a plankton or bucket-type

centrifuge, or by allowing the cultures to settle in a refrigerator for

approximately two-to-three weeks and siphoning off the supernatant.

2. The cell density (cells/mL) in the concentrate is measured with an

electronic particle counter, microscope and hemocytometer, fluorometer, or

spectrophotometer, and used to determine the roncentration required to

achieve a final cell count of 3.0 to 3.5 X 10 ImL.

3. Assuming a cell density of approximately 1.5 X 106 cells/mL in the algal

food cultures at 7 days, and 100% recovery In the concentration process, a

3-L, 7-10 day culture will provide 4.5 X 10 algal cells. This number of

cells would provide approximately 150 mL of algal cell concentrate (1500

feedings at 0.1 mL/feeding) for use as food. This would be enough algal

food for four Ceriodaphnia tests.

4. Algal concentrate may be stored in the refrigerator for one month.

3.11 FEEDING

3.11.1 Cultures should be fed daily to maintain the organisms in optimum

condition so as to provide maximum reproduction. Stock cultures which are

stressed because they are not adequately fed may produce low numbers of young,

large number of males, and ephippial females. Also, their offspring may

produce few young when used in toxicity tests.

1. If YCT is frozen, remove a bottle of food from the freezer 1 h before

feeding time, and allow to thaw.

2. Mass cultures are fed daily at the rate of 7 mL YCT and 7 mL algae

concentrate/L culture.

3. Individual cultures are fed at the rate of 0.1 mL YCT and 0.1 mL algae

concentrate per 15 mL culture.

4. YCT and algal concentrate should be thoroughly mixed by shaking before

dispensing.

5. Return unused YCT food mixture and algae concentrate to the refrigerator.

Do not re-freeze YCT. Discard unused portion after one week.

3.12 FOOD QUALITY

3.12.1 The quality of food prepared with newly acquired supplies of yeast,

trout chow, dried cereal leaves, or algae, should be determined in side-by-side

147
comparisons of Cerjodaphnja survival and reproduction, using the new food and

food of known, acceptable quality, over a seven-day period in control medium.

4. TEST ORGANISMS

4.1 Neonates, or first instar Cerjodaphnja less than 24 hours old, taken from

the 3rd or 4th brood, are used in toxicity tests. To obtain the necessary

number of young for an acute toxicity test, it is recommended that the animals

be cultured in individual 30 mL beakers or plastic cups for seven days prior to

the beginning of the test. Neonates are used from broods of at least eight

young. Fifty adults in individual cultures will usually supply enough neonates

for one toxicity test.

4.2 Use a disposable, widemouth pipette to transfer Cerjodaphnja. The diameter

of the opening should be approximately 4 mm. The tip of the pipette should be

kept under the surface of the water when the Cerjodaphnja are released to

prevent air from being trapped under the carapace. Liquid containing adult

Cerjodaphnja can be poured from one container to another without risk of

injuring the animals.

148
SELECTED REFERENCES

Beckett, D.C., and P.A. Lewis. 1982. An efficient procedure for slide

mounting of larval chironomids. Trans. Am. Microsc. Soc. 101(1):96-99.

Brandl, Z., and C.H. Fernando. 1971. Microaggregation of the cladoceran

Ceriodaphnia affinis Lilleborg with a possible reason for

microaggregation of zooplankton. Can. J. Zool. 49:775.

Cowgill, U.M., K.I. Keating, and I.T. Takahashi. 1985. Fecundity and

longevity of Ceriodaphnia dubia/affinis in relation to diet at two

different temperatures. J. Crust. Biol. 5(3):420-429.

Devaux, J. 1980. Contribution to limnological study of Lake Pavin, France.

2: Relationship between abiotic parameters, phytoplankton and

zooplankton in the 0-20 meter zone. Hydrobiologia 68(1):17-34.

Fairchild, G.W. 1981. Movement and micro distribution of 5idia crystalline and other littoral micro Crustacea. Ecology 62(5):1341-1352.

Geller, W., and H. Mueller. 1981. The filtration apparatus of Cladocera

filter mesh sizes and their implications on food selectivity. Oecologia

(Berl.) 49(3):316-321.

Gophen, M. 1976. Temperature dependence of food intake, ammonia excretion,

and respiration in Ceriodaphnia reticulata, Lake Kinneret, Israel.

Freshwat. Biol. 6(5):451-455.

Kititsyna, L.A., and O.A. Sergeeva. 1976. Effect of temperature increase on

the size and weight of some invertebrate populations in the cooling

reservoir of the Kinakhovsk State Regional Electric Power Plant.

Ekologyia 5:99-102.

Lynch, M. 1978. Complex interactions between natural coexploiters - Daphnia and Ceriodaphnia. Ecology 59(3):552-564.

Lynch, M. 1979. Predation, competition, and zooplankton community

structure: An experimental study. Limnol. Oceanogr. 24(2):253-272.

McNaught, D.C., and 0.1. Mount. 1985. Appropriate durations and measures for

Ceriodaphnia toxicity tests. In: R.C. Bahner and D.J. Hansen (eds.),

Aquatic TOXicology and Hazard Assessment: Eighth Symposium. ASTM STP

891, American Society for Testing and Materials, Philadelphia, PA.

pp. 375-381.

Norberg, T.J., and 0.1. Mount. 1985. Diets for Ceriodaphnia reticulata

life-cycle tests. In: R.D. Cardwell, R. Purdy and R.C. Bahner (eds.),

Aquatic Toxicology and Hazard Assessment: Seventh Symposium. ASTM STP

854. American Society for Testing and Materials, Philadelphia, PA.

pp. 42-52.

149
Pal, G. 1980. Characterization of water quality regions of the Lake of

Velence, Hungary with planktonic crustaceans. Allattani Kozl.

67(1-4):49-58.

Pennak, R.W. 1989. Fresh-water invertebrates of the United States. 3rd ed.

Protozoa to Mollusca. John Wiley &Sons, New York, NY.

Shuba, T., and R.R. Costa. 1972. Development and growth of Ceriodaphnia

reticu7ata embryos. Trans. Am. Microsc. Soc. 91(3):429-435.

Smagowicz, K. 1976. On the zooplankton of Lake Zeribar, Western Iran. Acta

Hydrobiol. 18(1):89-100.

USEPA. 1986. Taxonomy of Ceriodaphnia (Crustacea: Cladocera) in U.S.

Environmental Protection Agency cultures. D.B. Berner. U. S.

Environmental Protection Agency, Environmental Monitoring and Support

Laboratory, Cincinnati, OH. EPA/600/4-86/032.

USEPA. 1989. Culturing of Ceriodaphnia dubia. Supplemental report for video

training tape. U. S. Environmental Protection Agency, Washington, D.C.

EPA/505/8-89/002a.

Vigerstad, T.J., and L.J. Tilly. 1977. Hyper-thermal effluent effects on

heleo planktonic Cladocera and the influence of submerged macrophytes. Hydrobiologia 55(1):81-86.

Wong, C.K. 1981. Predatory feeding behavior of Epischura 7acustris

(Copepoda: Calanoida) and prey defense. Can. J. Fish. Aquat. Sci.

38:275-279.

150
APPENDIX A

DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS

A.2. DAPHNIA (D. MAGNA AND D. PULEX)

1. SYSTEMATICS

1.1 MORPHOLOGY AND TAXONOMY

1.1.1 The generalized anatomy of a parthenogenetic female is shown in

Figure 1. Daphnja pu7ex is an extremely variable species consisting of

several reproductively isolated clonal groups and is often not distinguishable

from other species (such as D. obtusa) that have large teeth on the middle

pecten of the postabdomenal claw (Figure 2C) (Lynch, 1985; Dodson, 1981).

Probably the most distinctive feature of the parthenogenetic female D. pu7ex

is the long second abdominal process of the abreptor (postabdomen) that

extends beyond the base of the anal setae (Figure 2A).

1.1.2 D. pulex is a wide ranging species that shows little variation

throughout its range. Two of its most distinctive characteristics are the

deeply sinuate posterior margin of the abreptor (Figures 3A and 3D) and the

ridges on the head which run parallel to the mid-dorsal line (Figure 3B).

1.1.3 D. pulex is much smaller than D. magna, attaining a length of up to

3.5 mm compared to 5.0 or 6.0 mm for D. magna. Although the two species can

often be separated by size, they can be differentiated with certainty only by

examining the postabdominal claws for size and number of spines using a

compound microscope. D. pulex has 5-7 stout teeth on the middle pecten

(Figure 2C) while D. magna has a uniform row of 20 or more small teeth

(Figure 3E). Another characteristic for separating the neonates of the two

species is the location of the nuchal organ which is higher up on the

posterior margin of the head in D. magna than in D. pulex (Schwartz and

Hebert, 1984). For a more complete taxonomic discussion of the two species

see Brooks (1957).

2. DISTRIBUTION

2.1 D. magna has a worldwide distribution in the northern hemisphere. In

North America it appears to be absent from the eastern United States (except

for Northern New England) and Alaska (Figure 4). D. pUlex occurs over most of

North America except the tropics and high arctic (Figure 5), and probably

occurs in Europe and South America as well. Both species often occur in the

same pools but D. pu7ex usually out-competes D. magna in mixed populations and

takes over as the sole inhabitant by summer's end (Modlin, 1982; Lynch, 1983).

151
Figure 1. Generalized anatomy of a female Daphnja, X70; A, antenna; AS, anal

setae; BC, brood chamber; H, heart; INT, intestine; L, legs; OV,

ovary; P, postabdomen; PC, postabdominal claw. (From Pennak, 1989).

c

Figure 2. Female Daphnja pu7ex. A, lateral aspect (note smoothly rounded

posterior margin of postabdomen); B, ephippial female; C,

postabdomen showing large spines on the claw. (From Brooks, 1957).

152
E

c

B

D

A

Figure 3. Female D. magna. A. Lateral aspect; B. dorsal aspect; c.

ephippial female; D. postabdomen showing sinuate posterior margin;

E. postabdominal claw. (From Brooks, 1957).

153
Figure 4.

Figure 5.

Map showing the North American distribution of D. magna.

Map showing the North American distribution of D. pulex.

154
3. ECOLOGY AND LIFE HISTORY

3.1 GENERAL ECOLOGY

3.1.1 D. magna is principally a lake dweller and is restricted to waters in

northern and western North America exceeding a hardness of 150 mg/L (as CaCO~)

(Pennak, 1989). In the Netherlands, D. magna are found in shallow ponds witn

muddy bottoms rich in organic matter and with low oxygen demand (3 to 4 mg/L).

D. pulex is principally a pond dweller where the oxygen content is higher, but

is also found in lakes. It is generally considered a clean water species

being dominant in nature during periods of low turbidity. However, Scholtz,

et al. (1988) found that high turbidity had little effect on survival and

reproduction in laboratory studies.

3.1.2 Daphnia populations are generally sparse in winter and early spring,

but as water temperatures reach 6°C to 12°C, they increase in abundance and

subsequently may reach population densities as high as 200 to 500

individuals/L (Pennak, 1989). Populations in ponds decline to very low

numbers during the summer months. In autumn there may be a second population

pulse, followed by a decline to winter lows.

3.1.3 During most of the year, populations of Daphnia consist almost entirely

of females, the males being abundant only in spring or autumn when up to 56%

of the offspring of D. magna may be males (Barker and Hebert, 1986). Males

are distinguished from females by their smaller size, larger antennules,

modified postabdomen, and first legs, which are armed with a stout hook used

in clasping. Production of males appears to be induced principally by low

temperatures or high densities and subsequent accumulation of excretory

products, and/or a decrease in available food. These conditions may induce

the appearance of sexual (resting) eggs (embryos) in cases called ephippia

(Figures 2B and 3C), which are cast off during the next molt. It appears that

the shift toward male and sexual egg production is related to the metabolic

rate of the parent. Any factor which tends to lower metabolism may be

responsible. Ruvinsky et al. (1978) suggested that the genome of the animal

has two developmental programs based on identical sets of chromosomes. The

female program consistently functions under a wide range of conditions,

whereas the male program is turned on by specific ecological stimuli. The

eggs from which the males and females develop have identical chromosome sets.

Sex determination is based on changes in chromatin structure when the mother

receives a specific signal that sexual reproduction is needed for adaptation

to extreme conditions.

3.1.4 D. magna reproduce only by cyclic parthenogenesis in which males

contribute to the genetic makeup of the young during the sexual stage of

reproduction, whereas D. pulex may reproduce either by cyclic or obligate

parthenogenesis in which the zygotes develop within the ephippium by ameoitic

parthenogenesis with no genetic contribution from the males. Thus, the

ephippial and live-born offspring are genetically identical to their mothers.

Both forms may be present in the same population resulting in cyclic

populations exhibiting considerable genetic variation early in the year and an

obligate population with a low range of genotypic values. After 25

generations of asexual reproduction the variation in the cyclic

155
parthenogenesis group becomes about the same as that in the obligate group (Lynch, 1984). These populations exhibiting a low range of genotypic values

are much more vulnerable to perturbations such as nutrient introduction or

toxic discharges. The clonal makeup of a Daphnia population is effected by

food, oxygen, temperature and predation (Weider, 1985; Brookfield, 1984).

3.1.5 Ephippia are small and lightweight and can be dried and stored for long periods making them easy to transport. They may be shipped in envelopes like

seeds. Upon arrival at the new location the ephippia can be hatched in a few

days when placed in water at the proper temperature (Schwartz and Hebert,

1987).

3.1.6 Daphnia are preyed upon by many predators and have developed behavioral

and morphological anti predator defenses to make themselves more difficult to

catch and consume. Dodson (1988) showed that D. pulex responded to a possible chemical stimuli released by the predator which resulted in the daphnids retreating from the vicinity of the predators. Certain clones of D. pulex may develop morphological changes when predators are present but not when they are

absent from the pond. Some of these changes are of such magnitude that they have been described as separate species. D. minnehaha is a morphological variation of D. pulex which develops spines in response to the stimuli of

predators (Krueger and Dodson, 1981). Different genotypes of D. pulex react

in different ways to the predator (Chaoborus) factor and to temperature

(Havel, 1985).

3.2 FOOD AND FEEDING

3.2.1 Both D. pulex and D. magna are well adapted to live in algal blooms,

which are high in proteins and carbohydrates, where they feed on algae and

bacteria. D. magna prefers bacteria to algae as food (Ganf, 1983; Hadas et

al., 1983) while D. pulex uses bacteria as food only when algal biomass

declines (Borsheim and Olsen, 1984). Food type and abundance affect the

sensitivity of Daphnia to pollutants and their reproduction rate. Keating and

Dagbusan (1986) showed that both D. pulex and D. magna fed diatoms were more

tolerant of pollutants than those fed only green algae. Lipid reserves are a

good indication of the nutritional condition of the animals (Holm and Shapiro,

1984; Tessier and Goulden, 1982).

3.3 LIFE HISTORY

3.3.1 Four distinct periods may be recognized in the life history of Daphnia:

(1) egg, (2) juvenile, (3) adolescence, and (4) adult (Pennak, 1989). The

life span of Daphnia, from the release of the egg into the brood chamber until

the death of the adult, is highly variable depending on the species and

environmental conditions (Pennak, 1989). Generally the life span increases as

temperature decreases, due to lowered metabolic activity. The average life

span of D. magna is about 40 days at 25°C, and about 56 days at 20°C. The

average life span of D. pulex at 20°C is approximately 50 days. Typically, a

clutch of 6 to 10 eggs is released into the brood chamber, but as many as 57

have been reported. The eggs hatch in the brood chamber and the juveniles, which are already similar in form to the adults, are released in approximately

two days when the female molts (casts off her exoskeleton or carapace). The

156
time required to reach maturity (produce their first offspring) in D. pulex

varies from six to 10 days (mean = 7.78 days) and also appears to be dependent

on body size. The growth rate of the organism is greatest during its juvenile

stages (early instars), and the body size may double during each of these

stages. D. pulex has three to four juvenile instars, whereas D. magna has

three to five instars. Each instar stage is terminated by a molt. Growth

occurs immediately after each molt while the new carapace is still elastic.

3.3.2 Following the juvenile stages, the adolescent period is very short, and

consists of a single instar. It is during the adolescent instar that the

first clutch of eggs reaches full development in the ovary. Generally, eggs

are deposited in the brood chamber within minutes after molting, and the young

which develop are released just before the next molt.

3.3.3 D. magna usually has 6-22 adult instars, and D. pulex has 18-25. In

general, the duration of instars increases with age, but also depends on

environmental conditions. A given instar generally lasts approximately two

days under favorable conditions, but when conditions are unfavorable, may last

as long as a week.

3.3.4 Four events take place in a matter of a few minutes at the end of each

adult instar: (1) release of young from the brood chamber to the outside, (2)

molting, (3) increase in size, and (4) release of a new clutch of eggs into

the brood chamber. The number of young per brood is highly variable for

Daphnia, depending primarily on food availability and environmental

conditions. D. magna and D. pulex may both produce as many as 30 young during

each adult instar, but more commonly the number is six to 10. The number of

young released during the adult instars of D. pulex reaches a maximum at the

tenth instar, after which there is a gradual decrease (Anderson and Zupancic,

1937). Scholtz et al. (1988) reported that nearly all of the eggs that are

oviposited by D. pulex became neonates, indicating a highly successful

hatching rate. The maximum number of young produced by D. magna occurs at the

fifth adult instar, after which it decreases (Anderson and Jenkins, 1942).

4. CULTURING METHODS

4.1 SOURCES OF ORGANISMS

4.1.1 Daphnia are available from commercial biological supply houses. Only a

small number of organisms (20-30) are needed to start a culture. D. pulex is

preferred over D. magna by some biologists because it is more widely

distributed, is tolerant of a wider range of environmental conditions, and is

easier to culture. However, the neonates are smaller, swim faster and are

more difficult to count, and produce more "floaters" than D. magna and,

therefore, are somewhat more difficult to use in toxicity tests. Guidance on

the source and species of Daphnia to be used by a permittee for effluent

toxicity tests should be obtained from the permitting authority.

4.1.2 Cultures of test organisms should be started at least three weeks

before the brood animals are needed, to ensure an adequate supply of neonates

for the test.

157
4.1.3 Starter animals may be obtained from an outside source by shipping in

polyethylene bottles. Approximately 20-30 animals and 3 mL of food (see

below) are placed in a l-L bottle filled full with culture water. Animals

received from an outside source should be transferred to new culture media

gradually over a period of 1-2 days to avoid mass mortality.

4.1.4 It is best to start the cultures with one animal, which is sacrificed

after producing young, embedded, and retained as a permanent microscope slide

mount to facilitate identification and permit future reference. The species identification of the stock culture should be verified by a taxonomic

authority. The following procedure is recommended for making slide mounts of

Daphnia (Beckett and Lewis, 1982):

1. Pipet the animal onto a watch glass.

2. Reduce the water volume by withdrawing excess water with the pipet.

3. Add a few drops of carbonated water (club soda or seltzer water) or

70% ethanol to relax the specimen so that the post-abdomen is

extended. (Optional: with practice, extension of the postabdomen

may be accomplished by putting pressure on the cover slip).

4. Place a small amount (one to three drops) of mounting medium on a

glass microscope slide. The recommended mounting medium is

CMCP-9/9AF Medium, prepared by mixing two parts of CMCP-9 with one

part of CMCP-9AF. For more viscosity and faster drying, CMC-10

stained with acid fuchsin may be used. CMCP-9 and 9AF are available

from Polysciences, Inc., Paul Valley Industrial Park, Warrington, PA

18976 (215-343-6484).

5. Using a forceps or a pipet, transfer the animal to the drop of

mounting medium on the microscope slide.

6. Cover with a cover slip and exert minimum pressure to remove any air

bubbles trapped under the cover slip. Slightly more pressure will

extend the postabdomen.

7. Allow mounting medium to dry.

8. Make slide permanent by placing CMC-I0 around the edges of the

coverslip.

9. Identify to species (see Pennak, 1989).

10. Label with waterproof ink or diamond pencil.

11. Store for permanent record.

4.2 CULTURE MEDIA

4.2.1 Although Daphnia stock cultures can be successfully maintained in some

tap waters, well waters, and surface waters, use of synthetic water as the

culture medium is recommended because (1) it is easily prepared, (2) it is of

known quality, (3) it yields reproducible results, and (4) allows adequate growth and reproduction. Reconstituted hard water (total hardness of 160 -180

mg/L as CaC03 ) is recommended for D. magna culturing, and reconstituted

moderately hard water (total hardness of 80-90 mg/L CaC03 ) is recommended for

D. pulex culturing. The quality of the dilution water is important in Daphnia

culture. The use of MILLIPORE MILLI-Q® or SUPER-Q®, or equivalent, to prepare

reconstituted water is highly recommended. The use of diluted mineral water

(DMW) for culturing and testing is widespread due to the ease of preparation.

158
4.2.2 The chemicals used and instructions for preparation of reconstituted

water are given in Section 7, Dilution Water. The compounds are dissolved in

distilled or deionized water and the media are vigorously aerated for several

hours before using. The initial pH of the media is between 7.0 and 8.0, but

it will rise as much as 0.5 unit after the test is underway.

4.3 CULTURE CONDITIONS

4.3.1 Daphnia can be cultured successfully over a wide range of temperatures, but should be protected from sudden changes in temperature, which may cause

death. The optimum temperature is approximately 20°C, and if ambient

laboratory temperatures remain in the range of 18-26°C, normal growth and

reproduction of Daphnia can be maintained without special temperature control

equipment. D. magna can survive when the DO concentration is as low as 3 mg/L but D. pulex does best when the DO concentration is above 5 mg/L. Therefore

it is recommended that the DO concentration in the culture be maintained at

5 mg/L or above. Unless the cultures are too crowded or overfed, aeration is

usually not necessary.

4.3.2 Illumination

4.3.2.1 The variations in ambient light intensities (10-20 pE/m2/s, or 50-100

ft-c) and prevailing day/night cycles in most laboratories do not seem to

affect Daphnia growth and reproduction significantly. However, a minimum of

16 h of illumination should be provided each day.

4.3.3 Culture Vessels

4.3.3.1 Culture vessels of clear glass are recommended since they allow easy

observation of the Daphnia. A practical culture vessel is an ordinary 4-L

glass beaker, which can be filled with approximately 3 L medium (reconstituted

water). Maintain several (at least five) culture vessels, rather than only

one. This will ensure back-up cultures so that in the event of a population

"crash" in one or several chambers, the entire Daphnia population will not be

lost. If a vessel is stocked with 30 adult Daphnia, it will provide

approximately 300 young each week.

4.3.3.2 Initially, all culture vessels should be washed well (see Section 5,

Facilities and Equipment). After the culture is established, clean each

chamber weekly with distilled or deionized water and wipe with a clean sponge

to rid the vessel of accumulated food and dead Daphnia (see section on culture

maintenance below). Once per month, wash each vessel with detergent during

medium replacement. Rinse three times with tap water and then with culture

medium to remove all traces of detergent.

4.3.4 Weekly Culture Media Replacement

4.3.4.1 Careful culture maintenance is essential. The medium in each stock

culture vessel should be replaced three times each week with fresh medium.

This is best accomplished by changing solutions Monday, Wednesday, and Friday,

as follows:

159
1. Place about 300 mL of the old media in a temporary holding vessel.

2. Transfer about 25 or 30 adults from the old culture vessel to the

holding vessel using a wide bore pipette. 3. Discard the remaining Daphnia along with the media.

4. Clean the culture vessel as described above.

5. Fill the newly-cleaned vessel with fresh medium.

6. Gently transfer (by pouring) the contents of the temporary holding vessel (old medium with the Daphnia) into the vessel containing the np.w

medium making sure that none of the animals stick to the sides of the

vessel.

7. Feed the animals

4.3.4.2 If the medium is not replaced three times weekly, waste products will

accumulate, which could cause a population crash or the production of males

and/or sexual eggs.

4.3.4.3 Daphnia cultures should be thinned whenever the population exceeds

200 individuals per stock vessel to prevent over-crowding, which may cause a

population crash, or the production of males and/or ephippia. A good time to

thin the populations is on Monday, Wednesday, and Friday, before feeding. To

transfer Daphnia, use a IS-em disposable, jumbo bulb pipette, or 10-mL "serum"

pipette which has had the delivery tip cut off and fire polished. The

diameter of the opening should be approximately 5 mm. A serum pipette, a

pipette bulb, such as a PROPIPETTE@, or (MOPET@) portable, motorized pipettor, will provide the controlled suction needed when selectively collecting Daphnia.

4.3.4.4 Liquid containing adult D. pu7ex and D. magna can be poured from one

container to another without risk of air becoming trapped under their

carapaces. However, the very young Daphnia are much more susceptible to air

entrapment and for this reason should be transferred from one container to

another using a pipette. The tip of the pipette should be kept under the

surface of the liquid when the Daphnia are released.

4.3.4.5 Each culture vessel should be covered with a clear plastic sheet or

glass plate to exclude dust and dirt, and minimize evaporation.

4.4 FOOD PREPARATION AND FEEDING

4.4.1 Feeding the proper amount of the right food is extremely important in

Daphnia culturing. The key is to provide sufficient nutrition to support

normal reproduction without adding excess food which may reduce the toxicity of the test solutions, clog the animal's filtering apparatus, or greatly

decrease the 00 concentration and increase mortality. YCT, a combination of

Yeast, CEROPHYLL@, and Trout chow (or flake food), along with the unicellular

green alga, Se7enastrum capricornutum, will provide suitable nutrition if fed

daily.

160
4.4.2 The YCT and algae are prepared as follows:

4.4.2.1 Digested trout chow (or flake food):

10 The preparation requires one week. Use starter or No.1 pellets

prepared according to current U.S. Fish and Wildlife Service

specifications, or flake food. Suppliers of trout chow include Zeigler

Bros., Inc., P.O. Box 95, Gardners, PA 17324 (717-780-9009); Glencoe

Mills, 1011 Elliott, Glencoe, MN 55336 (612-864-3181); and Murray Elevators, 118 West 4800 South, Murray, UT 84107 (800-521-9092).

2. Add 5.0 g of trout chow pellets or flake food to 1 L of MILLI-Q® water.

Mix well in a blender and pour into a 2-L separatory funnel. Digest prior to use by aerating continuously from the bottom of the vessel for

one week at ambient laboratory temperature. Water lost due to

evaporation is replaced during digestion. Because of the offensive odor

usually produced during digestion, the vessel should be placed in a fume

hood or other isolated, ventilated area.

3. At the end of digestion period, place in a refrigerator and allow to

settle for a minimum of 1 h. Filter the supernatant through a fine mesh

screen (i.e., NITEX® 110 mesh). Combine with equal volumes of

supernatant from CEROPHYLL® and yeast preparations (below). The

supernatant can be used fresh, or frozen until use. Discard the

sediment.

4.4.2.2 Yeast:

1. Add 5.0 g of dry yeast, such as FLEISCHMANN'S® to 1 L of MILLI-Q® water.

2. Stir with a magnetic stirrer, shake vigorously by hand, or mix

with a blender at low speed, until the yeast is well dispersed. 3. Combine the yeast suspension immediately (do not allow to settle) with

equal volumes of supernatant from the trout chow (above) and CEROPHYLL®

preparations (below). Discard excess material.

4.4.2.3 CEROPHYLL® (Dried, Powdered, Cereal Leaves):

1. Place 5.0 g of dried, powdered, cereal leaves in a blender. (Available

as "CEREAL LEAVES," from Sigma Chemical Company, P.O. Box 14508, St.

Louis, MO 63178 (800-325-3010); or as CEROPHYLL®, from Ward's Natural

Science Establishment, Inc., P.O. Box 92912, Rochester, NY 14692-9012

(716-359-2502). Dried, powdered, alfalfa leaves obtained from health

food stores have been found to be a satisfactory substitute for cereal

leaves.

2. Add 1 L of MILLI-Q® water.

3. Mix in a blender at high speed for 5 min, or stir overnight at medium

speed on a magnetic stir plate.

4. If a blender is used to suspend the material, place in a refrigerator

overnight to settle. If a magnetic stirrer is used, allow to settle for

1 h. Decant the supernatant and combine with equal volumes of

supernatant from trout chow and yeast preparations (above). Discard

excess material.

161
4.4.2.4 Combined YCT Food:

1. Mix equal (approximately 300 mL) volumes of the three foods as described

above.

2. Place aliquots of the mixture in small (50-mL to 100-mL) screw-cap

plastic bottles and freeze until needed.

3. Freshly prepared food can be used immediately, or it can be frozen until

needed. Thawed food is stored in the refrigerator between feedings, and

is used for a maximum of one week.

4. It is advisable to measure the dry weight of solids in each batch of YCT

before use. The food should contain 1.7- 1.9 g solids/L. Cultures or

test solutions should contain 12-13 mg solids/L.

4.4.3 Algal (Selenastrum) Food

4.4.3.1 Algal Culture Medium

1. Prepare (five) stock nutrient solutions using reagent grade chemicals as

described in Table 1.

2. Add 1 mL of each stock solution, in the order listed in Table 1, to

approximately 900 mL of MILLI-Q® water. Mix well after the addition of

each solution. Dilute to 1 L and mix well. The final concentration of

macronutrients and micronutrients in the culture medium is given in

Table 2.

3. Immediately filter the medium through a 0.45 ~m pore diameter membrane

at a vacuum of not more than 380 mm (15 in.) mercury, or at a pressure

of not more than one-half atmosphere (8 psi). Wash the filter with 500

mL deionized water prior to use.

4. If the filtration is carried out with sterile apparatus, filtered medium

can be used immediately, and no further sterilization steps are required

before the inoculation of the medium. The medium can also be sterilized

by autoclaving after it is placed in the culture vessels.

5. Unused sterile medium should not be stored more than one week prior to

use, because there may be substantial loss of water by evaporation.

4.4.3.2 Algal Cultures

4.4.3.2.1 Two types of algal cultures are maintained: (1) stock cultures,

and, (2) "food" cultures.

4.4.3.2.2 Establishing and Maintaining Stock Cultures of Algae

1. Upon receipt of the "starter" culture (usually about 10 mL), a stock

culture is initiated by aseptically transferring one milliliter to each

of several 250-mL culture flasks containing 100 mL algal culture medium

(prepared as described above). The remainder of the starter culture can

be held in reserve for up to six months in a refrigerator (in the dark)

at 4°C.

2. The stock cultures are used as a source of algae to initiate "food"

cultures for Daphnia toxicity tests. The volume of stock culture

maintained at anyone time will depend on the amount of algal food

required for the Daphnia cultures and tests. Stock culture volume may

162
TABLE 1. NUTRIENT STOCK SOLUTIONS FOR MAINTAINING ALGAL STOCK CULTURES

AND TEST CONTROL CULTURES.

STOCK

SOLUTION

COMPOUND AMOUNT DISSOLVED IN

500 ML MILLI-Q® WATER

MgCl z·6H2O 6.08 9

CaC1 2· 2H2O 2.20 9

NaN03 12.75 9

MgS04·7H2O 7.35 9

K2HP04 0.522 9

NaHC03 7.50 9

B.

C.

D.

2. MICRONUTRIENTS

1. MACRONUTRIENTS

A.

92.8 mg 208.0 mg 1.64 mga

79.9 mg 0.714 mg b

3.63 mgC

0.006 mgd

150.0 mg 1.196 mge

BZnC1 2 - Weigh out 164 mg and dilute to 100 mL. Add 1 mL of this solution

to Stock #1.

bCoC12.6H20 - Weigh out 71.4 mg and dilute to 100 mL. Add 1 mL of this

solution to stock #1.

CNaaMo04.2H~O - Weigh out 36.6 mg and dilute to 10 mL. Add 1 mL of this

solution to stock #1.

dCuCl .2H20 - Weigh out 60.0 mg and dilute to 1000 mL. Take 1 mL of this

solulion and dilute to 10 mL. Take 1 mL of the second dilution and add to

Stock #1.

eNaaSe04 - Weigh out 119.6 mg and dilute to 100 mL. Add 1 mL of this

sOlution to Stock #1.

163
TABLE 2. FINAL CONCENTRATION OF MACRONUTRIENTS AND MICRONUTRIENTS IN THE

CULTURE MEDIUM

MACRONUTRIENT CONCENTRATION ELEMENT CONCENTRATION

(MG/L) (MG/L)

NaN03 25.5 N 4.20

MgC1 2 ·6H2O 12.2 Mg 2.90

CaC1 2 ·2H2O 4.41 Ca 1. 20

MgS04·7H2O 14.7 S 1. 91

K2HP04 1.04 P 0.186

NaHC03 15.0 Na 11.0

K 0.469

C 2.14

MICRONUTRIENT CONC ENTRATI ON ELEMENT CONCENTRATION

(J.!G/L) (J.!G/L)

H3B03 185 B 32.5

MnC1 2 ·4H2O 416 Mn 115

ZnC1 2 3.27 Zn 1.57

CoC1 2 • 6H2O 1.43 Co 0.354

CuC1 2 ·2H2O 0.012 Cu 0.004

Na2Mo04·2H2O 7.26 Mo 2.88

FeC1 3 ·6H2O 160 Fe 33.1

NazEDTA.2HzO 300

Na 2Se04 2.39 Se 1.00

164
be rapidly "scaled up" to several liters, if necessary, using 4-L serum

bottles or similar vessels, each containing 3 L of growth medium.

3. Culture temperature is not critical. Stock cultures may be maintained

in environmental chambers with cultures of other organisms if the

illumination is adequate (cont~nuous "cool-white" fluorescent lighting

of approximately 86 ± 8.6 ~E/m Is, or 400 ft-c).

4. Cultures are mixed twice daily by hand or stirred continuously.

5. Stock cultures can be held in the refrigerator until used to start

"food" cultures, or can be transferred to new medium weekly. One-to-three milliliters of 7-day old algal stock culture, containing

approximately 1.5 X 106 cells/mL, are transferred to each 100 mL of

fresh culture medium. The inoculum should provide an initial cell

density of approximately 1,000-30,000 cells/mL in the new stock

cultures. Aseptic techniques should be used in maintaining the stock

algal cultures, and care should be exercised to avoid contamination by

other microorganisms. 6. Stock cultures should be examined microscopically weekly, at transfer,

for microbial contamination. Reserve quantities of culture organisms

can be maintained for 6-12 months if stored in the dark at 4°C. It is

advisable to prepare new stock cultures from "starter" cultures obtained

from established outside sources of organisms every four to six months.

4.4.3.2.3 Establishing and Maintaining "Food" Cultures of Algae

1. "Food" cultures are started seven days prior to use for Daphnia cultures

and tests. Approximately 20 mL of 7-day-old algal stock c~lture

(described in the previous paragraph), containing 1.5 X 10 cells/mL,

are added to each liter of fresh algal culture medium (i.e., 3 L of

medium in a 4-L bottle, or 18 L in a 20-L bottle). The inoculum should

provide an initial cell density of approximately 30,000 cells/mL.

Aseptic techniques should be used in preparing and maintaining the

cultures, and care should be exercised to avoid contamination by other

microorganisms. However, sterility of food cultures is not as critical

as in stock cultures because the food cultures are terminated in 7-10

days. A one-month supply of algal food can be grown at one time, and

the excess stored in the refrigerator.

2. Food cultures may be maintained at 25°C in environmental chambers with

the algal stock cultures or cultures of other organisms if the

illumination is adequate (cont~nuous "cool-white" fluorescent lighting

of approximately 86 ± 8.6 ~E/m Is, or 400 ft-c).

3. Cultures are mixed continuously on a magnetic stir plate (with a medium

size stir bar) or in a moderately aerated separatory funnel, or are

mixed twice daily by hand. If the cultures are placed on a magnetic

stir plate, heat generated by the stirrer might elevate the culture

temperature several degrees. Caution should be exercised to prevent the

culture temperature from rising more than 2-3°C.

4.4.3.3 Preparing Algal Concentrate for Use as Daphnia Food

1. An algal concentrate containing 3.0 to 3.5 X 107 cells/mL is prepared

from food cultures by centrifuging the algae with a plankton or

bucket-type centrifuge, or by allowing the cultures to settle in a

165
refrigerator for approximately two-to-three weeks and siphoning off the

supernatant. 2. The cell density (cells/mL) in the concentrate is measured with an

electronic particle counter, microscope and hemocytometer, fluorometer,

or spectrophotometer, and used to determine the ~oncentration required

to achieve a final cell count of 3.0 to 3.5 X 10/,mL.

3. Assuming a cell density of approximately 1.5 X 106 cells/mL in the algal food cultures at 7 days, and 100% recovery in the concentration process,

a 3-L, 7-10 day culture will provide 4.5 X 109 algal cells. This number

of cells would provide approximately 150 mL of algal cell concentrate.

4. Algal concentrate may be stored in the refrigerator for one month.

4.5 FEEDING

4.5.1 Feeding rate and frequency are important in maintaining the organisms

in optimal condition so that they achieve maximum reproduction. Stock cultures

which are stressed because they are not adequately fed may produce low numbers

of young, large numbers of males, and ephippial females. When the young taken

from these inadequately fed Daphnia cultures are used in toxicity tests, they

may show higher than acceptable mortality in controls and greater than normal

sensitivity to toxicants. Steps to follow when feeding the YCT and algal diet

are as follows:

1. If YCT is frozen, remove a bottle of the food from the freezer at least

1 h before feeding time, and allow to thaw.

2. Mass cultures are fed Monday, Wednesday, and Friday at the rate of

4.5 mL YCT and 2 mL of algae concentrate per 3-L culture.

3. On Tuesday and Thursday the culture water is stirred to re-suspend the

settled algae and another 2 mL of algal concentrate is added.

4. The YCT and algal concentrate is thoroughly mixed by shaking before

dispensing.

5. Return unused YCT food mixture and algal concentrate to the

refrigerator. Do not re-freeze the YCT. Discard unused portion of YCT

after one week.

4.5.2 The quality of food prepared with newly acquired supplies of yeast,

trout chow, and dried cereal leaves, or algae, should be determined in

side-by-side comparisons of Daphnia survival and reproduction tests, using the

new food and food of known, acceptable quality, over a seven-day period in

control medium.

166
Adema, D.M.M. 1978.

toxicity tests.

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171
APPENDIX A

DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS

A.3. MYSIDS (HYSIDOPSIS BAHIA)

1. DISTRIBUTION

1.1 Mysids (Figure 1) are small shrimp-like crustaceans found in both the

marine and freshwater environments. The mysid that currently is of primary interest in the NPDES program is the estuarine species, Mysidopsis bahia.

It occurs primarily at salinities above 15~. Stuck et al. (1979a) and Price

(1982) found it in greatest abundance at salinities near 30~. Three

sympatric species of Mysidopsis, M. a7myra, M. bahia, and M. bige7owi, have

been cultured and used in toxicity testing. The distribution of Mysidopsis

species has been reported by Stuck et al. (1979b), Price (1982), and Heard et

al. (1987).

1.2 Other marine mysids that have been used in toxicity testing and held or

cultured in the lab include Metamysidopsis e7ongata, Neomysis americana,

Neomysis awatschensis, Neomysis intermedia, and recently for the Pacific

coast, Ho7mesimysis scu7pta and Neomysis mercidis. A freshwater species,

Mysis re7icta, presently not used in toxicity testing, but found in the same

habitat as Daphnia pu7ex, might be considered in the future for toxicity

testing.

2. LIFE CYCLE

2.1 In laboratory culture, Mysidopsis bahia reach sexual maturity in 12 to 20

days, depending on water temperature and diet (Nimmo et al., 1977). Normally, the female will have eggs in the ovary at approximately 12 days of age. The

lamellae of the marsupium pouch have formed or are in the process of forming

when the female is approximately 4 mm in length (Ward, 1993). Unlike Daphnia, the eggs will not develop unless fertilized. Mating takes place at night and

lasts only a few minutes (Mauchline, 1980).

2.2 Brood pouches are normally fully formed at approximately 15 days (approximately 5 mm in body length), and young are released in 17 to 20 days

(Ward, 1993). The number of eggs deposited in the brood and the number of

young produced per brood are a direct function of body length as well as

environmental conditions. Mature females have produced as many as 25 Stage I

larvae (egg-shaped embryo) per brood (8-9 mm in body length) in natural and

artificial seawater (FORTY FATHOMS®) but average 11 ± 6 Stage III larvae

(final stage before larvae are released), with increasing numbers correlated

with increasing body length (Ward, 1993). A new brood is produced every 4 to

7 days.

2.3 At time of emergence, juveniles are immobile, making them susceptible to

predation by adult mysids. The juveniles are planktonic for the first 24-48

hours and then settle to the bottom, orient to the current, and actively

pursue food organisms such as Artemia. Carr et al. (1980) reported that the

172
statocyst

/ dorsal process

3

...e~~~ftiii;~~~:-.1.~II:;'""l..-~~~:;~- endopod

thoracic segments exopod

8th thoracic limb

antennule --~~~~-Y:::~~Q~fr-r--

antenna -----'

antennal scale ---

Figure 1. Lateral and dorsal view of a mysid with morphological features

identified (From Stuck et al., 1979a).

stage in the life cycle of M. almyra most sensitive to drilling mud was the

juvenile molt, which occurs between 24 and 48 hours after release from the

brood pouch. Ward (1989) found a relationship between CaC03 level and growth

and reproduction and that M. bahia were more sensitive to cadmium during

molting (24-72 h post release) in high or low levels of CaC03 • Work done by

Lee and Buikema (1979) for Daphnia pulex also showed increased sensitivity

during molting.

3. MORPHOLOGY AND TAXONOMY

3.1 Since Mysidopsis bahia occur with two other species of Mysidopsis, an

understanding of the taxonomy of M. almyra, M. bahia, and M. bigelowi is

173
important for culturing and testing practices. The taxonomic key of Heard et

al. (1987) is suggested (see Table 1 for morphological guide to Mysidopsis).

3.2 Adults of M. bahia range in length from 4.4 mm to 9.4 mm (Molenock,

1969), measured from the anterior margin of the carapace to the end of

uropods. The mature females are normally larger than the males and the

pleopods of the female are smaller than those of the male (Ward, 1993)

(Figure 2). Mysidopsis bahia can be positively identified as male or female

when they are 4 mm in body length (Ward, 1993). Living organisms are usually

transparent, but may be tinted yellow, brown or black. Mysidopsis bige70wi

can be readily distinguished from M. a7myra and M. bahia by the morphology of

the second thoracic leg. Mysidopsis bige70wi has a greatly enlarged endopod of the thoracic limb 2 ("first leg") and the limb has a distinctive row of

6 to 12 spiniform setae on the inner margin of the sixth segment (Heard et

al., 1987). Mysidopsis bahia can also be distinguished from other species of

Mysidopsis by the number of apical spines on the tel son (4-5 pairs) and the

number of spines on the inner uropods distal to the statocyst (normally 2-3)

(Figure 2).

3.3 Heard et al. (1987) state that the most reliable character for

separating adult M. a7myra and M. bahia is the number of spines on the inner

uropods (M. a7myra will always have a single spine). Further, Price (1982)

found that for all stages of development for both species, the shape of the

anterior margin of the carapace (rostral plate) could be used to distinguish

M. a7myra (broadly rounded) from M. bahia (more produced). Figure 2

illustrates the morphological features most useful in identifying M. bahia

(redrawn from Molenock, 1969; Heard et al., 1987).

4. CULTURE METHODS

4.1 SOURCE OF ORGANISMS

4.1.1 Starter cultures of mysids can be obtained from commercial sources,

particularly in the Gulf of Mexico region for M. a7myra and M. bahia.

4.1.2 Mysids of different species can also be collected by plankton tows or

dip nets (approximately 1.0 mm mesh size) in estuarine systems. Heard et al.

(1987) have identified specimens of M. bahia along the eastern coast, however,

it has been principally identified as a subtropical species found in the Gulf

of Mexico and along the east coast of Florida. Since many species of mysids

may be present at a given collection site, the identification of the organisms

selected for culture should be verified by an experienced taxonomist. The

permittee should consult the permitting authority for guidance on the source

of test organisms (indigenous or laboratory reared) before use.

4.2 CULTURING SYSTEM

4.2.1 Stock cultures can be maintained in continuous-flow or closed

recirculating systems. In laboratory culture of M. bahia, recirculating

systems are probably the most common practice. During the past ten years, a

number of closed recirculating systems have been described. Since no single recirculating technique is the best in all respects, the system adopted will

174
a

b

c

d

e f

Figure 2. Morphological features most useful in identifying Mysidopsis bahia.

a. male; b. female; c. thoracic leg 2; d. tel son; e. right uropod,

dorsal; f. male, dorsal (redrawn from Molenock, 1969; Heard et al.,

1987). Note gonad in area where marsupium is located on female and

length of male pleopods as compared to female. Also note the 3

spines on the endopod of the uropod (e).

175
TABLE 1. GENUS MYSIDOPSIS: COMMON AND SPECIFIC CHARACTERS OF SPECIES KNOWN FROM U.S

GULF OF MEXICO WATERS (HEARD, ET AL., 1987 AND MODIFIED IN PART FROM BRAT

M.

eel ipes

M. M.

brattstroemi mortenseni

M.

furca

M.

almyra

M.

bahia

M.

bigelowi (Inshor

Anterior dorsal margin Triangular

of carapace

Presence of distal seg- yes

ment on antennal

Length/breadth ratio of 2-2.5

antennal scale

# segments in carpo· 3

propodus of thoracic

endopods 3-8

Triangular

yes

3-3.5

3

Triangular Rounded Rounded Triangular Triangular Triangul

yes yes no no no no 

3.5-4 4 6 6 5.4 5.4 

3 322 2 2 

(2 in juv.)

# segmen1:s in exopod ?

of male pleopod 4

6-7 7 7 7 7-8 7 

.....

'-l

C'I # spines on uropodal

endopod

8-9

6

10·20 18-31 20-40 1·4 5 3-4 

(occ. 3-4) (occ. 2 

Length of terminal pair(s) Almost

of telson spines relative twice

to laterlill margin spines as long

Slightly

longer

Sl ightly

longer

> twice Gradually Gradually Abruptly Abrupt

as long increasing increasing increasing increas

# of pairs of apical

telson spines

# setae on inner margin

of segment 6 of second

thoracic endopod

# setae on inner margin

of segment 5 of second

thoracic endopod

ca 3

ca 5

ca 4ca 4

ca 3

ca 2

2

ca 3

ca 3

4-8

2-3

7-18

3·6

2·3

7-18

3

6-12

2

3-4

6-12

2
depend on the facilities and equipment available and the objectives of the

culturing activities. Two other species of mysid, M. almyra and M. bigelowi,

have also been successfully reared in the system described in this section

(Ward, 1991). Further, there now exist a number of review papers (Venables,

1987; and Lussier et al., 1988) that describe in detail techniques developed

by others that will be very helpful in culturing Mysidopsis.

4.2.2 Closed recirculating systems are unique because the re-used seawater

they contain develops an unusual set of characteristics caused primarily by

metabolic waste produced by the mysids. The accumulation of waste products

and suspended particles in the water column is prevented by passing the

seawater through a biological filtration system, in which ammonia and nitrite

are oxidized by nitrifying bacteria.

4,3 CULTURE TANKS

4.3.1 Stock cultures of mysids are maintained in a closed recirculating

system. The system should consist of four 200-L glass aquaria. However,

smaller tanks, such as 80-L glass aquaria, can be used. When setting up a

system, it is important to consider surface to volume ratio since this will

determine how many mysids can be held in each aquarium. If smaller tanks must

be used, the 20-gallon "high" form is recommended. Figure 3 (Ward, 1984;

1991) illustrates the main components of the biological filtration system.

The flow rate through the filter is controlled by the water valve and is

maintained between 4-5 L/min. This flow will be sufficient to establish a

moderate current (from the filter return line) in the aquarium to allow the

mysids (which are positively rheotactic) to align themselves with the current

formed.

4.3.2 The filtration system consists of commercially-available under-gravel

filter plates and external power filter. Each aquarium has two filter plates,

forming a false bottom on each side of the tank, on which 2 cm of crushed

coral are placed. The external power filter (Eheim, model 2017) canister is

layered as shown in Figure 3 with a thin layer of filter fiber between each

layer of carbon and crushed oyster shells. There has been some modification

of the original filtration system (Ward, 1984), with crushed coral instead of

oyster shells used on the filter bed, because crushed coral does not dissolve

in seawater as readily as crushed oyster shells. If the system described

above cannot be used, an acceptable alternative is an airlift pumping

arrangement (Spotte, 1979). Crushed coral and oyster shells are commercially available and should be washed with deionized water and autoclaved before use.

4.4 CULTURE MEDIA

4.4.1 A clean source of filtered natural seawater (0.45 ~m pore diameter)

should be used to culture Mysidopsis bahia, however, artificial seasalts

(FORTY FATHOMS®) have also been successfully used (Ward, 1993). A salinity

range between 20 and 30~ can be used (25~ is suggested) to culture M. bahia.

Leger and Sorgeloos (1982) reported success in culturing M. bahia in a formula

following Dietrich and Kalle (Kalle, 1971), and still report continued use of

this formula (Leger et al., 1987b). Other commercial brands have also been

used (Reitsema and Neff, 1980; Nimmo and Iley, 1982; Nimmo et al., 1988) with

177
Charcoal

\

Power filter

Water valve

\

Oyster shells

- ... -

-

Filter return line

Power filter feed line

_________ rr========~====~

:= -/

-------:-~- 4"-'"

-------""'" II" I-- - f

...

F'I b d { Crushed coral Iter e . Filter plates

Figure 3. Closed recirculating system showing the two phases of the biological filtration system which consists of the filter bed and external

power filter (from Ward, 1984; Ward, 1991).

178
varying degrees of success. The culture methods presented in Ward (1984;

1991) have been tried with a number of commercial brands of artificial

seawater listed in Bidwell and Spotte (1985). Commercial brands of seasalts

can be extremely variable in the amount of NaHCO~. they provide, which, if not

controlled, can affect growth and reproduction (ward; 1989, 1991). In a

comparative study, Ward (1993) found normal larval development within the

marsupium using both natural seawater and FORTY FATHOMS® (i.e., Stage I -

embryo; Stage II - eyeless larva; Stage III - eyed larva which is the final

stage before release) and stressed the importance of proper preparation of the

seasalts and monitoring of conditions in the tank.

4.4.2 The culture media should be aged to allow the build-up of nitrifying

bacteria in the filter substrate. To expedite the aging process, 15 mL of a

concentrated suspension of Artemia should be added daily. If using natural or

artificial seawater, the carbonate alkalinity level should be maintained

between 90 and 120 mg/L. It is also important to establish an algal community, Spiru7ina subsa7sa, in the filter bed (Ward, 1984) and a healthy

surface dwelling diatom community, Nitzchia sp., on the walls (Ward, 1991) in

conjunction with the transfer of part of the biological filter from a healthy

tank, when possible. After seven days, the suitability of the medium is

checked by adding 20 adult mysids. If the organisms survive for 96 hours, the

culture should be suitable for stocking.

4.4.3 If brine solutions are used, 100~ salinity must not be exceeded. This

corresponds to a carbonate alkalinity value of approximately 50 mg/L, which

will allow relatively normal physiological mechanisms associated with CaC03 to

occur during certain phases of the life cycle for M. bahia (Ward, 1989).

4.5 ENVIRONMENTAL FACTORS

4.5.1 Temperature must be maintained within a range of 24°C to 26°C. Twelve

to sixteen hours illumination should be provided daily at 50 to 100 ft-c. The

daily light cycle can be provided by combining overhead room lights, coolwhite fluorescent bulbs (approx. 50 ft-c, 12L:12D), with individual Grow-lux

fluorescent bulbs placed horizontally over each tank (approx. 65 ft-c,

10L:14D). This procedure will avoid acute illumination changes by allowing

the room lights to turn on one hour before and one hour after the aquaria

lights. A timing device, such as an electronic microprocessor-based timer

(CHRONTROL®, model CD, or equivalent) can be used to control the light cycle.

These procedures are fully outlined in Ward (1984; 1991).

4.5.2 Good aeration (~60% saturation by vigorous aeration with an air stone),

a 10-20 percent exchange of seawater per week, and carbonate in the filtration

system are essential in helping to control pH drops caused by oxidation of

NH 4 -N and NOz-N by bacteria.

4.5.3 The single most important environmental factor when culturing

Mysidopsis bahia or other organisms in recirculators is the conversion of

ammonia to nitrite, and nitrite to nitrate by nitrifying bacteria. Spotte

(1979) has suggested upper limits of 0.1 mg total NH4-N/L, 0.1 mg NOz-N/L and

20 mg N03 -N/L for good laboratory operation of recirculating systems. For the

recirculating system and techniques described here for mysids, the levels of

179
ammonia, nitrite and nitrate never exceeded 0.05 mg of total ammonia-NIL

(NH3(aq,and NH4+) , 0.08 mg NO -NIL and 18 mg N03-N/L (Ward, 1991). The toxicity of ammonia is based primarily on unionized ammonia (NH~) and the proportion of

NH3 species to NH4+ species is dependent on pH, ionic strength and

temperature. It is strongly recommended that the concentrations of total

ammonia, nitrite and nitrate do not exceed those reported here. The ammonia,

nitrite, and nitrate levels can be checked by using color comparison test kits

such as those made by LaMotte Chemical or equivalent methods.

4.5.4 Bacterial oxidation of excreted ammonia by two groups of autotrophic nitrifying bacteria (Nitrosomonas and Nitrobacter) , results in an increase of

hydrogen ions, which causes a drop in pH and subsequent loss of buffering

capacity. Typically, the culturist responds to the change in pH by adding

Na2C03 or NaHC03 • However, such efforts to buffer against a drop in pH will

result in an increase in alkalinity and the uncontrolled use of carbonates can

affect reproduction, especially at higher alkalinity values (Ward; 1989,

1991). Therefore, when using carbonates to buffer against pH changes~

alkalinity values should not exceed 120 mg/L, which is easily measured by

using a titrator kit such as that available from LaMotte Chemical or

equivalent methods.

4.5.5 Figure 4 (Ward, 1991) depicts juvenile production per aquarium, no

buffer added, over a period of 24 weeks. A regression line was calculated for

these data and the slope and correlation coefficient were analyzed by

Student's t test. The data showed that even when the pH dropped as low of

7.5, there was a significant increase (P < 0.001) in juvenile production.

However, the pH should be maintained above 7.8 by the controlled use of NaHC03 and frequent water exchanges.

4.6 FEEDING

4.6.1 Frequent feeding with live food is necessary to prevent cannibalism of

the young by the adults. McKenny (1987) suggests feeding densities of 2-3

Artemia per mL of seawater and Lussier et al. (1988) suggest a feeding rate of

150 Artemia nauplii per mysid daily.

4.6.2 In the M. bahia-Artemia predator-prey relationship, it is also

important to provide sufficient quantities of nutritionally viable freeswimming stage-I nauplii (Ward, 1987); final hatching from the membranous-sac

(pre-nauplii) into stage-I nauplii does not always occur. Artemia cysts that

have been incubated for 24 h should be periodically examined with a stereozoom

microscope to enumerate free-swimming stage-I nauplii and prenauplii (membranous-sac stage).

4.6.3 It has also been found that heavy metals can affect the hatchability of

Artemia (Rafiee et al., 1986; Liu and Chen, 1987), therefore, when using

natural seawater the level of metals should always be checked.

4.6.4 Ward (1987; 1991) has tried different brands of Artemia from different

geographic origins and lot numbers; many achieved stage I nauplii and still

caused variability in production of mysids which suggests that they were

nutritionally lacking. Leger et al. (1985; 1987a) have drawn attention to

180
1

12°°1 r0-

o 900 ...........

c

o

Y = 19.24 + 13.99 X

r = 0.728

n= 377

.. , .., , II , .., '1' .'

., '

I I I I

20 22 24 26

, ,

.:. ..

, " ,

, '.

• I

• a. •••• •

.. ., '. .' . . r .'. .'• I' ••

I • • • .' . • • • • .. "II .' .. -.' . " a. I' • '" .' • • II ... ·· .':. :. ldl''"! 1 ' 'I- - ,---, ,I l' •• • .. • •

•I" , o I 'I - I I I I I I

o 2 4 6 8 10 12 14 16 18

Time (weeks)

300

:;:;

g 600

-0

o

ln.

Q)

c

Q)

>

:J

J

Figure 4. Juvenile production per aquarium over time (from Ward, 1991).

181
poor larval survival of M. bahia and low levels of certain polyunsaturated fatty acids found in the Artemia fed. The enhancement of Artemia has also

been studied and there are numerous techniques that have been successful

(Leger et a1., 1986).

4.6.5 Ward (1987; 1991) has found that it is important to control the flow of

seawater in recirculating systems (keep below 5 L/min) so that Artemia does

not become limiting to the mysid. Newly hatched Artemia should be fed to

mysids at. least twice a day. To supply Artemia to the mysid population on the

weekend and prevent cannibalism of newly released mysids, an automatic feeder

such as described by Schimmel and Hansen (1975) or Ward (1984; 1991) could be

used. Ward (1991) designed a system to hatch Artemia when personnel were not

available to set up Artemia for the following morning and afternoon feeding,

such as Monday. Cysts were placed in two 4-L Erlenmeyer flasks (dry), an

airstone was placed in each flask, and two vessels overhead were filled with

3500 mL of 30~ seawater each. The previously described timer (ChronTrol®,

Model CD) was used to open the normally closed solenoids, allowing the

seawater to gravity feed and hydrate the cysts.

4.6.6 It is possible that a surface dwelling diatom community acts as a

secondary food that supplements deficient brands of Artemia, especially for

newly released juveniles. Ward (1991) has observed that a strong fertilizing

action is caused by the excretory products of the mysid population. As the

concentration of nitrate increases (nitrification) to about 5 mg/L (in

approximately 7-10 weeks in an aquarium), a bloom of surface dwelling diatoms,

principally Nitzschia, but including Amphora and Cocconeis, occurs in natural

or artificial seawater (Ward, 1993). It is interesting to note that, at the

same time, there is a dramatic increase in the number of juveniles observed in

the aquaria (Figure 4). The diatoms form layers on the walls of the aquarium

and swarms of newly released juveniles have been found among them, possibly feeding upon them.

4.6.7 Nitzschia has been identified as a food source for the marine mud

snail, 17yanassa obso7eta (Collier, 1981), and the sea urchin, Lytechinus

pictus (Hinegardner and Tuzzi, 1981). The diatom, Ske7etonema, has also been

used as a supplemental food for M. bahia (Venables, 1987). DeLisle and

Roberts (1986) reported on the use of rotifers, Brachionus plicatilis, as a

superior food for juvenile mysids. Rotifers are active swimmers, ranging in

size from 100-175 ~m as compared to 420-520 ~m for Artemia, and would provide

a good alternative food source if their fatty acid profile is adequate.

4.7 CULTURE MAINTENANCE

4.7.1 To avoid an excessive accumulation of algal growth on the internal

surfaces of the aquaria, the walls and internal components should be scraped

periodically and the shell substrate (coral or oyster) turned over weekly.

Also, the filter plates must be completely covered so that the biological

filter functions properly. After a culture tank has been in operation for

approximately 2-3 months, detritus builds up on the bottom, which is removed

with a fish net after first removing the mysids. The rate of water flow

through the tanks should be maintained between 4-5 L/min, and 10-20% of the

seawater in each aquarium should be exchanged weekly.

182
4.7.2 Some culturists have noted problems with hydrozoan pests in their

cultures and there are procedures for their eradication, if necessary (Lawler

and Shepard, 1978; Hutton et al., 1986).

4.8 PRODUCTION LEVEL

4.8.1 At least four aquaria should be maintained to insure a sufficient

number of organisms on a continuing basis. If each 200-L aquarium is

initially stocked with between 200 and 500 adults (do not exceed 500 adults),

they will provide sufficient numbers of test organisms (Figure 4) each month.

If the cultures are correctly maintained, at least 20 percent of the adult

population should consist of gravid females (have a visible oostegite brood

pouch with young). It is also advantageous to cull older mysids in the

population every 4-6 weeks and to move mysids among the four aquaria to

diversify the gene pool.

5. VIDEO TRAINING TAPE AVAILABLE FOR CULTURING METHODS

5.1 A video training tape and supplemental report (USEPA, 1990) on culturing

Mysidopsis bahia are available from the National AudioVisual Center, Customer

Services Section, 8700 Edgeworth Drive, Capitol Heights, MD 20743-3701, (Phone

301-763-1891), as part of a video package on culturing and short-term chronic

toxicity test methods (Order No. A18657; cost $75.00).

6. TEST ORGANISMS

6.1 Juvenile Mysidopsis bahia, one to five days old, are used in the tests.

To obtain the necessary number for a test, there are a number of techniques

available. A mysid generator such as the one described by Reistsema and Neff

(1980) has been successfully used. Another method to obtain juveniles is to

take approximately 200 adult females (bearing embryos in their brood pouches)

from the stock culture and place them in a large (10 cm X 15 cm) standard fish

transfer net (2.0 to 3.0 mm openings) that is partially submerged in an 8-L

aquarium containing 4 L of clean culture medium. As the juveniles are

released from the brood pouches, they drop through the fish net into the

aquarium. The adults and juveniles in the aquarium are fed twice daily

24-hour post hydrated Artemia. The adults are allowed to remain in the net

for 48 h, and are then returned to the stock tanks. The juveniles that are

produced in the small tank may be used in the toxicity tests over a five-day

period. Another method for obtaining juveniles (Ward 1987; 1989) is simply to

remove juveniles from the stock culture with a fine mesh net, place them in

2-L PYREX® crystalline dishes with media, positioned on a light table that has

an attached viewing plate (2 mm squares), and remove juveniles less than 2 mm

in length (approximately 24 hold).

183
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an estuarine mysid. In: Vernberg, F.J., A. Calabrese, F.P. Thurberg, and

W.B. Vernberg, eds., Biological Monitoring of Marine Pollutants. Academic

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Nimmo, D.R., T.L. Hamaker, E. Matthews, and W.T. Young. 1982. The long-term

effects of suspended particulates on the survival and reproduction of the

mysid shrimp, Mysidopsis bahia, in the laboratory. In: G.F. Mayer, ed.,

Ecological Stress and the New York Bight: Science and Management.

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Nimmo, D.R., T.L. Hamaker, J.C. Moore, and R.A. Wood. 1980. Acute and chronic

effects of Dimilin on survival and reproduction of Mysidopsis bahia. In:

J.G. Eaton, P.R. Parrish, and A.C. Hendricks, eds., ASTM STP 707, American

Society for Testing and Materials, Philadelphia, PA. pp. 366-376.

Nimmo, D.R., R.J. Mirenda, and C.A. Carlson. 1991. Culturing the estuarine

mysid, Mysidopsis bahia: a synopsis of three case studies. In: T.P. Nesler

and E. P. Bergersen, eds., Mysids in fisheries: hard lessons from headlong

introductions. Proceedings of a symposium held at the 118th Annual Meeting

of the American Fisheries Society, September 9-15, 1988, Toronto, Ontario.

Amer. Fish. Soc. Symp. 9, pp. 160-168.

Nimmo, D.R., R.A. Rigby, L.H. Bahner, and J.M. Sheppard. 1978. The acute and

chronic effects of cadmium on the estuarine mysid, Mysidopsis bahia. Bull.

Environm. Contam. Toxicol. 19(1):80-84.

Price, W.W. 1982. Key to the shallow water Mysidacea of the Texas coast with

notes on their ecology. Hydrobiol. 93(1/2):9-21.

Price, W.W., A.P. McAllister, R.M. Towsley, and M. DelRe. 1986. Mysidacea from

continental shelfwaters of the northwestern Gulf of Mexico. Contrib.

Marine Sci. 29:45-58.

187
Rafiee, P., C.O. Matthews, J.C. Bagshaw, and T.H. MacRae. 1986. Reversible

arrest of Artemja development by cadmium. Can. J. Zool. 64:1633-1641.

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almyra (Crustacea; Mysidacea) in relation to temperature, salinity, and

hydrocarbon exposure. Ph.D. thesis, Texas A&M University, College Station,

TX.

Reitsema, L.A., and J.M. Neff. 1980. A recirculating artificial seawater

system for the laboratory culture of Mysjdopsjs bahja (Crustacea;

Pericaridea). Estuaries 3:321-323.

Roberts, M.H., J.E. Warinner, C.F. Tsai, D. Wright, and L.E. Cronin. 1982.

Comparison of estuarine species sensitivities to three toxicants. Arch.

Environm. Contam. Toxicol. 11:681-692.

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Shuba, P.J., H.E. Tatem, and J.H. Carro". 1978.

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Spotte, S. 1979. Fish and invertebrate culture. John Wiley and Sons, New

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238.

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Tattersall, W.M., and Tattersall. 1951. The British Mysidacea. Royal Soc.

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Environmental Research Laboratory, Gulf Breeze, FL. EPA-600/9-78-010. pp. 61-63.

188
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estuaries: Mysid shrimps. Ches. Sci. 13(4):254-262.

190
APPENDIX A

DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS

A.4. BRINE SHRIMP (ARTEMIA SALINA)

1. SYSTEMATICS

1.1 MORPHOLOGY AND TAXONOMY

1.1.1 The taxonomic status of Artemia has long been controversial because

there is considerable morphological variability over parts of its range. The

present consensus is that there is a single cosmopolitan species, Artemia

salina, which has numerous intergrading physiological and morphological

varieties (Pennak, 1989). Brine shrimp belong to the subclass Branchiopoda

which is characterized by many pairs of flattened appendages on the thorax

(Figure 1), in contrast to other members of the Crustacea that have no more

than six pairs. Probably the most distinctive feature of Artemia salina is

the compressed, triangular, and blade-shaped distal segment of the second

antenna of the male (Figure 2). The mature adult is 8 to 10 mm long with a

stalked lateral eye, sensorial antennulae,a linear digestive tract and 11

pairs of thoracopods. In the male the antennae are transformed into muscular

claspers used to secure the female during copulation.

2. DISTRIBUTION

2.1 Artemia are found nearly worldwide in saline lakes and pools. In North

America, they have been reported throughout the western United States, in

Nebraska and Connecticut and in Saskatchewan, Canada. They are probably more

widely distributed than indicated because of limited effort in collecting from

many areas of the country. They are absent from many suitable habitats,

probably because of their limited dispersal methods.

3. ECOLOGY AND LIFE HISTORY

3.1 GENERAL ECOLOGY

3.1.1 The ecological conditions under which brine shrimp live are highly

variable. The salinity can exceed 300~, where most other life cannot

survive. Favored by the absence of predators and food competitors in such

places, Artemia develop very dense populations. Although not a marine

species, they sometimes occur in bays and lagoons where brines are formed by

evaporation of seawater (salt pans). They are more commonly found in highly

saline lakes, such as the Great Salt Lake, where the shoreline may become

ringed with brown layers of accumulated brine shrimp cysts. Brine shrimp are

also common in evaporation basins used for the commercial production of salt. _

3.1.2 The reproductive habits of different populations vary considerably. In

parts of Europe parthenogenesis is the rule, males being rare or absent, but

in North America most Artemia populations seem to be diploid with males

common.

191
3.1.3 The principal mechanism of Artemia dispersion is transportation of the

cysts by wind or waterfowl and by deliberate or accidental human inoculation.

3.1.4 Growth of brine shrimp is influenced by many factors and the tolerance

of these factors is strain dependent. Optimum temperature for most strains

ranges between 25 and 35°C but strains have been reported thriving at 40°C.

Most geographical strains do not survive temperatures below 6°C except as

cysts. These cysts are tolerant of temperatures from far below DoC to near

the boiling point of water. Although Artemia can survive and reproduce under

a wide range of salinity, they are seldom found in nature in salinities below 45~ or above 2DD~. The pH tolerance of Artemia varies from neutral to

highly alkaline but the cysts will hatch best at a pH of 8 or higher.

3.1.5 Many predators including many zooplankton that populate natural salt

waters, many salt water fish, several insect groups (odonates, hemipterans and

beetles), and birds feed on brine shrimp in situations where they can tolerate

the conditions.

3.2 FOOD AND FEEDING

3.2.1 Brine shrimp are typically filter-feeders that consume organic

detritus, microscopic algae and bacteria. Blooms of microscopic algae are

favorite habitats of Artemia, and large populations develop in such areas

where they feed on the algae and heterotrophic bacteria that are produced by

these blooms. Brine shrimp populations have done well in cultures when fed

algae, rice bran (Sorgeloos et al., 1979), soybean meal or whey powder

(Bossuyt and Sorgeloos, 1979). The nauplii do not need food for four days after hatching.

3.3 LIFE HISTORY

3.3.1 Most strains of Artemia produce cysts that float (cysts from the Mono

Lake, California strain sink). These cysts remain in diapause as long as they

are kept dry or under anaerobic conditions. Upon hydration, the embryo in the

cyst becomes activated. After several hours the outer membrane bursts and the

embryo emerges still encased in the hatching membrane. Soon the hatching

membrane is ruptured and the free-swimming nauplius is born. The first instar

is brownish-orange colored and has three pairs of appendages (Figure 3). The

larva grows through about 15 molts and becomes differentiated into male or

female after the tenth molt. Copulation is initiated when the male grasps the

female with its modified antennae (Figure 4). The fertilized eggs develop

either into free-swimming nauplii, or they are surrounded by a thick shell and

deposited as cysts which are in diapause.

192
,

Figure 1. Drawi ng of male (A) and

female (B) brine shrimp

(From Kuenen and BassSeeking, 1938).

Figure 2. Head of adult male

showing triangular

distal segment of

antennae modified as

claspers (From Persoone

et al., 1980a).

Figure 3. Pre-nauplius and freshly

hatched first instar

(From Persoone et al.,

1980a).

193

Figure 4. Male and female brine

shrimp nauplius preparing to copulate

(From Persoone et al.,

1980a).
4. METHODS FOR HATCHING ARTEMIA CYSTS

4.1 SOURCES OF CYSTS

4.1.1 Brine shrimp cysts are available from many commercial sources,

representing several geographical strains. The cysts from any source can vary

from batch to batch in terms of nutritional quality for the test organisms.

Therefore, it is recommended that each new batch purchased should be analyzed

chemically, and that a side-by-side feeding test be performed on their

nutritional suitability by comparing the response of the test organisms with

the new cysts and cysts of known quality (ASTM, 1993). A list of sources of

cysts is provided at the end of this chapter.

4.2 STORAGE OF CYSTS

4.2.1 Sealed cans of Artemia cysts can be stored for years at room

temperature, but once opened, should be used up within two months. After each

use, the can should be tightly covered with a plastic lid and stored in the

refrigerator. If the entire contents of a can will not be used up in two

months, it is recommended that the portion that is expected to be unused be

placed in a tightly closed container and frozen until needed.

4.3 HATCHING OF CYSTS

4.3.1 A 2-L separatory funnel makes a convenient brine shrimp hatching

vessel, but nearly any transparent or translucent (preferably colorless)

conical shaped container that will hold water may be used. A satisfactory

apparatus can be prepared by removing the bottom of a 2-L plastic soft drink

bottle and inserting a rubber stopper with a flexible tube and pinch cock.

The hatching chambers must be clean and free from toxic material. All

detergents should be completely removed by rinsing well with deionized water.

4.3.2 Salinity of the water used for hatching brine shrimp cysts should be

between 25 and 35~. Natural sea water or water made up from artificial sea

salts may be used. The hatching medium can be prepared by placing 1800 mL of

deionized water in the hatching chamber and adding 50-70 g non-iodized salt.

After the salt is added, lower a 1 mL pipette or glass tube fitted to an air

supply into the vessel,so that the tip rests on the bottom, and bubble air

vigorously through it to dissolve the salt.

4.3.3 Add the desired quantity of cysts to the vessel. Approximately 15 mL

of cysts in a 2-L hatching vessel will provide enough brine shrimp nauplii to

feed three large stock cultures of mysids in 76-L aquaria, or 1000 to 1500

newly hatched fish in four to six 8-L tanks.

4.3.4 Continue the aeration to keep the cysts and newly hatched naupl ii from

settling to the bottom where the DO would quickly be depleted and the newly

hatched animals would die.

4.3.5 The area in wh~ch the cysts are hatched should be provided with

approximately 20 ~E/m Is (100 ft-c) of illumination.

194
4.3.6 The cysts will hatch in about 24 h at a temperature of 25°(. Hatching

time varies with incubation temperature and the geographic strain of Artemia

used.

4.4 HARVESTING THE NAUPLII

4.4.1 When the brine shrimp nauplii first emerge from the cyst, they are

enclosed in a membranous sac (Figure 3), To be taken as food by'the test

organisms, the pre-nauplii must emerge from the sac and swim about (Stage-lor

first instar nauplius).

4.4.2 The first instar (Stage-I) nauplij do not feed. Their value as food

for the test organisms decreases from birth until they begin feeding. Because

they do not feed in the hatching vessels. it is important to harvest and use

the nauplii soon after hatching. The nauplii can be easily harvested in the

following manner:

1. After approximately 24 h at 25°(, remove the pipet supplying air and

allow the nauplii to settle to the bottom of the hatching chamber. The

empty egg shells will float to the top and the newly hatched naupll i and

unhatched eggs will settle to the bottom. A light trained on the bottom

of the separatory funnel wi n has ten the settling process.

2. After approximately 5 min, using the stopcock. drain off the naupl i j

into a 250 ml beaker.

3. After another 5 min, again drain the nauplii into the beaker.

4. The nauplii are further concentrated by pouring the suspension into a

small cylinder which has one end closed with #20 plankton netting or

they may be washed through a 150-~m net or screen.

5. The concentrate is resuspended in 50 mLof appropriate culture water,

mixed well, and dispensed with a pIpette. (Mysids require approximately

100 to 150 nauplii/mysid/day).

6. Discard the remaining contents of the hatching vessel and wash the

vessel with hot soap and water.

7. Prepare fresh salt water for each new hatch.

4.4.3 To have a fresh supply of Artemia nauplii daily, at least two hatching

vessels should be used. so that the newly-hatched can be harvested daily.

4.5 FEEDING ASSAY

4.5.1 Before using brine shrimp naupljj from a new batch of cysts for

routine feeding of cultures and test organisms, they should be tested for

their ability to support life, growth, and reproduction of the test animals.

Two treatments with four replicates each are required for this test. In

Treatment (A). the test organisms are fed the nauplii from the new batch of

Artemia cysts, and in Treatment (B), the test organisms are fed nauplii of

known, good quality, such as from the reference Artemia cysts or from a batch

of Artemia cysts that have been successfully used in culturing and testing.

4.5.2 If there is no significant difference in the survival, growth, and/or

reproduction of the organisms in the two treatments at the end of a 7-day

period, it is assumed that the new batch of Artemia cysts is satisfactory. If

195
the survival, growth, and/or reproduction in treatment A is significantly less

than the response in treatment 8 over a 7-day test period it is assumed that

the new batch of brine shrimp cysts are unsuitable for use as a food source

for the organisms tested.

4.5.3 Test chambers and all test conditions during the feeding assay should

be similar to those planned for use in the subsequent toxicity tests.

196
4,6 LIST OF COMMERCIAL SOURCES OF ARTEMIA CYSTS

Aquafauna Biomarine

P.O. Box 5

Hawthorne, CA 90250

Tel. (213) 973-5275

Fax (213) 676-9387

(Great Salt lake, North Arm

San Francisco Bay)

Argent Chemi cal

8702 152nd Ave. NE

Redmond, WA 98052

Tel. (800) 4266258

Tel. (206) 855-3777

Fax (206)885-2112

(Platinum label - San Francisco Bay; Gold lable - San Francisco Bay,

Brazil, other; Silver Label - Great

Salt Lake, Australia, other; Bronze

label - China, Canada, other]

Bonneville Artemia International, Inc.

P.O. Box 511113

Salt Lake City, UT 84151-1113

Tel. (801) 972-4704

Fax (801) 972-4795

Ocean Star International

P.O. Box 643

Snowville, UT

Tel. (801) 872-8217

Fax (801) 872-8272

(Great Salt Lake)

Sanders Brine Shrimp Co.

3850 South 540 West

Ogden, UT 84405

Tel. (801) 393-5027

(Great Salt Lake)

Sea Critters Inc.

P.O. Box 1508

Tavernier, FL 33070

Tel. (305) 367-2672

197

Aquarium Products

180l Penrod Court

Glen Burnie, MD 21061

Tel. (800) 368-2507

Tel. (301) 761-2100

(Colombia)

Artemia Systems

Wiedauwkaai 79

B-9000 Ghent, Belgium

Tel 011-32-91-534142

Fax 011-32-91-536893

(For marine species - AF grade

[small nauplii], UL grade [large

nauplii], for freshwater species - IH grade [small nauplii], EG grade

[large nauplii]

Golden West Artemia

411 East 100 South

Salt Lake City, UT 84111

Tel. (801) 532-1400

Fax (801) 531-8160

Pennsylvania Pet Products

Box 191

Spri ng City, PA

(Great Salt Lake)

San Francisco Bay Brand

8239 Enterprise Drive

Newark, CA 94560

Tel. (415) 792-7200

(Great Salt lake, San Francisco

Bay)

Western Brine Shrimp

957 West South Temple Salt Lake City, UT 84104

Tel. (801) 364-3k642

Fax (801) 534-0211

(Great Salt lake)
brine shrimp nauplii as food for

Standard EI203-87, Annual Book of

Society for Testing and Materials,

SELECTED REFERENCES

ASTM. 1993. Standard practice for using

test animals in aquatic toxicology.

ASTM Standards, Vol. 11.04, American

Philadelphia, PA.

Beck, A.D., and D.A. Bengtson. 1982. International study on Artemia XXII:

Nutrition in aquatlC toxlcology - Diet quality of geographical strains of

the brine shrimp, Artemia. In: J.G. Pearson, R.B. Foster, and W.E. Bishop

(eds.), Aquatic Toxicology and Hazard Assessment: Fifth Conference. ASTM

STP 766, American Society for Testing and Materials, Philadelphia, PA.

pp. 161-169.

Beck, A.D., D.A. Bengtson, and W.H. Howell. 1980. International study on

Artemia. V. Nutritional value of five geographical strains of Artemia:

Effects of survival and growth of larval Atlantic silversides, Menidia

menidia. In: G. Persoone, P. Sorgeloos, D.A. Roels, and E. Jaspers, eds.

The- brine shrimp, Artemia. Vol. 3. Ecology, culturing, use in aquaculture.

Universa Press, Wetteren, Belgium. pp. 249-259.

Bengtson, D.A.S., A.D. Beck, S.M. Lussier, D. Migneault, and C.E. Olney.

1984. International study on Artemia. XXXI. Nutritional effects in

toxicity tests: Use of different Artemia geographical strains. In: G.

Persoone, E. Jaspers, and C. Claus, (eds.). Ecotoxicological testing

for the marine environment, Vol. 2. State Univ. Ghent and Inst. Mar.

Sci. Res., Bredene, Belgium. pp. 399-416.

Bossuyt, E., and P. Sorgeloos. 1979. Technological aspects of the batch

hatching of Artemia in high densities. In: G. Persoone, P. Sorgeloos,

O. Roels and E. Jaspers (eds.), The brine shrimp Artemia. Vol. 3.

Ecology, culturing, use in aquaculture. Universa Press, Wetteren,

Belgium. pp. 133-152.

Browne, R.A. 1982. The cost of reproduction in brine shrimp. Ecology

63(1):43-47.

Johns, D.M., W.J. Berry, and W. Walton. 1981. International study on

Artemia. XVI. Survival, growth and reproductive potential of the

mysid, Mysidopsis bahia Molenock fed various geographical strains of

the brine shrimp, Artemia. J. Exp. Mar. Biol. Ecol. 53:209-219.

Kuenen, D.J., and L.G.M. Baas-Becking. 1938. Historical notes on Artemia

sa7ina (L.). Zool. Med. 20:222-230.

Leger, P., D.A. Bengtson, K.L. Simson, and P. Sorgeloos. 1986. The use and

nutritional value of Artemia as a food source. Oceanogr. Mar. 8iol.

Ann. Rev. 24:521-623.

198
Lenz, P.H. 1980. Ecology of an alkali-adapted variety of Artemia from Mono

lake, California, U.S.A. In: G. Persoone, P. Sorgeloos, O. Roels and E.

Jaspers (eds.), The brine shrimp Artemia. Vol. 3. Ecology, culturing, use

in aquaculture. Universa Press, Wetteren, Belgium, pp. 79-96.

Nikonenko, Y.M. 1986. Adaptation of Artemia salina to toxicants.

Hydrobiol. J. 22(5):94-98.

Pennak, R.W. 1989. Fresh-water invertebrates of the United States.

Protozoa to mollusca. John Wiley and Sons, New York, NY. pp. 358-359.

Persoone, G., P. Sorgeloos, O. Roels, and E. Jaspers, (eds.). 1980a. The

brine shrimp Artemia. Vol. 1. Morphology, genetics, radiobiology,

toxicology. Universa Press, Wetteren, Belgium. 318 pp.

Persoone, G., P. Sorgeloos, O. Roels, and E. Jaspers, (eds.). 1980b. The

brine shrimp Artemia. Vol. 2. Physiology, biochemistry, molecular

biology. Universa Press, Wetteren, Belgium. 636 pp.

Persoone, G., P. Sorgeloos, O. Roels, and E. Jaspers, (eds.). 1980c. The

brine shrimp Artemia. Vol. 3. Ecology, culturing, use in aquaculture.

Universa Press, Wetteren, Belgium. 428 pp.

Sorgeloos, P. 1980. Life history of the brine shrimp Artemia. In: G.

Persoone, P. Sorgeloos, D.A. Roels, and E. Jaspers (eds.), The brine

shrimp, Artemia. Vol. 1. Morphology, genetics, radiobiology,

toxicology. Universa Press, Wetteren, Belgium. pp. ixx-xxii.

Sorgeloos, P., M. Baesa-Mesa, E. Bossuyt, E. Bruggeman, J. Dobbeler, D.

Versichele, E. Lavina, and A. Bernardine. 1979. Culture of Artemia on

rice bran: The conversion of waste-products into highly nutritive

animal protein. Aquaculture 21:393-396.

Usher, R.R., and D.A. Bengtson. 1981. Survival and growth of sheepshead

minnow larvae and juveniles on diet of Artemia nauplii. Prog. Fish-Cult. 43:102-105.

199
APPENDIX A

DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS

A,5, FATHEAD MINNOW (PIMEPHALES PROMELAS)

1, MORPHOLOGICAL AND ANATOMICAL CHARACTERISTICS

1.1 Fathead minnows vary greatly in many characteristics throughout their

wide geographic range. The morphology and characters for identification are

taken from Clay (1962), Hubbs and Lagler (1964), Eddy and Hodson (1961), Scott

and Crossman (1973), and Trautman (1981). Adults (Figure 1) are small fish,

typically 43 mm to 102 mm, and averaging about 50 mm, in total length. The

standard lengths are usually less than four and one-half times the body depth.

The first rudimentary ray of the dorsal fin is more or less thickened and

distinctly separated from the first well-developed ray by a membrane. The

lateral line is usually incomplete, but may be complete in specimens from some

geographic areas. The scales are cycloid and moderate in size. Andrews

(1970), reporting on fish collected in Colorado, noted that no scales were

found on fish smaller than 14 mm, and the average length for first scale

formation was 16.3 mm. The scales in the lateral series number 41 to 54.

1.2 The mouth is terminal. The snout does not extend beyond the upper lip

and is decidedly oblique. Nuptial tubercles occur on mature males only, are

large and well-developed on the snout, and rarely extend beyond the nostrils.

Figure 1. Fathead minnow: adult female (left) and breeding male (right). (From

Eddy and Hodson, 1961).

They occur in three main rows, with a few on the lower jaw. In addition to

nuptial tUbercles, the males have an elongate, fleshy, or spongy pad extending

in a narrow band from the nape to the dorsal fin. The pad is wide anteriorly,

and narrows to engulf the first dorsal ray. In addition, the sides of the

body become almost black except for two wide vertical bars which are light in

color. In contrast to the males, the mature females remain quite drab.

1.3 The peritoneum is brownish-black, and the intestine is long and coiled

one or more times.

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1.4 Some external markings occur infrequently. Young occasionally have a

dusky band on the snout and opercules. Other young and adults, from clear and

weedy waters, have a distinct, lateral band across the body. The band may be

absent in breeding males or, if present, becomes very diffuse anteriorly.

This band is usually most apparent on preserved specimens. Dymond (1926),

Trautman (1981), and others described the saddle-like pattern often associated

with breeding males in which a light area develops just behind the head and

another beneath the dorsal fin, the areas between producing a saddle affect.

A dark spot is usually present in front of the dorsal fin in mature males, and

a narrow, dark, vertical bar or spot is present at the base of the caudal fin,

but often is not very distinct.

2. TAXONOMY

2.1 The specific name (Pimepha7es prome7as) appears to be incorrectly applied

to this fish because the fathead minnow does not fit the description

originally given by Rafinesque (1820) (Lee et al., 1980). Common names

include "northern fathead minnow", and "blackhead minnow," in addition to

fathead minnow. The holotype was collected near Lexington, Kentucky.

2.2 Some geographic variations have been noted in the morphology of the

fathead minnow. Vandermeer (1966) indicated that the introduction of this

species outside its native range may have resulted in some local deviations

from the broad patterns of geographic variation in taxonomic characters. Some

populations have been designated as subspecifically distinct: Pimepha7es

prome7as prome7as, the northern form; P. p. harveyensis, the Harvey Lake form,

from Isle Royal in Lake Superior and P. p. confertus, the southern form (Hubbs

and Lagler, 1949, 1964). However, Taylor (1954), Vandermeer (1966), and

others expressed doubt concerning the validity of assigning subspecific status

to the variants and recommended against their recognition. Vandermeer (1966),

in a statistical analysis of the geographic variations in taxonomic

characters, stated that two of the three described subspecies intergrade clinally.

2.3 Of the eight characters measured, two showed a north-south trend; (1) eye

diameter, with the northern fish having smaller eyes, and (2) completeness of

the lateral line, with the northern fish having the least complete lateral

line. However, Scott and Crossman (1973), indicated that some Canadian

populations exhibit a nearly complete lateral line. The American Fisheries

Society (1980) does not recognize any of the fathead minnow subspecies.

201
3. DISTRIBUTION

3.1 The fathead minnow is widely distributed in North America (Figure 2). It

is a popular bait fish, and the ease with which it is propagated has led to

its widespread introduction both within and outside the native range of the

species. It has been so widely distributed in the eastern and southwestern

United States by bait transportation that it is difficult to determine its

original range. The presumed native distribution (Vandermeer, 1966; Scott and

Crossman, 1973; Lee, et al., 1980) extended from the Great Slave Lake in the

northwest to New Brunswick, in eastern Canada, southward throughout the

Mississippi valley in the United States, to southern Chihuahua in Mexico.

Distribution records for this species also now include Oregon (Andreasen,

1975), and the Central Valley (Kimsey and Fisk, 1964) and other locations in

California (Andreasen, 1975), but there are no records for British Columbia.

Figure 2. Map showing the distribution of the fathead minnow in North America.

Open circles represent transplanted populations. Most Atlantic

slope records are probably transplanted populations. (From Lee et

a1., 1980).

3.2 This species is found in a wide range of habitats. It is most abundant

in muddy brooks, streams, creeks, ponds, and small lakes, is uncommon or

absent in streams of moderate and high gradients and in most of the larger and

202
deeper impoundments, and is tolerant of high temperature and turbidity, and

low oxygen concentrations.

3.3 Species associated with the fathead minnow seem to vary greatly

throughout its range (Scott and Crossman, 1973; Trautman 1981). Trautman

(1981) reported that fathead minnows and bluntnose minnows, Pimepha7es notatus

(Rafinesque), were competitors, and that fathead minnows occurred in greatest numbers only where bluntnose minnows were absent or comparatively few in

number. He also stated that the fathead minnow may hybridize with the

bluntnose minnow

3,4 The fathead minnow is primarily omnivorous, although Coyle (1930)

reported algae to be one of its main foods in Ohio. Elsewhere in the United

States, young fish have been reported to feed on organic detritus from bottom

deposits, and unicellular and filamentous algae and planktonic organisms. Adults feed on aquatic insects, worms, small crustaceans, and other animals.

Scott and Crossman (1973) and others regard the fathead minnow as a highly

desirable forage fish, providing food for other fishes and birds.

4. GENERAL LIFE HISTORY

4.1 The natural history and spawning behavior (Markus, 1934; Flickinger,

1973; Andrews and Flickinger, 1974; and others) of the fathead minnow are well

known because of the early interest in raising the fish for bait and for

feeding other pond fish, such as bass. Sexual dimorphism occurs at maturity.

Breeding males develop a conspicuous, narrow, elongated, gray, fleshy pad of

spongy tubercles on the back, anterior to the dorsal fin, and two or three

rows of strong nuptial tubercles across the snout. The sides of the body

become almost black except for two wide vertical bars which are light in

color. In contrast, the females remain quite drab.

4.2 The initiation of spawning varies with temperature throughout its

geographic range. Isaak (1961), Carlander (1969), and others reported that,

in the wild, fathead minnows begin spawning in the spring, when the water

temperature reaches 16-18°C, and continue to spawn throughout most of the

summer. The minimum spawning temperature, however, may vary with population and latitude.

4.3 Markus (1934) reported that spawning always occurred at night, whereas

Isaak (1961) observed spawning during the day, as well as at night. Gale and

Buynak (1982) and others reported that spawning often began before dawn and

usually was completed before noon. Observations of the fathead minnow

cultures at EPA's Newtown Facility also indicate the majority of fathead

minnows spawn in early morning.

4.4 Breeding males are very territorial and select sites for spawning, such

as the underside of a log or branch, rock, board, tin can, or almost any other

solid inanimate object, usually in water from 7 cm to 1 m in depth. A

receptive female is sought out and brought into position below the nest site.

After circling below the nesting site, the female is nudged and lifted on the

male's back until she lies on her side immediately below the undersurface of

the spawning substrate, where she releases a small number of eggs (usually 100

203
to 150) at a time. The eggs are adhesive and attach to the underside of the

spawning substrate. The females have a urogenital structure (ovipositor) to

help deposit the eggs on the underside of objects. Flickinger (1966)

indicated that the ovipositor is noticeable at least a month prior to

spawning. The reported size of the eggs varies from 1.15 mm (Markus, 1934) to

1.3 mm in diameter (Wynne-Edwards, 1932).

4.5 Immediately after the eggs are laid, they are fertilized by the male, and

the female is driven off. Once eggs are deposited in the nest, the male

becomes very aggressive and will use the large tubercles on his snout to help

drive off all intruding small fishes. In addition to fertilizing and guarding the eggs, the male agitates the water around the eggs, which ventilates them

and keeps them free of detritus. Some males will spawn with several females

on the same substrate, so that the nest may contain eggs in various stages of

development. The number of eggs per nest may vary from as few as nine or 10

to as many as 12,000.

4.6 The ovaries of the females contain eggs in all stages of development, and

they spawn repeatedly as the eggs mature. A female may deposit eggs in more

than one nest. Although the average number of eggs per spawn is generally 100

to 150, large females may lay 400 to 500 eggs per spawn.

4.7 Gale and Buynak (1982), in a study using five captive pairs of fathead

minnows in separate outdoor pools, observed that each pair produced 16 to 26

clutches of eggs between May and August. The time between spawns, which

ranged from two to 16 days, was affected by water temperature. As the

temperature increased, the intervals between spawning sessions become shorter

and more uniform. In their study, from nine to 1,136 (mean of 414) eggs were

deposited per spawn. The average number of eggs deposited per spawn ranged from 371 to 480, and the total number of eggs spawned per female ranged from

6,803 to 10,164 (mean of 8,604). The length of the spawning period during a

given season also varied greatly between females. The authors suggested that

the fecundity of fathead minnows is much higher than has generally been

recognized, but they noted that fecundity of fish in the natural environment,

where conditions might be more or less favorable, might differ from that of

captive fish.

4.8 The incubation time depends on temperature, and is 4.5 to 6 days at 25°C.

The newly-hatched young (larvae) are about 5 mm long, white in color, and have

large black eyes. The general appearance and typical pigmentation of the

various larval stages are illustrated in Figures 3A-3M. In a warm, food-rich

environment, growth is rapid. Markus (1934) stated that fish hatched in

May in Iowa reached adult size and were spawning by late July. Hubbs and

Cooper (1935) and others noted that such rapid growth is unlikely in more

northerly waters, and that the young do not spawn the first year. In cooler

water the adult size is probably not reached until the second year. The males

generally grow faster than the females, a characteristic of minnow species.

4.9 The fathead minnow is short lived, and rarely survives to the third year.

However, Scott and Crossman (1973) stated that longevity varies throughout the geographic range of the species. Post-spawning mortality was reported to

be great by several authors, but was not observed by Gale and Buynak (1982).

204
A

B

D

E

F .-:.>.~.'.

G

Figure 3. Fathead minnow (Pimepha7es prome7as) larvae: A. protolarva, lateral

view, 4.3 mm TL; B. protolarva, dorsal view, 5.6 mm TL; C.

protolarva, lateral view, 5.6 mm TL; O. protolarva, ventral view,

5.6 mm TL; E. mesolarva, lateral view, 6.9 mm TL; F. mesolarva,

dorsal view, 7.9 mm TL; G. mesolarva, lateral view, 7.9 mm TL; (From

Snyder et al., 1977).

205
H ....\

...........:,,-.'..

J

K

L '-~ .::.-:-'

M

Figure 3. Fathead minnow (Pimepha7es prome7as) larvae. H. mesolarva, ventral

view, /.Y mm TL; I. metalarva, lateral view, 9.3111I11 TL; J.

metalarva, dorsal view, 14.3 mm TL; K. metalarva, lateral view, 14.3

mm TL; L. metalarva, ventral view, 14.3 mm TL; M. late metalarva,

lateral view, 19.6 mm TL (CONTINUED) (from Snyder et al., 1977).

206
However, in defending their territory, male fish, may become weakened by a

lack of food over a prolonged period and their resistance to disease may be

lowered. Also, at spawning time, many waters are warm and somewhat stagnate,

favoring the spread of fish parasites and disease.

5. CULTURE METHODS

5.1 OUTSIDE SOURCES OF FATHEAD MINNOWS

5.1.1 Fathead minnows are available from commercial biological supply houses.

Fish obtained from outside sources for use as brood stock or in toxicity tests

may not always be of suitable age and quality. Fish provided by supply houses

should be guaranteed to be of (1) the correct species, (2) disease free, (3)

in the requested age range, (4) and in good condition. The latter can be done

by providing the record of the date on which the eggs were laid and hatched,

and information on LC50 of contemporary fish using reference toxicants.

5.2 INHOUSE SOURCES OF FATHEAD MINNOWS

5.2.1 Problems in obtaining suitable fish from outside laboratories can be

avoided by developing an inhouse laboratory culture facility. Fathead minnows

can be easily cultured in static, recirculating, or flow-through systems.

5.2.2 Flow-through systems require large volumes of water and may not be

feasible in some laboratories. The culture tanks should be shielded from

extraneous disturbances using opaque curtains, and should be isolated from

toxicity testing activities to prevent contamination.

5.2.3 To avoid the possibility of inbreeding of the inhouse brood stock, fish

from an outside source should be introduced yearly into the culture unit.

5.2.4 The inhouse culture facility consists of the following components:

5.2.4.1 Water Supply

5.2.4.1.1 Water Quality

5.2.4.1.1.1 Reconstituted (synthetic) water or dechlorinated tap water can

be used, but natural water may be preferred. To determine water quality, it

is desirable to analyze the water for toxic metals and organics quarterly (see

Section 4, Quality Assurance). Temperature, dissolved oxygen, pH, hardness,

and alkalinity should also be measured periodically.

5.2.4.1.1.2 If a static or recirculating system is used, it is necessary to

equip each tank with an outside activated carbon filter system, similar to

those sold for tropical fish hobbyists (or one large activated carbon filter

system for a series of tanks) to prevent the accumulation of toxic metabolic

wastes (principally nitrite and ammonia) in the water.

207
5.2.4.1.2 Dissolved oxygen

5.2.4.1.2.1 The DO concentration in the culture tanks should be maintained

near saturation, using gentle aeration with 15 cm air stones if necessary. Brungs (1971), in a carefully controlled long-term study, found that the

growth of fathead minnows was reduced significantly at all DO concentrations

below 7.9 mg/L. Soderberg (1982) presented an analytical approach to the

re-aeration of flowing water for culture systems.

5.2.4.2 Maintenance

5.2.4.2.1 Adequate procedures for culture maintenance must be followed to

avoid poor water quality in the culture system. The spawning and brood stock

culture tanks should be kept free of debris (excess food, detritus, waste,

etc.) by siphoning the accumulated materials (such as dead brine shrimp nauplii or cysts) from the bottom of the tanks daily with a glass siphon tube

attached to a plastic hose leading to the floor drain. The tanks are more

thoroughly cleaned as required. Algae, mostly diatoms and green algae, growing on the glass of the spawning tanks are left in place, except for the

front of the tank, which is kept clean for observation. To avoid excessive

build-up of algal growth, the walls of the tanks are periodically scraped.

The larval culture tanks are cleaned once or tWice a week to reduce the mass

of fungus growing on the bottom of the tank.

5.2.4.2.2 Activated charcoal and floss in the tank filtration systems should

be changed weekly, or more often if needed. Culture water may be maintained

by preparation of reconstituted water or use of dechlorinated tap water.

Distilled or deionized water is added as needed to compensate for evaporation.

5.2.4.2.3 Before new fish are placed in tanks, salt deposits are removed by

scraping or with 5% acid solution, the tanks are washed with detergent,

sterilized with a hypochlorite solution, and rinsed well with hot tap water

and then with laboratory water.

5.2.5 SPAWNING TANKS AND CULTURE CONDITIONS

5.2.5.1 For breeding tanks, it is convenient to use 60 L (15 gal) or 76 L

(20 gal) aquaria. The spawning unit is designed to simulate conditions in

nature conductive to spawning, such as water temperature and photoperiod.

Spawning tanks must be held at a temperature of 25 ± 2°C. Each aquarium is

equipped with a heater, if necessary, a continuous filtering unit, and

spawning substrates. The photoperiod for the culture system should be

maintained at 16 h light and 8 h darkness. For the spawning tanks, this

photoperiod must be rigidly controlled. A convenient photoperiod is 5:00 AM

to 9:00 PM. Fluorescent lights should be suspended about 60 cm above the

surface of the water in the brood and larval tanks. Both DURATEST® and coolwhite fluorescent lamps have beeQ used, and product similar results. An

illumination level of 10-20 ~E/m Is (50 to 100 ft-c) is adequate.

208
5.2.6 SPAWNING BEHAVIOR AND CONDITIONS

5.2.6.1 To simulate the natural spawning environment, it is necessary to

provide substrates (nesting territories) upon which the eggs can be deposited and fertilized, and which are defended and cared for by the males. The

recommended spawning substrates consist of inverted half-cylinders, such as

7.6 cm X 7.6 cm (3 in. X 3 in.) sections of schedule 40, PVC pipe. The

substrates should be placed equi-distant from each other on the bottom of the

tanks.

5.2.6.2 To establish a breeding unit, 15-20 pre-spawning adults six to eight months old are taken from a "holding" or culture tank and placed in a 76-L

spawning tank. At this point, it is not possible to distinguish the sexes.

However, after less than a week in the spawning tank, the breeding males will

develop their distinct coloration and territorial behavior, and spawning will

begin. As the breeding males are identified, all but two are removed,

providing a final ratio of 5-6 females per male. The excess spawning substrates are used as shelter by the females.

5.2.6.3 Sexing of the fish to ensure a correct female/male ratio in each tank

can be a problem. However, the task usually becomes easier as experience is

gained (Flickinger, 1966). Sexually mature females usually have large bellies

and a tapered snout. The sexually mature males are usually distinguished by their larger overall size, dark vertical color bands, and the spongy nuptial

tubercles on the snout. Unless the males exhibit these secondary breeding characteristics, no reliable method has been found to distinguish them from

females. However, using the coloration of the males and the presence of an

enlarged urogenital structures and other characteristics of the females, the

correct selection of the sexes can usually be achieved by trial and error.

5.2.6.4 Sexually immature males are usually recognized by their aggressive

behavior and partial banding. These undeveloped males must be removed from

the spawning tanks because they will eat the eggs and constantly harass the

mature males, tiring them and reducing the fecundity of the breeding unit.

Therefore, the fish in the spawning tanks must be carefully checked

periodically for extra males.

5.2.6.5 A breeding unit will remain in their spawning tank about four months.

Thus, each brood tank or unit is stocked with new spawners about three times a

year. However, the restocking process is rotated so that at anyone time the

spawning tanks contain different age groups of brood fish.

5.2.7 EMBRYO COLLECTION

5.2.7.1 Fathead minnows spawn mostly in the early morning hours. They should

not be disturbed except for a morning feeding (approximately 8:00 AM) and

daily examination of substrates for eggs in late morning or early afternoon.

In nature, the male protects, cleans, and aerates the eggs until they hatch.

In the laboratory, however, it is necessary to remove the eggs from the tanks

to prevent them from being eaten by the adults, and for ease of handling for

purposes of recording embryo count and hatchability, and for the use of the

newly hatched for young fish for toxicity tests.

209
5.2.7.2 Daily, beginning six to eight hours after the lights are turned on

(i.e., 11:00 AM - 1:00 PM), the substrates in the spawning tanks are each

lifted carefully and inspected for embryos. Substrates without embryos are

immediately returned to the spawning tank. Those with embryos are immersed in

clean water in a collecting tray, and replaced with a clean substrate. A

daily record is maintained of each spawning site and estimated number of

embryos on the substrate.

5.2.8 EMBRYO INCUBATION

5.2.8.1 Three different methods are described for embryo incubation.

5.2.8.1.1 Incubation of Embryos on the Substrates: Several (2-4) substrates

are placed on end in a circular pattern (with the embryos on the inner side)

in 10 cm of water in a tray. The tray is then placed in a constant

temperature water bath, and the embryos are aerated with a 2.5 cm airstone

placed in the center of the circle. The embryos are examined daily, and the

dead and fungused embryos are counted, recorded, and removed with forceps. At

an incubation temperature of 25°C, 75-100% hatch occurs in five days. At

22°C, embryos incubated on aerated tiles require seven days for 50% hatch.

5.2.8.1.2 Incubation of Embryos in a Separatory Funnel: The embryos are

removed from the substrates with a rolling action of the index finger ("rolled

off")(Gast and Brungs, 1973), their total volume is measured, and the number

of embryos is calculated using a conversion factor of approximately 430

embryosjmL. The embryos are incubated in about 1.5 L of water in a 2 L

separatory funnel maintained in a water bath. The embryos are stirred in the

separatory funnel by bubbling air from the tip of a plastic micro-pipette

placed at the bottom, inside the separatory funnel. During the first two

days, the embryos are taken from the funnel daily, those that are dead and

fungused are removed, and those that are alive are returned to the separatory funnel in clean water. The embryos hatch in four days at a temperature of

25°C. However, usually on day three the eyed embryos are removed from the

separatory funnel and placed in water in a plastic tray and gently aerated

with an air stone. Using this method, the embryos hatch in five days.

5.2.8.1.2.1 Hatching time is greatly influenced by the amount of agitation of

the embryos and the incubation temperature. If on day three the embryos are

transferred from the separatory funnel to a static, unaerated container, a 50%

hatch will occur in six days (instead of five) and a 100% hatch will occur in

seven days.

5.2.8.1.3 Incubation in Embryo Incubation Cups: The embryos are "rolled off"

the substrates, and the total number is estimated by determining the volume.

The embryos are then placed in incubation cups attached to a rocker arm

assembly (Mount, 1968). Both flow-through and static renewal incubation have

been used. On day one, the embryos are removed from the cups and those that

are dead and fungused are removed. After day one, only dead embryos are

removed from the cups. Most of the embryos will hatch in five days if

incubated at 25°C.

210
5.2.8.1.4 During the incubation period, the eggs are examined daily for

viability and fungal growth, until they hatch. Unfertilized eggs, and eggs

that have become infected by fungus, should be removed with forceps using a

table top magnifier-illuminator. Non-viable eggs become milky and opaque, and

are easily recognized. The non-viable eggs are very susceptible to fungal

infection, which may then spread throughout the egg mass. Removal of fungused

eggs should be done quickly, and the spawning substrates should be returned to

the incubation tanks as quickly as possible so that the good eggs are not

damaged by desiccation.

5.2.9 LARVAE REARING TANKS

5.2.9.1 Newly-hatched larvae are transferred daily from the egg incubation

apparatus to small rearing tanks, using a large bore pipette, until the hatch

is complete. New rearing tanks are set up on a daily basis to separate fish

by age group. Approximately 1500 newly hatched larvae are placed in a 60-L

(IS-gal) or 76-L (20-gal) all-glass aquarium for 30 days. A density of 150

fry per liter is suitable for the first four weeks. The water temperature in

the rearing tanks is allowed to follow ambient laboratory temperatures of

20-25°C, but sudden, extreme, variations in temperature must be avoided.

5.2.10 HOLDING OR CULTURE TANKS FOR REPLACEMENT SPAWNERS

5.2.10.1 Replacement spawners (brood stock) are cultured from larvae produced

in the spawning tanks. After 30 days in a larval rearing tank, a number of

juveniles, equivalent to 2-4 days hatch are transferred to brood stock tanks

for a 30- to 60-day growth period. The sub-adults then are transferred to

500-L brood stock tanks to provide about 500 sub-adult fish per month for the

brood tank rotation. The surplus fish are transferred to 2000-L fiber glass,

or equivalent, holding tanks.

5.2.10.2 Surplus young males removed from spawning tanks, and other surplus

mature males, are placed in all-male holding tanks for future use as spawners.

Similarly, young and surplus mature females are held in all-female holding

tanks until needed as spawners. Tanks holding replacement spawners need not

be temperature-controlled, but for ease of transfer to the spawning tanks, it

is preferable to hold the water temperature close to that of the spawning tanks (25 ± 2°e).

5.2.11 FOOD AND FEEDING

5.2.11.1 Newly hatched brine shrimp nauplii or frozen adult brine shrimp and

commercial fish starter are fed to the fish cultures in volumes based on age,

size, and number of fish in the tanks. The amount of food and feeding schedule

affects both growth and egg production.

5.2.11.2 Fish from hatch to 30 days old are fed starter food at the beginning

and end of the work day, and newly hatched brine shrimp nauplii (from the

brine shrimp culture unit) twice a day, usually mid-morning and mid-afternoon.

Utilization of older (larger) brine shrimp nauplii may result in starvation of

the young fish because they are unable to ingest the larger food organisms

(see Appendix A.4 for instructions on the preparation of brine shrimp

211
nauplii). Avoid introducing Artemia cysts and empty shells when the brine

shrimp nauplii are fed to the fish larvae. Some of the mortality of the

larval fish observed in cultures could be caused from the ingestion of these

materials.

5.2.11.3 Fish older than four weeks are fed frozen brine shrimp and

commercial fish starter (#1 and #2), which is ground fish meal enriched with

vitamins. As the fish grow, larger pellet sizes are used, as appropriate. (Starter, No.1 and N. 2 granules, U.S. Fish and Wildlife Service Formulation

Specification Diet SD9-30, can be obtained from Zeigler Bros., Inc., P.O. Box

90, Gardners, PA 17324 (717-780-9009); Glencoe Mills, 1011 Elliott, Glencoe,

MN 55336 (612-864-3181); and Murray Elevators, 118 West 4800 South, Murray, UT

84107 (800-521-9092).

5.2.11.4 The spawning fish and pre-spawners in holding tanks usually are fed

all the adult frozen brine shrimp and tropical fish flake food or dry

commercial fish food (No.1 or No.2 granules) that they can eat (ad libitum)

at the beginning of the work day and in the late afternoon (i.e., 8:00 AM and

4:00 PM). The fish are feed twice a day, twice with dry food and once with

adult shrimp, during the week, and once a day on weekends.

5.2.12 DISEASE CONTROL

5.2.12.1 Fish are observed daily for abnormal appearance or behavior.

Bacterial or fungal infections are the most common diseases encountered.

However, if normal precautions are taken, disease outbreaks will rarely, if

ever, occur. Hoffman and Mitchell (1980) have put together a list of some

chemicals that have been used commonly for fish diseases and pests.

5.2.12.2 Treatment of individual lots of infected fish should be carried out

separate from the main culture. Use of treated fish should be avoided, if

possible, and diseased cultures should be replaced.

5.2.12.3 In aquatic culture systems where filtration is utilized, the

application of certain antibacterial agents should be used with caution.

A treatment with a single dose of antibacterial drugs can interrupt nitrate

reduction and stop nitrification for various periods of time, resulting in

changes in pH, and in ammonia, nitrite and nitrate concentrations (Collins et

al., 1976). These changes could cause the death of the culture

organisms.

5.2.12.4 To prevent possible rapid spread of disease, do not transfer

equipment from one tank to another without first disinfecting tanks and nets.

If an outbreak of disease occurs, any equipment, such as nets, airlines,

tanks, etc., which has been exposed to diseased fish should be disinfected

with sodium hypochlorite. Also to avoid the contamination of cultures or

spread nf disease, each time nets used to remove live or dead fish from tanks,

they are first sterilized with sodium hypochlorite or formalin, and rinsed in

hot tap water. Before a new lot of fish is transferred to culture tanks, the

tanks are cleaned and sterilized as described above.

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5.2.13 RECORD KEEPING

5.2.13.1 Records are kept in a bound notebook, include: (1) type of food and

time of feeding for all fish tanks; (2) time of examination of the tiles for

embryos, the estimated number of embryos on the tile, and the tile position

number; (3) estimated number of dead embryos and embryos with fungus observed

during the embryonic development stages; (4) source of all fish; and (5) daily

observation of the condition and behavior of the fish.

5.2.14 REFERENCE TOXICANTS

5.2.14.1 It is recommended that static acute toxicity tests be performed monthly with a reference toxicant. Fathead minnow larvae one to 14 days old

are used to monitor the acute toxicity of the reference toxicant to the test

fish produced by the culture unit.

6. VIDEO TRAINING TAPE AVAILABLE FOR CULTURING METHODS

6.1 A video training tape and supplemental report (USEPA, 1989) on culturing

fathead minnows are available from the National AudioVisual Center, Customer

Services Section, 8700 Edgeworth Drive, Capitol Heights, MD 20743-3701, (Phone

301-763-1891), as part of a video package on short-term chronic toxicity tests

for freshwater organisms (Order No. EPA18036), which costs $45.00.

7. TEST ORGANISMS

7.1 Fish one to 14 days old are used in acute toxicity tests.

7.2 If the fish are kept in a holding tank or container, most of the water

should be siphoned off to concentrate the fish. The fish are then transferred

one at a time randomly to the test chambers until each chamber contain

10 fish. Alternately, fish may be placed one to two at a time into small

beakers or plastic containers until they each contain five fish. Two of these

beakers/plastic containers (total of 10 fish) are then assigned to each

randomly-arranged control and exposure chamber.

7.3 The fish are transferred directly to the test vessels or intermediate

chambers using a large-bore, fire-polished glass tube (6 mm to 9 mm I.D. X 30

cm long) equipped with a rubber bulb, or a large volumetric pipet with tip

removed and fitted with a safety type bulb filler. The glass or plastic

containers should only contain a small volume of dilution water.

7.4 It is important to note that larvae should not be handled with a dip net.

Dipping small fish with a net may result in damage to the fish and cause

mortality.

213
SELECTED REFERENCES

American Fisheries Society. 1980. A list of common and scientific names of

fishes from the United States and Canada. 4th ed. American Fisheries

Society, Committee on Names of Fishes. 174 pp.

ASTM. 1993. Standard practice for using brine shrimp nauplii as food for test

animals in aquatic toxicology. Designation: E 1203-87, Annual Book of

ASTM Standards, Vol. 11.04, American Society for Testing and Materials,

Philadelphia, PA.

Andreasen, J.K.

in Oregon.

1975. Occurrence of the fathead minnow, Pimepha7es prome7as,

Calif. Fish Game 6(3):155-156.

Andrews, A.K.

prome7as.

1970. Squamation chronology of the fathead minnow, Pimepha7es

Trans. Amer. Fish. Soc. 99(2):429-432.

Andrews, A.K. 1971. Altitudinal range extension for the fathead minnow

(Pimepha7es prome7as). Copeia 1:169.

Andrews, A., and S. Flickinger. 1974. Spawning requirements and

characteristics of the fathead minnow. Proc. Ann. Conf. Southeastern

Assoc. Game Fish Comm. 27:759-766.

Benoit, D.A. and R.W. Carlson. 1977. Spawning success of fathead minnows on

selected artificial substrates. Prog. Fish. Cult. 39(2):67-69.

Brown, B.E. 1970. Exponential decrease in a population of fathead minnows.

Trans. Am. Fish. Soc. 99(4):807-809.

Brungs, W.A. 1971a. Chronic effects of elevated temperature on the fathead

minnow (Pimepha7es prome7as Rafinesque). Environmental Research

Laboratory, U.S. Environmental Protection Agency, Duluth, MN.

EPA/600/8-81/011.

Brungs, W.A. 1971b. Chronic effects of low dissolved oxygen concentrations

on fathead minnows (Pimepha7es prome7as). J. Fish. Res. Bd. Can.

28:1119-1123.

Buttner, J.K. and S.W. Duda. 1988. Maintenance and reproduction of fathead

minnows in the laboratory. Aquatic Ecology Section, Department of

Biological Sciences, SUNY College at Brockport, Brockport, NY 14420.

Carl ander, K. 1969. Handbook of freshwater fishery biology, Vol. 1. Iowa

State Univ. Press, Ames, lA.

Chiasson, A.G. and J.H. Gee. 1983. Swim bladder gas composition and control

of buoyancy by fathead minnows (Pimepha7es prome7as) during exposure to

hypoxia. Can. J. Zool. 61(10):2213-2218.

Clay, W. 1962. The Fishes of Kentucky. Kentucky Dept. Fish and Wildlife

Res., Frankfort, KY.

214
APPENDIX A

DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS

A.6. RAINBOW TROUT, ONCORHYNCHUS HYKISS AND

BROOK TROUT, SALVELINIUS FONTINALIS

1, RAINBOW TROUT

1.1 SYSTEMATICS AND TAXONOMY

1.1.1 Rainbow trout are native to the streams of the Pacific coast where

several varieties or strains have developed. The seagoing form is known as

the steel head trout and is thought to be identical to the strictly freshwater

rainbow form. Many other strains, for example, the inland lake form (Kamloops

trout) are found in other watersheds. Because of the ease with which the eggs

can be transported, different strains have been distributed allover the

world.

1.1.2 Rainbow trout are a variable species that differ considerably over the

whole of their range. Populations in different regions and watersheds of

North America have been referred to over the years by different scientific

names (e.g. species, distinct subspecies, or variants of a single species and

different regional common names). In recent years the validity of the generic

name, Sa7mo, for some western North American trout species has been

questioned. Fish taxonomists agree that native "Sa7mo" trouts of the northern

Pacific Ocean drainage are closely related with Pacific salmon Oncorhynchus

spp. The American Society of Ichthyologists and Herpetologists and the

American Fisheries Society have accepted Oncorhynchus as the appropriate

generic name for all native Pacific drainage trouts that are presently called

Sa7mo, based on new data and evidence by Smith and Stearly (1989).

Furthermore, the Names of Fishes Committee of the American Fisheries Society

has adopted the specific name, Oncorhynchus mykiss, for the rainbow trout and

its anadromous form, steel head trout. The new names for the other North

American species affected are the following: Apache trout (0. apache),

cutthroat trout (0. clarki), Gila trout (0. gi7ae), golden trout

(0. aguabonita) , and Mexican golden trout (0. chrysogaster).

1.2 DISTRIBUTION

1.2.1 The native range of the rainbow trout group (all varieties) in North

America is west of the Rocky Mountains and along the eastern Pacific Ocean,

but the species (Oncorhynchus mykiss) has now been introduced into many parts

of the continent (Figure 1). Except for the northern and southern extremes of

the rainbow trout range, anadromous populations occur in all coastal rivers.

This species, under all its common names (rainbow trout, Kamloops trout,

steelhead trout, steelhead, coast rainbow trout, and silver trout), has been

so widely introduced in North America outside its natural range as to suggest

it may occur throughout the United States in all suitable habitats. Rainbow

trout are widely introduced and established in appropriate cold water habitats

allover the world.

219
Figure 1. Map showing the distribution of the rainbow trout in North America.

(Modified from Lee et al., 1980).

1.3 GENERAL LIFE HISTORY

1.3.1 In its natural environment of flowing streams of the western mountains,

the rainbow trout (Figure 2) thrives best at temperatures ranging from 3°C in

the winter to 21°C in the summer, but the optimum temperature is between 10-

16°C. The rainbow trout can withstand higher and lower temperature if it is

acclimated gradually. However, the rainbow trout's growth is impeded by

extremes of temperature, for example, above 27°C which it can tolerate only for short periods of time.

1.3.2 Rainbow trout are basically spring spawners, but they can spawn at the

beginning of summer or early winter, depending on climate, elevation, and

genetic strain. If the spawning occurs in late fall or in winter, the eggs do

not hatch until spring. Prior to the spawning season adult males develop a

kype (elongated hooked snout) on the lower jaw and their colors intensify. Males and females usually migrate upstream and select spawning sites in beds

of fine, clean gravel in riffles or runs above pools in streams. Long journeys may be made by lake-dwelling rainbow (or Kamloops) and steelhead

trouts or anadromous, ocean-run rainbow steel heads. If the rainbow trout are

confined to land-locked lakes, they move into shallow shoals or reefs of

gravel and sand for spawning. Females dig out pits or sweep out depressions

(redds) in the gravel or sand and later spawn with males. Males are capable

220
Figure 2. Rainbow trout (Modified from Eddy and Underhill, 1974).

of displaying aggressive behavior on the spawning grounds and can drive other

males away from a redd occupied by a female. In general, one or more males

court the digging female by sliding along side and crossing over her body and

rubbing their snout against her caudal peduncle with body pressing and body

vibrations. The female deposits her eggs, which are 3-5 mm in diameter,

demersal, and pink to orange in color. The eggs are immediately fertilized by

one or more males, fall into spaces between the gravel, and are covered with

loose gravel or sand to depths of 20 cm or more by the female. Females are

capable of digging and spawning in several redds with the same male or

different males. The number of eggs released can range from 400-3000,

depending on the size of the female.

1.3.3 Eggs usually hatch in approximately four to seven weeks. The time of

hatching, however, varies greatly with region and habitat. If the stream

temperature averages 7°C, eggs will hatch in about 48 days. The newly hatched

fish, called alevins, have a yolk sac, which is absorbed in three to seven

days. After the yolk sac is absorbed, the young are called fry, and begin

feeding in 10-15 days. In general, rainbow trout feed on a variety of

invertebrates. Also, depending on their size and the habitat in which they

live, other fishes and fish eggs, especially salmon, can be important food.

The fry of lake-resident spawners move up or down the spawning river to the

lake, or they may spend as much as one to three years in the streams. The

stream-resident spawners remain in the streams, whereas the steelhead trout,

which are stream-spawners, migrate to the sea, usually after 1-4 years in

freshwater.

1.3.4 The growth of rainbow trout is highly variable with the area, habitat,

type of life history, and quantity and type of food. Some males may be good

breeders at two years of age, but few females produce eggs until their third

year of life. Rainbow trout young attain fingerling size of about 76 mm by

the end of their first summer. The length may range between 178-204 mm at the

end of the second year, 279-382 mm after the third year, 356-406 mm after the

fourth year, and 406 mm or more after the fifth year. Lake- and ocean-run

rainbows may grow over twice as fast as this. However, the average length of

221
rainbow trout (or Kamloops trout) is 305-458 mm and that of steel head trout is

508-762 mm. Under favorable conditions of artificial propagation, yearlings

average about 28 g, 2-year-olds about 255 g, 3-year-olds between .45-9 kg, and

4-year-olds between 1.4-1.8 kg. Returning sea-run individuals weigh up to

18 kg, or even more, but usually between 1.4-9 kg with the majority weighing

less than 5.4 kg. Some western varieties weight up to 23 kg, but the midwest

rainbows are much smaller. Those in streams are rarely over 1.4 kg, but in

some large lakes (e.g.; Lake Superior) and in some western lakes they may

reach 7 kg or much larger. The life expectancy of rainbow trout can be as low

as three or four years in many streams and Lake populations, but that of

seagoing steel head rainbow trout and Great lakes populations would appear to

be 6 to 8 years (Scott and Crossman, 1973).

1.4 GENERAL DESCRIPTION

1.4.1 Adult rainbow trout are bluish or olive green above and silvery on the

sides, with a broad pink lateral stripe that is enhanced during the spawning

season. The back, the sides, and the dorsal and caudal fins are profusely dotted with small dark spots. Their color is variable with habitat, size, and

sexual condition. Stream forms and spawners are generally darker with color

more intense, lake forms lighter, brighter, and more silvery. Different color

types are often called by different names, e.g., darker stream fish often

called rainbows; larger, brighter, silvery fish in western lakes often called

Kamloops trout, and large silvery specimens returning from the sea and in the

Great Lakes or tributaries called steel head trout. The scales are large,

numbering 120 to 150 in the lateral line. The caudal fin is very slightly

forked. The dorsal fin has 11 rays, and the anal fin has from 10 to 12 rays.

1.4.2 Young rainbow trout are typically blue to green on the dorsal surface,

silver to white on the sides and white ventrally. There are 5-10 dark marks

on the back between the head and dorsal fin. Also, there are 5-10 short,

dark, oval parr marks widely spaced on the sides, straddling the lateral line

with some small dark spots above but not below the lateral line. The dorsal

fin has a white to orange tip and a dark leading edge, or a series of bars or

spots. The adipose fin is edged with black, and the anal fin has an orange to

white tip.

2. BROOK TROUT

2.1 SYSTEMATICS AND TAXONOMY

2.1.1 Brook trout can be found exhibiting some variation in growth rate and

color throughout its range, but is considered a stable and well-defined

species (American Fishery Society, 1980). Male brook trout may be crossed

with female lake trout (Sa7ve7inus namaycush) to produce fertile hybrids that

are known as splake. Trautman (1981) and other papers cited in this section

indicate that brook trout can naturally and artificially hybridize with brown

trout (Sa7mo trutta) and rainbow trout (Oncorhynchus mykiss). For additional

information and discussion on freshwater and anadromous brook trout stocks and

systematic notes of brook trout, see Scott and Crossman (1973) and other

papers cited in this section.

222
2.2 DISTRIBUTION

2.2.1 The native range of the brook trout (Figure 3) is eastern North America,

extending throughout much of eastern Canada from Hudson Bay and Ungava Bay drainages and Labrador; southward through the New England States and in the

Appalachian Mountains to the headwaters of the Savannah, Chattahoochee, and

Tennessee Rivers in the Carolinas and Georgia. In the Great Lakes, brook

trout are native to Lake Superior and tributaries to the northern tip of the

Lower Peninsula, the interior of the Great Lakes basin. They are also native

to a few far-northern headwaters of the upper Mississippi river system, and

western Minnesota and northeastern Iowa.

Figure 3. Map showing the distribution of the brook trout in North America.

(Modified from Lee et al., 1980).

2.2.2 The brook trout has been widely introduced to higher elevations in

western North America. This species is also found in temperate regions of

other continents. Inland forms are found in colder lakes and streams, and

sea-run (anadromous) forms are found in the northeastern North American

coastal water areas.

223
2.3 GENERAL LIFE HISTORY

2.3.1 Brook trout (Figure 4) are generally found in clear brooks, streams,

and rivers in which the mean temperature rarely exceeds 10°C. The optimum

temperature is reported as ranging from 7 to 13°C, but they may be found

living in waters with temperatures ranging from 1 to 22°C (Piper et al. 1982).

The brook trout usually inhabits waters which flow less swiftly than those

inhabited by the rainbow. Brook trout also thrive in the small cold-water

lakes of the Great Lakes region, provided that suitable spawning conditions

exist.

Figure 4. Brook trout. (Modified from Eddy and Underhill, 1974).

2.3.2 Brook trout spawn in late summer or autumn, the date varying with

latitude and temperature, usually from late October to December when the water

temperature is suitable although some may start spawning in September in

certain streams flowing into large lakes. Some females are capable of

spawning when they are a year old, while others do not mature until the second

year. When the spawning season occurs, brook trout move upstream into small

head waters or brooks where they select gravel and sand substrates usually in

shallow riffle areas or the tail-ends of pools for the spawning beds.

Spawning usually occurs during the day.

2.3.3 The female prepares a nest (redd), similar to those of the rainbow

trout, by sweeping out a depression in the gravel and sand substrate. During

preparation of the redd, the male starts courtship by quivering around the

female and driving off all intruders. When the female is ready to spawn, she

takes a position above and close to the redd. The male gets close to her side

and arches his body over hers, discharging milt as the female deposits her

eggs. Occasionally second male may join them in the spawning. After

spawning, the male leaves. .

2.3.4 The eggs are 3.5 to 5.0 mm in diameter, are adhesive, and adhere to the

gravel at the bottom of the redd. The female pushes loose gravel and sand to

224
the center, covering the entire redd, and then desert the nest. A female may

spawn several times, and the number of eggs can vary from 100 to 5000,

depending on the size of the female.

2.3.5 The eggs remain in the redd until the water temperature rises during

the following spring. If the level of DO is adequate, the eggs will hatch in

approximately 75 days at an average water temperature of 6.1°C, and in

approximately 50 days at an average temperature of 10°C. The upper lethal

temperature limit for developing eggs is about 11.7°C (Scott and Crossman,

1973).

2.3.6 After the eggs hatch, the larvae (sac fry) remain in the gravel of the

redd until the yolk is absorbed. Depending on the water temperature, it may take from one to three months for the yolk sac to be absorbed (Lagler, 1956).

While the yolk sac is absorbed, the fry work themselves free from the gravel and start feeding. They become free swimming at about 38 mm long. Under

natural conditions, newly hatched brook trout establish small feeding

territories in the stream and feed on small aquatic insects, insect larvae,

and other organisms.

2.3.7 Growth of brook trout is extremely variable, depending on the

suitability of the environment. The average length attained at various ages

may approximate 8.9 cm the first year; 15.2 cm the second year, 22.9 cm the

third year, 30.5 cm the fourth year, and 33 cm the fifth year. Brook trout

generally do not exceed a length of 54 cm and a weight of 1.5 kg (Trautman,

1981). However, Scott and Crossman (1973) reported a brook trout as large as

6.6 kg. Rumors of larger brook trout have been circulated, but none have been

verified. Brook trout may overpopulate small streams, resulting in large

numbers of small trout less than 25.4 cm long. Wild brook trout seldom live

longer than five years, and rarely live more than eight years.

2.4 GENERAL DESCRIPTION

2.4.1 The sides of large young and adult brook trout are dark olive,

sprinkled with light spots and red spots outlined with purplish or blue hue.

Some forms have red spots with light brown margins. The scales are cycloid,

small, in about 215 to 250 rows at the lateral line. The top of the head and

back is dark olive and heavily vermiculated. There are no black or brown

spots on the head, back, adipose, or caudal fin. The anterior rays of the

pectoral, pelvic, and anal fins are milk-white, bordered posteriorly with a

dusky hue and the remainder of the fins yellowish or reddish.

2.4.2 The back of young or immature brook trout is olive, the sides are

lighter and more silvery, and the belly is whitish. There are between 8-12

rectangular parr marks en the sides, also a few yellow and blue spots, but no

black spots.

2.4.3 The dorsal fin has 10 rays, and the anal fin has 9 rays. The belly of

breeding males is red, and some males may develop a hook (or kype) at the

front of the lower jaw. The tailor caudal fin is slightly notched in the

young but is generally square in older brook trout.

225
3. HOLDING AND ACCLIMATION PROCEDURES FOR TROUT STOCKS

3.1 SOURCES OF ORGANISMS

3.1.1 Trout fry are obtained from commercial hatcheries during March through

July. However, if trout are needed for toxicity testing, it is advisable to

contact the hatchery for its trout hatching and rearing schedule. If trout

must be ordered from out-af-state, the State Fish and Game Agency should be

contacted concerning regulations on fish importation. The recommended age for

test organisms is approximately 15-30 days (after yolk sac absorption to 30

days) for rainbow trout and 30-60 days for brook trout. Trout are purchased

36 to 48 h prior to their use as testing organisms, but they must have time to

stabilize over the acclimation period. Trout should appear disease-free and

unstressed, with fewer than 5% of the animals dying during the 24-48 hours

preceding use in a toxicity test.

3.1.2 Trout fry are usually transported in plastic bags of at least 4-mil

plastic or thicker in shipping containers. The bags are partially filled with

water saturated with oxygen. During warm weather the shipping containers are

cooled with ice or cold packs to prevent temperature increases which will

result in the loss of fish. Trout should be acclimated gradually from the

temperature of the transportation unit to that of holding environment. Upon

arrival at the destination the plastic bags should be allowed to float

unopened in the holding tank for about 30 minutes to acclimate the fish.

3.2 HOLDING CONDITIONS

3.2.1 Trout are held in 200-L (50-gal) or larger tanks supplied with a

flow-through water system, or with recirculated water and a biological

filtration system. The holding water should be moderately hard and free of

chlorine, have low concentrations of metals, and should have a pH between

6 and 9. Provide a daily pholoperiod of 16 hours light, 8 hours darkness with

an illumination at 10-20 ~E/m/s (50-100 ft-c, or ambient laboratory levels).

A IS-min dimmer timer should be used to gradually increase or decrease the

illumination when lights are turned on or off. The gradual increase and

decrease of illumination at the beginning and ending of the photoperiod is

important because trout tend to jump when startled by a sudden change in light

intensity. Holding water temperature is maintained at 12°C ± 2°C and is

aerated as close as possible to saturation. Measurements of temperature, DO,

pH, conductivity, and ammonia are made on holding water daily.

3.3 FEEDING

3.3.1 Trout are fed fine texture trout chow which can be obtained from

Zeigler Bros., Inc., P.O. Box 95, Gardners, PA 17324 (717-780-9009); Glencoe

Mills, 1011 Elliott, Glencoe, MN 55336 (612-864-3181); and Murray Elevators,

118 West 4800 South, Murray, UT 84107 (800-521-9092). The fry in the holding

tank are fed (ad libitum) up to 24 hours before the start of the acute

toxicity test. Dead or moribund fish should be removed from the holding tanks

every day. Excess food and feces are vacuum-siphoned off the bottom of the

tank daily.

226
3.3.2 Daily records should be maintained for organism survival, health, and

acclimation conditions.

4. TEST ORGANISMS

4.1 Rainbow trout fry 15 to 30 days old, and brook trout 30-60 days old, are

used in acute tests (see summary tables of test conditions in Section 9, Acute

Toxicity Test Procedures). The fry in the holding tank are not fed for 24

hours prior to the start of the test. The fry are caught carefully with a

fine mesh net and placed gently in the 5 L (4 L test solution volume) test

chambers, until 10 fish are reached per test chamber. Larger test chambers or

S fish/chambers may be necessary if DO or pH problems are encountered.

Placement of the test chambers is random.

4.2 After the fish are introduced, the behavior should be noted and recorded

throughout the test period. At the beginning and ending of the photoperiod, during the test, the light intensity should be raised and lowered gradually

over a IS-min period using a dimmer switch or suitable device. Between

observations the test vessels are covered to act as a dust barrier and to

prevent fish from jumping out.

227
SELECTED REFERENCES

American Fisheries Society. 1980. A list of common and scientific names of

fishes from the United States and Canada. Special Publication No. 12.

Amer. Fish. Soc., Bethesda, MD.

Bailey, R.M. and C.R. Robins. 1989. Changes in North American fish names,

especially as related to the International Code of Zoological Nomenclature,

1985. Bull. Zool. Nomencl. 45(2):92-103.

Eddy, S. and A.C. Hodson. 1970. Taxonomic keys to the common animals of the

north central states. Burgess Publ. Co., Minneapolis, MN.

Eddy, S. and J.C. Underhill. 1974. Northern fishes. Univ. Minnesota Press,

Minneapolis, MN.

Hubbs, C.L. and K.F. Lagler. 1967. Fishes of the Great Lakes Region. Univ.

Michigan Press, Ann Arbor, MI.

Lagler, K.F. 1956. Freshwater Fishery Biology. Wm. C. Brown Co., Publ.,

Dubuque, IA.

Lee, D.S., C.R. Gilbert, C.H. Hocutt, R.E. Jenkins, D.E. McAllistger, and R.

Stauffer, Jr. 1980. Atlas of North American freshwater fishes. Publ.

1980-12, North Carolina· State Museum Nat. Hist., Raleigh, NC.

Leitritz, E. and R.C. Lewis. 1976. Trout and salmon culture (Hatchery methods). California Dept. Fish and Game, Fish Bulletin 164. Sacramento,

CA.

National Academy of Sciences. 1974. Fishes - Guidelines for the breeding,

care, and management of laboratory animals. Printing and Publishing

Office, National Academy of Sciences, Washington, D.C.

Piper, R.G., I.B. McElwain, L.E. Orme, J.P.McCraren, L.G. Fowler, and J.R.

Leonard. 1982. Fish hatchery management. U.S. Dept. Interior, Fish and

Wildlife Service, Washington, D.C.

Scott, W.B. and E.J. Crossman. 1973. Freshwater fishes of Canada. Fisheries

Research Board of Canada, Ottawa, Canada.

Smith, G.R. and R.F. Stearly. 1989. The classification and scientific names of

rainbow and cutthroat trouts. Fisheries 14(1):4-10.

Trautman, M.B. 1981. The fishes of Ohio. Ohio State Univ. Press and Ohio Sea

Grant Program, Center Lakes Erie Area Research, Columbus, OH.

Willers, B. 1991. Trout Biology: A natural history of trout and salmon. Lyons and Burfoud, 31 West 21 Street, New York, NY 10010.

228
APPENDIX A

DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS

A.7. SHEEPSHEAD MINNOW (CYPRINODON VARlfGATUS)

1. MORPHOLOGY AND TAXONOMY

1.1 The sheepshead minnow (Cyprinodon variegatus) belongs to the family

Cyprinodonitidae (killifishes), which includes 45 genera and 300 species

worldwide, occurring on all continents except Australia. Most species are

freshwater, but some occur in brackish and coastal marine waters. There are

thirteen species in the Genus Cyprinodon in the United States (American

Fisheries Society, 1980). The sheepshead minnow is the only marine species,

and is widely distributed in the coastal waters of the Atlantic and Gulf of

Mexico.

1.2 Adult sheepshead minnows (see Hardy, 1978, for a complete description).

can attain a total length of 93 mm, but the average standard length report for

adults is 35-50 mm. The males are usually somewhat longer than females. The

fish have the folloWing morphological characteristics: lack a lateral line;

have 24-29 lateral scale rows; have a large elongate humeral scale just above

the pectoral base; the dorsal fin has nine to 13 rays; the anal fin has nine

to 12 rays; the caudal fin has 14-16 principal rays and a total of 28-29 rays;

the pectoral fin has 14-17 rays, and the ventral fin has five to seven rays.

1.3 The body of males is short, compressed, and deep. The depth increases

with age. The upper profile is evenly elevated. The males are olivaceous

above with a lustrous steel blue or bluish green area on the back from nape to

dorsal or beyond, and have a series of poorly defined dark bars on the sides

and a belly that is yellowish white to deep orange. The dorsal fin ocellus on

posterior rays is lacking or developed as faint dusky spot.

1.4 The females are light olive, brown, brassy, or light orange above with

14 dark crossbars on the lower sides alternating with seven to eight crossbars

on the back. The lower sides and belly are yelloWish or white. The dorsal

fin is olive or dusky and has one or two prominent ocelli on the posterior

rays.

2. LIFE HISTORY

2.1 DISTRIBUTION AND GENERAL ECOLOGY

2.1.1 Sheepshead minnows occur in estuaries along the Atlantic and Gulf coasts

(Figure 1). They are a schooling, euryhaline species that inhabit a variety

of shallow water habitats, such as coves, bays, ponds, inlets, harbors,

bayous, salt marshes, and along open beaches. In some cases, they may be very

abundant where the bottom is partially sandy, emergent vegetation lacking, and

little current or wave action are present. This species may establish

populations in inland lakes containing relatively high concentrations of

229
Figure 1. Map showing the distribution of the sheepshead minnow (Cyprinodon

variegatus) in North America. Open circles represent transplanted populations (From Lee et al., 1980).

dissolved salts. They are tolerant of extreme changes in water temperatures, ranging from 0-40°C, and in salinities, ranging from 0.1 to 149~ (Simpson and

Grunter, 1956; Nordlie, 1987).

2.1.2 This omnivorous fish is an important component of the estuarine

ecosystem serving as a link in transferring energy from lower trophic levels,

detritus and benthic plants and animals, to carnivores in higher trophic

levels (Hansen and Parrish, 1977). Sheepshead minnows serve as forage fish

for commercially and recreationally valued fish species, such as the black

drum (Pogonias cromis), red drum (Sciaenops oce77ata), bluefish (Pomatomus

sa7tatrix), spotted seatrout (Cynoscion nebu7osus) , striped bass (Morone

saxati7is), and snook (Centropomus undecima7is) (Gunter, 1945; Darnell, 1958;

Grant, 1962; Sekavec, 1974, and Carter et al., 1973).

2.2 GENERAL SPAWNING BEHAVIOR

2.2.1 Sheepshead minnows (Figures 2, 3, 4) spawn at depths of 2.5 to 60 cm in

shallow bays, tide pools, mangrove lagoons, and pools in shallow, gently flowing streams, and other similar habitats over bottoms of sand, black silt,

or mud. Males occupy territories up to 0.3-0.6 m in diameter and mayor may

not construct nest pits. Spawning may take place out of both pit and

230
D

G

J

Figure 2. Sheepshead minnow (Cyprinodon variegatus). A. unfertilized egg; B.

blastodisc stage; C-D. 8-cell stage; E. 16-cell stage; F. late

cleavage; G. germ ring formed; H. blastoderm over 1/4 of yolk; I.

early embryo; J. embryo 48-hours old; K. tail-free embryo. (From

Kuntz, 1916).

231
A

B

c

D

E

F

G

3.4 mmTL

4.0mm

9.0mm

12.0mm

Figure 3. Sheepshead minnow (Cyprjnodon varjegatus). A-E. yolk-sac larvae; F.

larvae; G. juvenile; (B-C, E-G, from Kuntz, 1916; A, D, from Foster,

1974).

232
Figure 4. Sheepshead minnow (Cyprinodon variegatus). A. juvenile; B. adult

(From Jordan and Evermann, 1896-1900).

233
territory. Besides temperature) Martin (1972) reported that sudden changes in

salinity can initiates spawning activities. Eggs (Figure 2) are demersal,

adhesive or semi-adhesive with very minute attachment filaments (threads) more

or less evenly distributed over the chorion. They stick to a variety of

substrates, such as plants, sand, rocks, logs, and to each other. Sometimes

they stick to plants near the surface, and at other times become partially

buried in the bottom. The yolk contains one very large and many minute oil

globules. Adults spawn possibly throughout the year on the Gulf coast of the

United States. Hansen and Parrish (1977) reported that in an estuary near

Pensacola, Florida, spawning may occur during any month of the year. Ripe

females are found April to October in North Carolina, throughout the summer in

the Chesapeake Bay, May to August in Delaware Bay, May to September in New

Jersey and New York, and June to mid-July in Massachusetts.

3. CULTURE METHODS AND FACILITIES

3.1 SOURCES OF ORGANISMS

3.1.1 Juvenile and adult sheepshead minnows (Figure 4) for use as brood stock

spawners may be obtained from commercial biological supply houses or taken by

seine in coastal estuaries of the Atlantic coast and Gulf of Mexico. They may also be obtained from young fish raised to maturity in the laboratory. Feral

brood stock and first generation laboratory fish may be preferred, to minimize

inbreeding. A continuous supply of wild stock, however, may be more cost

effective. Neither fish nor eggs of feral stock should contain excessive

contaminants nor exhibit excessive mortality, and the fish should demonstrate

normal behavior. Before being used as a source of gametes, field-caught

adults should be maintained and observed in the laboratory for at least one

week to permit detection of disease and to allow time for acute mortality

resulting from stress of capture. Injured or diseased fish should be

discarded.

3.2 LABORATORY CULTURE FACILITIES

3.2.1 Sheepshead minnows can be cultured in a static, recirculated, or

flow-through systems. Flow-through systems require large volumes of water and

may not be feasible in some laboratories.

3.3 LABORATORY YEAR-ROUND SPAWNING

3.3.1 In the laboratory, adults may be kept in breeding condition year round.

Females may spawn a number of times at intervals of one to seven days, and

will generally produce an average of 10 to 30 eggs per spawning (USEPA,

1978a). To obtain large number of eggs at one particular time, adult fish of

27 mm standard length or greater should be used. If fish are taken in the

field, they should be acclimated for at least one to two weeks in 20-30~

salinity, a water temperature of 25-28°C, and a photoperiod of 16 h light and

8 h dark.

3.3.2 Sheepshead minnows can be continuously cultured in the laboratory from

eggs to adults. The eggs (embryos), larvae, juveniles, and adults (Figures 2,

3, 4) should be kept in rearing and holding tanks of appropriate size and

234
maintained at ambient laboratory temperature. The larvae should be fed

sufficient newly-hatched Artemia nauplii daily to assure that live nauplii are

always present. At the juvenile stage, they are fed frozen adult brine shrimp and a commercial flake food, such as TETRA SM-80® (available from Tetra Sales,

(U,S.A.), 201 Tabor Rd, Morris Plains, NJ 07950, phone: 800-526-0650), MARDEL

AQUARIAM® Tropical Fish Flakes (available from Marde1l Laboratories, Inc.,

1958 Brandon Court, Glendale Heights, IL 60139, phone: 312-351-0606), or

equivalent. Adult fish are fed flake food two or three times daily,

supplemented with frozen adult brine shrimp.

3.3.3 Sheepshead minnows normally reach sexual maturity three to five months

after hatching, and have an average standard length of approximately 27 mm for

females and 34 mm for males, if held at a temperature of 25-30°C in rearing

tanks of adequate size, and fed adequately. 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 to obtain large number of embryos by

natural spawning, the brood stock should be kept in a temperature controlled

system at 18-20°C. To initiate spawning, the spawners are moved to spawning

tanks with a temperature of 25°(. Adults can be maintained in natural or

artificial seawater in a flow-through, static, or recirculating, aerated

system consisting of an all-glass aquarium, or a LIVING STREAM® fiberglass,

circular or rectangular tank (Figid Unit, Inc., 3214 Sylvania Ave., Toledo, OH

43613, phone 419-474-6971), or equivalent (see USEPA, 1985 and USEPA, 1987).

3.3.4 Static systems are equipped with an undergrave1 filter. Recirculating

systems are equipped with an outside biological filter constructed in the

laboratory using a reservoir system of crushed coral, crushed oyster shells,

or dolomite and gravel, charcoal, floss, (see Spotte, 1973; 1979, Bower, 1983

for information on filters and conditioning the biological filter), or a

commercially available cartridge filter, such as a MAGNUM® Filter, available

from Carolina Biological Supply Co., Burlington, NC 27215, phone 800-334-5551,

an EHEIM® Filter, available from Hawaiian Marine Imports Inc., P.O. Box

218687, Houston, TX 77218, phone 713-492-7864, or an equivalent system. The

culture conditions should include seawater at 20-30~, and a photoperiod of

16 h light and 8 h dark. Water temperature may be controlled or maintained at

ambient laboratory levels.

3.4 OBTAINING EGGS (EMBRYOS) FOR TOXICITY TESTS

3.4.1 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, available from United States

Biochemical Corp., Cleveland, OH 44128, phone, 216-765-5000. If the culturing

system for adults is temperature controlled, natural spawning can be induced

to obtain large number of embryos by raising the temperature to 25°(. Natural

spawning is preferred because repeated spawning can be obtained from the same

brood stock, whereas with hormone injection, the brood stock is sacrificed in

obtaining gametes. It should be emphasized that the injection and hatching

schedules given below are to be used only as gUidelines. Response to the

hormone varies with brood stock and temperature. Time-to-hatch and percent

235
hatch also vary among stocks and among batches of embryos obtained from the

same stock, and are dependent on temperature, DO, and salinity.

3.5 NATURAL SPAWNING

3.5.1 Adult fish should be maintained at 18-20°C in a temperature controlled

system. The number of spawning chambers and fish to be spawned should be

based on the requirements for providing sufficient numbers of viable embryos.

As indicated above, an adult female in spawning condition will generally produce an average 10 to 30 eggs per spawn. To obtain embryos for a test,

adult fish (generally, at least eight-to-ten females and three males) are

transferred to a spawning chamber in a 57 L (15 gal) aquarium with the correct

photoperiod and temperature (16 h light/8 h dark, and a temperature of 25°C),

seven to eight days before the larval fish are needed. The spawning tank is

fitted with a spawning chamber and an embryo collection tray. The spawning chamber consists of a basket of 3-5 mm NITEX® mesh, approximately 20 X 35 X 22

cm high (USEPA, 1978a), designed to fit into the aquarium. Spawning generally will begin within 24 h or less. The embryos will fall through the bottom of

the spawning chamber and lightly adhere onto a collecting screen or tray

placed on the bottom of the tank. 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 fish needed to perform a

toxicity test (generally two to four) as determined by experience. If the

collecting trays do not contain sufficient embryos after the first 24 h,

discard the embryos, replace the tray, and collect the embryos for another

24 h. To help keep the embryos clean, the adults are fed while the screens

are removed. Spawning fish should be shielded from excessive outside

disturbance, e.g. an opaque curtain should surround the entire culture system.

Care should also be taken so that outside light sources do not interfere with

the photoperiod.

3.5.2 The embryos are collected in a tray placed on the bottom of the tank.

The collecting trays are fabricated from plastic fluorescent light fixture

diffusors (grids), with cells approximately 14 mm deep X 14 mm square. A

screen consisting of 250-500 ~m 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. 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

gently with a fine brush. The embryos from several spawning units are

generally pooled in a single container to provide a sufficient number to

conduct the test(s). The embryos are transferred to 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). The embryos are then placed in

incubation dishes (e.g. KIMAX® or PYREX® crystallizing dishes, Carolina

culture dishes, or equivalent; see 3.8, Embryo Incubation and Hatching

Facility). It is recommended that the embryos be obtained from fish cultured

inhouse, rather than from outside sources, to eliminate the uncertainty of

damage caused by shipping and handling that may not be observable, but which

might affect the results of the test. After sufficient number embryos are

236
collected for the test, the adult fish are returned to the (18-20°C) culture

holding tanks.

3.6 SUSTAINED NATURAL EMBRYO PRODUCTION

3.6.1 Sustained (long-term), daily, embryo production can be achieved by maintaining mature fish (ratio of approximately 12-15 males to 50-60 females)

in tanks, such as a 285-L LIVING STREAM® tank, or equivalent, at a temperature

of 23-25°C. Embryos are collected seven or eight days prior to starting the

acute or chronic toxicity tests for less than 24 hour or older larvae.

Embryos are produced daily, and when needed, collecting trays are placed on

the bottom of the tank. The next morning, the embryo collectors are removed,

and the embryos are washed into a shallow glass culture dish using artificial

seawater. Four collecting trays, each approximately 20 cm X 45 cm, will cover

the bottom of a 285-L tank.

3.7 FORCED SPAWNING

3.7.1 Human chorionic gonadotrophin (HCG) is reconstituted with sterile

saline or Ringer's solution immediately before use. The standard HCG vial

contains 1,000 IU, which is reconstituted in 10 mL of saline. Freeze-dried

HCG, which comes with premeasured and sterilized saline, is the easiest to

use. The reconstituted HCG may be used for several weeks if kept in the

refrigerator.

3.7.2 Each female is injected with HCG on two consecutive days. The HCG is

injected into the peritoneal cavity, just below the skin, using the smallest

needle possible. A 50 IU dose (0.5 mL of reconstituted hormone solution) 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. 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. Injected females should be isolated from males.

3.7.3 With injections made on days one and two, females which are held at

25°C should be ready for stripping on Days 4, 5, or 6. Ripe females should

show pronounced abdominal swelling, and release at least a few eggs in

response to a gentle squeeze. Eggs are stripped from the ripe 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

of viable embryos.

3.7.4 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.

3.7.5 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

237
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 under-ripe eggs. Ripe

eggs are spherical, approximately 1.0-1.7 mm in diameter, and almost clear.

Place the testes in a fold of NITEX~ screen (250-500 ~m mesh), dampen with

seawater, and macerate while holding over the dish containing the eggs. Rinse

the testes with seawater to remove the sperm from the tissue, and wash the

remaining sperm and testes into the dish with the eggs. Let the eggs and

sperm stand together for 10-15 minutes, sWirling occasionally.

3.7.6 Pour the contents of the dish into a crystallizing dish or equivalent and insert an airstone. Aerate gently, so that the water moves slowly over

the eggs, and incubate at 25°C for 60-90 min. After this period of time, wash

the fertilized eggs on a NITEX~ screen, place them in clean seawater in an

incubation chamber.

3.8 EMBRYO INCUBATION AND HATCHING FACILITY

3.8.1 Embryos are incubated in KIMAX® or PYREX~ crystallizing dishes,

Carolina culture dishes, or equivalent, at a temperature of 25°C and 14-h

light/10-h dark photoperiod. An air stone is placed in each dish, and the

contents are gently aerated for the duration of the incubation. The water in

the incubation chambers is replaced daily. Approximately 24 h prior to

hatching, the salinity of the seawater in the incubation chambers is changed

to that of the test salinity, if different. The salinity must remain within

the 20 to 30~ range. The embryos should hatch in 6-7 days at 25°C, and in 4

to 5 days at 30°C.

3.9 FEEDING AND STOCKING DENSITY

3.9.1 The sheepshead minnow cultures should be provided a sufficient amount

of high quality nutrition without over-feeding. The adult and juvenile sheepshead minnows are fed, frozen adult brine shrimp and flake food,

ad libitum, daily. The larvae are fed newly hatched Artemia nauplii and

crushed flake food, ad libitum, daily. Methods for culturing brine shrimp are

discussed in Appendix A.4. The stocking of adult fish in the holding tanks

depends on the biological filter system (see Biological Filters and Substrate

Conditioning). A circular, 1.3 m (48 in.) diameter, 880 L (235 gal), fiberglass tank will hold approximately 30-50 adult fish with a varied sex

ratio. A stocking density of about 300 larvae is suitable in a 76 L aquarium. Brood stock should be replaced with feral fish annually, or whenever the

fecundity of the females diminishes, and they appear spent with age and from

frequent breeding.

3.10 CULTURE TANKS

3.10.1 Larvae, juvenile, and adult fish should be kept in holding and rearing

tanks of appropriate size. The tanks can be all-glass aquaria, fiberglass tanks, or equivalent. All tanks should have appropriate biological filtration

systems, and the culture filtration system should be conditioned properly before adding the fish (see Spotte, 1973, 1979; Bower, 1983).

238
3.11 BIOLOGICAL FILTERS AND SUBSTRATE CONDITIONING

3.11.1 Holding and rearing aquaria and tanks can accommodate as many fish as

its biological filter will permit. The substrate conditioning for the

undergravel or outside filters is also important to the life and health of the

fish. Substrate conditioning is the process to develop nitrifying bacteria

(Njtrosomonas and Njtrobacter) that can convert ammonia and nitrite to

nitrate. A conditioned filter bed is defined as one in which the capacity for

ammonia and nitrite oxidation is sufficient to keep pace with the production

of ammonia by the fish. Consult Spotte (1973; 1979) or Bower (1983) for a

thorough understanding of the biological filter and conditioning process.

3.12 CULTURE WATER

3.12.1 Artificial seawater is prepared by dissolving FORTY-FATHOMS® or

equivalent artificial sea salts in deionized water to a salinity of 20-30~. Synthetic sea salts are packaged in plastic bags and mixed with

deionized (MILLI-Q® or equivalent) water. The instructions on the package of

sea salts should be followed carefully, and the salts should be

mixed in a separate container, and 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 (see Spotte, 1973,

1979; Bower, 1983) before it is used for culturing by aerating mildly for at

least 24 h.

3.12.2 Adequate aeration will bring the Ph and concentration of dissolved

oxygen and other gases into equilibrium. The concentration of dissolved

oxygen in the water supply should be 90-100% saturation before it is used. If

a residue or precipitate is present, the solution should be filtered before

use. The seawater should be monitored periodically to insure a constant

salinity.

3.13 CULTURE CONDITIONS

3.13.1 Holding and rearing tanks and any area used for manipulating live

sheepshead minnows should be located in a room or space separated from that in

which toxicity test(s) are to be conducted. The salinity of the culture

systems should be between 20 and 30~. Water temperature for the brood stock shou~d be maintained at 18-20°(. A photoperiod of 14 h illumination (10-20 ~E/m Is, or 50-100 ft-c) and 10 h dark, should be provided. The holding and

rearing tanks should be aerated so that the DO is not less than 1.0 ppm below

saturation at any given temperature, with 5.0 ppm (60% saturation) being the

absolute lowest limit.

3.14 CULTURE MAINTENANCE

3.14.1 Replace approximately 10% of the culture water every two weeks, or 25%

monthly. The culture water should be clear. If the water appears cloudy or

discolored, replace at least 50% of it. Replacement water should be well

oxygenated and at the same temperature and salinity as the culture water.

Salinity is maintained at the proper level by adding deionized water to

compensate for evaporation. A replenisher, made of the trace elements, iodine

239
(KI) and bromine (KBr), is added (1 mL/400 L) to the culture water each week,

or commercial trace elements replenisher should be used as directed by the

artificial sea salt manufacturer.

3.14.2 To avoid excessive build up of algal growth, periodically scrape the

walls of culture system. Some of the algae will serve as a supplement to the

diet of the fish. A partial activated carbon "charcoal" change in the

filtration systems should be done monthly or as needed. The detritus (dead

brine shrimp nauplii and cysts, adult brine shrimp, other organic material

accumulation) should be siphoned from the bottom of rearing and holding

aquaria or tanks each week or as needed.

3.15 WATER QUALITY MONITORING

3.15.1 Checking the chemistry of the sea water is critical to the success of

the marine culture system. The water quality will determine whether the life

support processes in the filter bed work at reasonable and steady rates. The

culture water is checked routinely for temperature, alkalinity, pH, DO, total

ammonia, nitrite, and nitrate. More frequent monitoring of these parameters

is recommended during periods of organism procurement and starting new culture

systems with inside underground filters and outside-of-tank biological

filtration. The DO should be maintained at greater than 60% saturation. The

pH should not go below 7.5 with an acceptable range between 7.5 to 8.3. Low

pH levels can result from overcrowding, overfeeding, or waste accumulation,

especially in static or recirculating culture systems.

3.15.2 Acceptable pH levels can be re-established by siphoning off 50-75% of

the water and replacing it with conditioned artificial seawater of the same

temperature. Also, sodium bicarbonate or commercially available liquid

buffers can be added to the tanks whenever the pH falls below 7.5. Un-ionized

ammonia, total (NH3 + NH4), and nitrite ion (N02) levels should not exceed

0.1 ppm in the holding tanks. It is recommended that the ammonia and nitrite

concentrations be determined prior to starting new culture systems. It is

recommended that nitrate (N03 ) concentrations be determined prior to starting

new culture systems, and the nitrate ion concentrations should not exceed

20 mg/L.

3.15.3 A specific schedule for water quality monitoring should be established

for each culture system. All water quality measurements and data are recorded

in the culture and environmental conditions log books.

3.16 DISEASE CONTROL AND TREATMENT

3.16.1 Discussions of identification and treatment of common parasites of

marine fish culturing can be found in Spotte (1973), Sindermann (1970), and

Bower (1983). Several commercial companies, e.g. Aquatronics, P.O. Box 12107,

La Costa Station, Malibu, CA 09265; Marine Enterprises, Inc., Baltimore, MD

(301) 321-1189; and Hawaiian Marine Imports, Inc., Houston, TX (713) 492-7864,

sell various kinds of medication to treat common parasites of marine fish.

240
3.16.2 A colorless medication, FORMALITE II®, available from Aquatronics,

has been used successfully for the treatment of the protozoan parasites, Chj7odone77a, Costja, Trjchojna, Scyphjdja, Trjchophrya, and Ichyophjrjus.

4. VIDEO TRAINING TAPE AVAILABLE FOR CULTURING METHODS

4.1 A video training tape and supplemental report (USEPA, 1990) on culturing

sheepshead minnows are available from the National AudioVisual Center,

Customer Services Section, 8700 Edgeworth Drive, Capitol Heights, MD 20743-

3701, (Phone 301-763-1891), as part of a video package on short-term chronic

toxicity tests for marine organisms (Order No. A18545). The package includes

methods for sheepshead minnows, inland silversides, sea urchins, and Champja,

and costs $85.00.

5. TEST ORGANISMS

5.1 Sheepshead minnows 1-14 days old are used in the acute toxicity test. If

the larvae are used one or two days after hatching, they can be held in the

crystallizing or culture dishes. If they are to be used later, they should be

placed in larger holding aquarium or tanks. Prior to beginning the test, the

larvae can be transferred to small beakers or plastic cups, using a

large-bore, fire-polished glass tube (6 mm to 9 mm 1.0. X 30 cm long) equipped with a rubber bulb.

5.2 If the larvae are to be moved to holding aquaria, a large-bore,

fire-polished glass tube should also be used to move them. It is important to

note that larvae and fry should not be handled with a dip net. Dipping larvae

and fry with a net can result in very high mortality. Some of the water in

the holding aquarium or tank containing the larvae should be siphoned off

before they are transferred using the large-bore tube. This should make them

easier to catch. The same large-bore, fire-polished glass tube discussed

above should be used to gently transfer the fish from the holding vessels to

the test vessels. As the fish are counted, they can be transferred to small

plastic cups before they are added to the test vessels. It is more convenient

to first transfer five fish to each of several small beakers or plastic

containers with a few mL of 20-30~ saline dilution water. The appropriate

number of fish (multiples of five) can then be added to the test vessels.

241
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fishes from the United States and Canada. Special Publication No. 12.

American Fisheries Society, Bethesda, MD.

ASTM. 1993. Standard practice for using brine shrimp nauplii as food for test

animals in aquatic toxicology. Designation: E 1203-87, Annual Book of ASTM

Standards, Vol. 11.04, American Society for Testing and Materials,

Philadelphia, PA.

Andreasen, J.K., and R.W. Spears. 1983. Toxicity of Texan petroleum well brine

to the sheepshead minnow (Cyprinodon variegatus) , a common estuarine fish.

Bull. Environ. Contam. Toxicol 30(3):277-283.

Battalora, M.S.J., R.D. Ellender, and B.J. Martin. 1985. Gnotobiotic

maintenance of sheepshead minnow larvae. Prog. Fish-Cult. 47(2):122-125.

Bower, C.E. 1983. The basic marine aquarium. Charles C. Thomas, Publ.,

Springfield, IL.

Carter, M.A., L.A. Burns, T.R. Cavinder, K.R. Dugger, P.L. Fore, D.B. Hicks,

H.L. Revells, and T.W. Schmidt. 1973. Ecosystems analysis of the Big

Cypress Swamp and estuaries. Surveillance and Analysis Division and South

Florida Ecological Study, Region IV, U.S. Environmental Protection Agency,

Atlanta, GA. EPA/904/9-74/002.

Clark, J.R., P.W. Borthwick, L.R. Goodman, J.M. Patrick, Jr., E.M. Lores, and

J.C. Moore. 1987. Effects of aerial thermal fog applications of Fenthion

on caged pink shrimp, mysids, and sheepshead minnows. Am. Mosquito Control

Assoc. 3:466-472.

Darnell, R.M. 1958. Food habits of fishes and larger invertebrates of Lake

Pontchartrain, Louisiana, an estuarine community. Publ. Inst. Mar. Sci.

Univ. Texas 5:354-416.

Drummond, R.A., and W.F. Dawson. 1970. An inexpensive method for simulating

diel patterns of lighting in the laboratory. Trans Amer. Fish. Soc.

99(2):434-435.

Foster, W.R. 1974. Cyprinodontidae - Killifishes. In: A.J. Lippson and R. L.

Moran. Manual for identification of early developmental stages of fishes

of the Potomac River Estuary. Power Plant Siting Program, Md. Dep. Nat.

Resour. PPSP-MP-13, pp. 127-142.

Grant, G.S. 1962. Predation of bluefish on young Atlantic menhaden in Indian

River, Delaware. Chesapeake Sci. 3(1): 45-47.

Gunter, G. lY40. ~tuales of marine fishes of Texas.

Univ. Texas 1(1):1-190.

242

Publ. Inst. Mar.
Hansen, D.J., P.R. Parrish, J.I. Lowe~ A. Wilson. 1971. Chronic toxicity,

uptake, and retention of Aroclor 1254 in two estuarine fishes. Bull.

Environ. Contam. Toxicol. 6:113-119.

Hansen, D.J., S.C. Schimmel, and J. Forester. 1974. Aroclor@ 1254 in eggs of

sheepshead minnows (Cyprinodon variegatus). Effect of fertilization

success and survival of embryos and fry. Proc. 27th Ann. Conf. Southeast.

Assoc. Game Fish Comm. Oct. 1973. Hot Springs, AR. pp. 420-426.

Hansen, D.J., and P.R. Parrish. 1977. Suitability of sheepshead minnows

(Cyprinodon variegatus) for life-cycle toxicity tests. Aquatic Toxicology and Hazard Evaluation. ASTM STP 634. F.L. Mayer and J.L. Hamelink, Eds.

American Society of Testing and Materials, Philadelphia, PA. pp. 117-126.

Hansen, D.J., S.C. Schimmel, and J. Forrester. 1977. Endrin: Effects on the

entire life-cycle of a salt water fish. J. Toxicol. Environ. Health

3:721-733.

Hardy, J.D. 1978. Development of fishes of the Mid-Atlantic Bight. An atlas of

egg, larval, and juvenile stages. Vol. II. Anguillidae through Syngnathidae. Fish and Wildlife Service, U.S. Dept. Interior,

FWS/OBS-78/12, pp. 141-151.

Holland, H.T., and D.L. Coppage. 1970.

generations of sheepshead minnows.

362-367.

Sensitivity to pesticides in three

Bull. Environ. Contam. Toxicol. 5(1):

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to determine relationships between time and toxicity of an acidic effluent

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Parrish, and A.C. Hendricks, eds., Aquatic toxicology and hazard

assessment, ASTM STP 707, American Society for Testing and Materials,

Philadelphia, PA. pp. 251-265.

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America. A description catalogue of the species of fishlike vertebrates

found in the waters of North America, north of the isthmus of Panama. U.S.

Natl. Museum Bull. 47 (in 4 parts). 3313 pp.

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Kuntz. A. 1916. Notes on the embry()logy and larval development of five

species of teleostean fishes. Sullo U.S. Bur. Fish. 34(831):409-429.

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R. Stautter, Jr. 1980. Atlas of North American freshwater fishes. Publ.

1980-12, North Carolina State Museum Natural History, Raleigh, NC.

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Martin, F.D. 1972. Factors influencing local distribution of

Cyprinodonvariegatus (Pisces: Cyprinodontidae). Trans. Am. Fish. Soc.

101(1):89-93.

Martin, B.J. 1980. Effects of petroleum compounds on estuarine fishes. Govt.

Reports Announcements & Index (GRA&I), Issue 11.

Nordlie, F.G. 1987. Plasma osmotic Na+ and Cl· regulation under euryhaline

conditions in Cyprinodon variegatus Lacepede. Compo Biochem. Phys;ol. A,

86A(1):57-61.

Perschbacker, P.W., and K. Strawn. 1986. Feeding selectivity and standing

stocks of Fundulus grandis in an artificial brackishwater pond, with

comments on Cyprinodon variegatus. Contrib. Mar. Sci. 29: 103-111.

Sekavec, G. B. 1974. Summer Foods, length-weight relationship, and condition

factor of juvenile ladyfish, flops saurus Linnaeus, from Louisiana Coastal

Streams. Trans. Amer. Fish. Soc. 3: 472-476.

Schimmel, S.C., and D.J. Hansen. 1974. Effects of Aroclor~1254 on the embryo

and fry of sheepshead minnows. Trans. Amer. Fish. Soc. 103(3):522-586.

Schimmel, S.C., P.R. Parrish, D.J. Hansen, J.M. Patrick, Jr., and J. Forester.

1975. Endrin: effects on several estuarine organisms. Proc. 28th Ann.

Conf. Southeast. Assoc. Game Fish. Comm., pp. 187-194.

Schimmel, S.C., and D.J. Hansen. 1975. Sheepshead minnow (Cyprinodon

variegatus): An estuarine fish suitable for chronic (entire life-cycle)

bioassays. Proc. 28 Ann. Conf. Southeast. Assoc. Game Fish Comm.

pp. 392-398.

Schimmel, S.C., D.J. Hansen, and J. Forester. 1974. Effects of Aroclor~ 1254

on the embryo and fry of sheepshead minnow. Trans. Amer. Fish. Soc.

103(3):522-586.

Seligmann, E.B. Jr.

aquatic notes.

1951. Cyprinodon variegatus reverindi (Poey) and other

Aquarium 20(9):234-236.

Sindermann, C.J. 1970. Principal diseases of marine fish and shellfish.

Academic Press, New York, NY.

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and life histories of Texas salt water cyprinodontes. Tulane Stud. Zool.

4:113-134.

Spotte, S. 1973. Marine aquarium keeping. John Wiley and Sons, New York, NY.

Spotte, S.H.

systems.

NY.

1979. Fish and invertebrate culture: water management in closed

Wiley-Interscience Publication, John Wiley and Sons, New York,

244
USEPA. 1973. Ecosystem analysis of the Big Cypress Swamp and estuaries. M.A.

Carter, L.A. Burns, T.R. Cavinder, K.R. Dugger, P.L. Fore, D.B.Hicks, H.L.

Revells, and T.W. Schmidt. Surveillance and Analysis Division, U.S.

Environmental Protection Agency, Athens, GA. EPA/904/9-74-002.

USEPA. 1978a. Laboratory culture of sheep shead minnow (Cyprinodon variegatus).

D.J. Hansen. In: Bioassay procedures for the ocean disposal permit

program. U.S. Environmental Protection Agency, Environmental Research

Laboratory, Gulf Breeze, FL. EPA-600/9-78-010, pp. 107-108.

USEPA. 1978b. Bioassay procedures for the ocean disposal permit program.

Environmental Research Laboratory, Gulf Breeze, FL. EPA-600/9-78-010.

USEPA. 1978c. Chronic toxicity of chlordane, Trifluralin, and

pentachlorophenol to sheepshead minnows (Cyprinodon variegatus). P.R.

Parrish, E.E. Dyar, J.M. Enos, and W.G. Wilson. Environmental Research

Laboratory, U.S. Environmental Protection Agency, Gulf Breeze, FL 32561.

EPA-600/3-78-010.

USEPA. 1978d. Life-cycle toxicity test using sheepshead minnows (Cyprinodon

variegatus). D.J. Hansen, P.R. Parish, S.C. Schimmel, and L.R. Goodman.

In: Bioassay procedures for the ocean disposal permit program,

Environmental Research Laboratory, U.S. Environmental Protection Agency,

Gulf Breeze, FL. EPA/600/9-78-010, pp. 109-117.

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eds. Methods for measuring the acute toxicity of effluents to freshwater

and marine organisms. Environmental Monitoring and Support Laboratory,

U.S. Environmental Protection Agency, Cincinnati, OH. EPA/600/4-85/013.

pp. 126-137.

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water larval fish growth-survival studies with the sheepshead minnow

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conduct and interpretation of complex effluent toxicity tests at

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Laboratory, U.S. Environmental Protection Agency, Narragansett, RI.

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with early life stages of four atherinid fishes: The inland silverside,

Menidia bery77ina, Atlantic silverside, M. menidia, tidewater silverside,

M. peninsu7ae, and California grunion, Leuresthes tenuis. D.P. Middaugh,

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Environmental Protection Agency, Washington, D.C. 20460. EPA/600/8-87-004.

245
USEPA. 1988. Short-term methods for estimating the chronic toxicity of

effluents and receiving water to marine and estuarine organisms. C.I.

Weber, W.B. Horning, II, D.J. Klemm, T.W. Neiheisel, P.A. Lewis, E.L.

Robinson, J. Menkedick, and F. Kessler. eds. Environmental Monitoring and

Support Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH

45268. EPA-600/4-87-028.

USEPA. 1990. Sheepshead minnow and inland silverside larval survival and

growth toxicity tests. Supplemental report for training videotape. Office of Research and Development, U. S. Environmental Protection Agency,

Washington, D.C. EPA/600/3-90/075.

Usher, R.R., and D.A. Bengtson. 1981. Survival and growth of sheepshead minnow

larvae and juveniles on diet of Artemia nauplii. Prog. Fish-Cult.

43(2):102-105.

Ward, G.S., and P.R. Parrish. 1980. Evaluation of early life-stage toxicity

tests with embryos and juveniles of sheepshead minnows (Cyprinodon

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Parrish, and A.C. Hendricks, Eds. American Society for Testing and

Materials, Philadelphia, PA. pp. 243-247.

246
APPENDIX A

DISTRIBUTION, LIFE CYCLE, TAXONOMY, AND CULTURE METHODS

A.B. SILVERSIDES: INLAND SILVERSIDE (HENIDIA BERYLLINA) , ATLANTIC SILVERSIDE, (H. HENIDIA), AND

TIDEWATER SIlVERSIDE (H. PENINSULAE)

1. MORPHOLOGY AND TAXONOMY

1.1 Adult Atlantic si1versides attain a total length of up to 117 mm (Figure

lA and IS). Females in general are slightly larger than males. The first

dorsal fin has three to seven, usually four or five spines. The second dorsal

fin has one spine and eight or nine rays; the anal fin has one spine and 19 to

29, usually 21 to 26, rays; and the pectoral fin has 12 to 16, usually 14 or

15, rays (Robbins, 1969). Atlantic silverside embryos are easily

distinguished from those of the closely related inland si1verside, Menidia

bery77ina. The former have a bundle of elastic filaments attached to the

chorion at one small area of insertion (Figure Ie and 10). These filaments,

typically longer than the diameter of the egg, are all the same diameter. In

contrast, inland silverside eggs posses one or two thick, elongated filaments,

up to 50 mm long and four to nine shorter, thinner filaments (Figure IE and

IF).

2. GENERAL LIFE HISTORY

2.1 DISTRIBUTION

2.1.1 Si1versides occur in estuaries along the Atlantic, Gulf, and Pacific

coasts (Figures 2-4). The Atlantic silverside, Menidia menidia, is a resident

of estuaries from Maine to northern Florida. It occurs at intermediate to

high salinities, typically of 12 to 30 parts per thousand (ppt), and remains

in Atlantic estuaries throughout most of the year (DeSylva et al., 1962;

Dahlberg, 1972). Recent evidence indicates an offshore migration at northern

latitudes in the fall and reappearance of adults in estuaries in late spring

(Conover and Kynard, 1984). This species is an important component in

estuarine ecosystems, serving as forage fish for commercially and

recreationally valued species such as striped bass, bluefish and spotted

seatrout (Merriman, 1941; Bayliff, 1950; Middaugh, 1981).

2.1.2 Although the culturing methods described in this section were written

primarily for Menidia menidia, they are also suitable for the inland

silverside, M. bery77ina, and the tidewater silverside, M. peninsu7ae (USEPA,

1987). The staff of the Environmental Research Laboratory, Gulf Breeze,

Florida, have developed procedures for spawning, culturing, and testing of

other fishes, including the California grunion, Leuresthes tenuis, and the

topsmelt, Atherinops affinis. The availability of these fishes as test

organisms will permit the use of indigenous fish in toxicity tests of

wastes discharged along the entire coast line of the contiguous United States

and Alaska.

247
A

B

c

Figure 1. Silverside (Menidia): A-D, M. menidia, (Atlantic silverside); A,

adult; ca. 95 mm SL (Massachusetts); B, adult, ca 102 mm SL

(Florida); C, unfertilized egg (diagrammatic); D. developing embryo

(note that filaments are all equal in diameter); E-F, M. bery77ina

(inland silverside); E, unfertilized egg (diagrammatic); F,

developing embryo (note one thick filament and several thin

fil aments). (A, B from Kendall, 1902; C from Wang, 1974; D from

Ryder, 1883; E from Wang, 1974; F from Hildebrand, 1922.)

248
Figure 2. Biographical Distribution: A, inland silverside, Menidia bery77ina; B, Atlantic silverside, M. menidia. (From USEPA, 1987).

249
-----.

_J

B

Figure 3. Biogeographical

peninsu7ae; B,

USEPA, 1987).

Distribution: A, tidewater silverside, Menidia

California grunion, Leuresthes tenuis. (From

250
Figure 4. Biogeographical Distribution: Topsmelt, Atherinops affinis.

(From USEPA, 1987).

2.2 SPAWNING BEHAVIOR

2.2.1 The Atlantic silverside spawns during spring and summer. Spawning runs

generally occur during April - June or July at northern latitudes, and March

through July or August at southern latitudes (Bayliff, 1950; Hildebrand and

Schroeder, 1928; Middaugh and Lempesis, 1976). Spawning occurs in the upper

intertidal zone during daytime high tides (Middaugh, 1981). Eggs are

deposited on a variety of substrates which provide protection from thermal

stress and desiccation (Middaugh et al., 1981; Conover and Kynard, 1981).

Females typically release 200 to 800 eggs, 1.0-1.2 mm diameter, as they spawn.

Individuals may spawn up to five or six times, at two week intervals, during

the reproductive season. The life span is generally 12-15 months, although

year class-2 fish are occasionally found (Beck, 1979).

3. CULTURING METHODS

3.1 SOURCES OF ORGANISMS

3.1.1 Menidia may be obtained from commercial biological supply houses or

collected in the field.

3.1.1.1 The optimal time for collecting ripe M. menidia in the field is just

prior to daytime high tides between 8:00 AM and noon (usually one to four days

after the occurrence of a new or full moon), when prespawning schools move

into the upper intertidal zone (Middaugh, 1981; Middaugh et al., 1981). Since

the Atlantic silverside prefers relatively high salinities, it is recommended

that collections be made in areas with salinities of 20~ or greater. Sandy

251
beaches, bordering open but protected estuarine bays, are suitable for

collecting adults. A 1 X 10-m bag seine with knotless 5-mm mesh is ideal for

collecting. Since Atlantic silversides typically reside in shallow water,

1.5 mdeep, they are easily captured by seining close to shore. It is

important to avoid total beaching of the bag seine when collecting M. menidia.

These fragile fish will quickly die if removed from water and, more

importantly, ripe females often abort their eggs if stranded. Ideally, the

bag portion of the seine, containing captured adults, should remain in water

5-15 cm deep (Middaugh and Lempesis, 1976).

3.1.1.2 It is possible to transport the spawn (fertilized eggs) or adults to

the laboratory. The following procedure is recommended for stripping, fertilizing and transporting eggs from the field to the laboratory:

1. Immediately after seining (while still on the beach) three to five ripe females should be dipped into a bucket of seawater to remove sand and

detritus.

2. Eggs are stripped into a glass culture dish containing seawater or onto

a nylon screen (0.45 to 1.0 mm mesh) (Figure 5), which is then gently lowered into a culture dish of seawater with the eggs on the upper surface of the screen (Barkman and Beck, 1976). If excessive pressure is required to strip the eggs, the female should be discarded. Mature

eggs, 1.0-1.2 mm in diameter, are clear, and have an amber hue.

3. Milt from several males can then be stripped into the culture dish and

mixed with the eggs by gently tilting the dish from side to side. Upon

contact with seawater, adhesive threads on mature eggs uncoil, making enumeration and separation difficult. If eggs are stripped directly

into the culture dish, one end of a nylon string may be dipped into the

dish and gently rolled so the embryos adhere (Middaugh and Lempesis,

1976). The Barkman and Beck (1976) technique for attaching the eggs to

nylon screening minimizes the natural clumping tendency due to

entanglement of the filaments on M. menidia eggs.

4. Strings of embryos or embryos on screens may be transported to the

laboratory by placing them in an insulated glass container filled with

seawater at the approximate temperature and salinity of fertilization.

If graVid fish are transported to the laboratory for subsequent spawning, care must be taken to avoid overcrowding of fish in transport containers. Continuous, vigorous aeration is required and any increase

in container water temperature should be minimized (Beck, 1979).

A mass culture system for incubating the screen-adhered eggs and

collecting the hatched larvae in a flOWing seawater system (Figure 5)

was described in detail by Beck (1979). A similar procedure utilizing a

recirculating system was described by Middaugh and Lempesis (1976).

3.2 Laboratory Year-round Spawning

3.2.1 Atlantic, inland, and tidewater silversides may be spawned in the

laboratory on a year-round basis. Procedures described by Middaugh and Takita

(1983), and Middaugh and Hemmer (1984), provide for maintenance of a brood

stock of 30 to 50 fish, sex ratio 1:1, in 1.3 mdiameter, circular holding

tanks which are part of a recirculating seawater system (Figure 6). The

photoperiod should be adjusted to 14 L:10 0 (lights on at 5:00 AM and off at

252
HAND STRIPPING EGGS HAND STRIPPING MALES

ONTO 500~ NYLON SCREEN

IMMERSED IN SEA WATER

'--..~~ __•• SPERM

SPERM SUSPENSION

IN SEAWATER

AERATION

FERTILIZED EGGS

ADHERED TO SCREEN

FILTERED

SEA WATER (FSW)

ADD SPERM SUSPENSION TO

TRAY CONTAINING EGGS

15 MINUTES

HATCHED LARVAE

~.'. .'.•~.=, :"'-~~ ..~.OISCHARGE 400, SCREENED

. .--... - . .. ~-.-.--

LARVAE HARVESTED

WITH NET WITH

PLASTIC FILM

BOnOM

LARVAE

STANDPIPE· SCREENED

LARVAL REARING TANK

FILLED WITH SEA WATER -

CONTINUOUS FLOW

'1.;'

'" I..i\~'::"-~"l' " ,.. 1 .11DAYSAT20·C '/''' ' ;', '-", If=jf:=J SCREEN~TH EGGS

EGG INCUBATION JAR

USE~ SCREENED OUTFLOW

Figure 5. Techniques for collection of silverside eggs in the field, and

production of larvae in the laboratory (From Beck, 1979).

253
7:00 PM, intensity 10-20 ~E/m2/s, or 50-100 ft c), with the water temperature

maintained at 18-20°C for fish from northern latitudes, and 20-25°C for

southern latitudes. Suitable salinities for the culture units would be 25-30

ppt for the Atlantic and tidewater silversides, and 7~ for the inland

silverside. Fish are fed 8 g TETRAMIN® each morning and afternoon, and

concentrated Artemia nauplii (hatch obtained from approximately 15 mL of eggs

after 48 h of incubation at 25°C) in mid-afternoon (see section on Artemia

culture). Excess food should be siphoned from the holding tanks weekly. Filter media (activated charcoal) located in a reservoir tray should be

changed weekly, immediately after cleaning the holding tanks. To induce

spawning by the Atlantic silverside, the circulation current velocity in the

holding tanks should be reduced to zero (from 8 to 0 cm/sec) twice daily by

turning off the seawater circulation pump from midnight to 1:00 AM, and from

noon to 1:00 PM. Atlantic silversides will spawn in response to interrupted

current velocities during daytime (noon to 1:00 PM). Spawning of the tidewater

silverside also is enhanced by reducing the current velocity twice daily, but

spawns primarily during nighttime. No interruption in current is necessary to

enhance spawning by the inland silverside.

3.2.2 A suitable spawning substrate can be made by cutting enough 25 cm

lengths of No. 18 nylon string to form a small bundle, and tying a string around the middle of the bundle to form a "mop." The mop is suspended just below the surface of the water, in contact with the side of the holding tanks.

Spawning fish will deposit eggs on this substrate. The mops are removed from

the holding tanks daily and suspended in incubation vessels. Typical egg production ranges from 300 to 1200 per spawn. Fish generally can be expected

to spawn three to four days each week.

3.2.3 It is essential that light-tight curtains surround the holding tanks.

These curtains should remain closed except during periodic feedings, tanks

cleaning, and during removal and replacement of spawning substrates.

3.2.4 Embryos attached to nylon screening or nylon string may be suspended in

a culture system such as shown in Figure 6. The culture chambers for embryos should be constructed of glass. Upon hatching, larvae may be transferred from

the collection container to a gO-cm diameter glass or fiberglass tank with a

volume of 350 L. Tanks receive a continuous flow of seawater at 2 L/min.

Water is introduced at the tank periphery causing a gentle current sufficient

to induce orientation to water movement and normal schooling behavior. Water

is discharged from the tank by two automatic siphons. Siphon openings are

protected by a 400 ~m nylon screen to prevent escape of larvae. An inverted

funnel is used at the siphon to decrease the velocity of discharge water, thus

preventing impingement of larvae.

3.2.5 Embryos can also be incubated in small (4-10 L) glass aquaria, by placing the nylon screening or strings just below the surface of the water.

Gentle aeration should be prOVided by an airstone positioned near the bottom

of the holdina aauaria. J •

254
I.

"

,.

"

"

"

"

".: I I

t. I' ~It

.~~ , , --...) :

, --_.-

"

,,

"

,.

Figure 6. Holding and spawning system utilized in the culture of silversides

(Menidia). A, 1.3 m diameter tanks; B, circulation pump; C,

reservoir; 0, seawater distribution system; E, by-pass line; F,

seawater return 1ine; and G, reservoir filter system. (From

Middaugh and Hemmer, 1984).

255
3.3 CULTURE MEDIA

3.3.1 Use natural seawater if it is available and unpolluted. Otherwise use

synthetic seawater prepared by adding artificial marine salts, such as FORTY

FATHOMS®, to deionized water. If synthetic seawater is used, it should be

aged for a least one week before being utilized in culture aquaria.

3.4 CULTURE CONDITIONS

3.4.1 The salinity maintained during incubation should be similar to that of

the water from which the adults were taken, if collected in the field, or at

which the adults are being maintained in the laboratory, if the embryos originate from laboratory brood stock. Water temperature should be maintained

at 20 to 25°C depending upon the latitude where fish are c9llected. Provide a

photoperiod of 12-14 h of illumination daily at 10-20 pE/m Is, or 50-100 ft-c

(12 h minimum light/24 h). Embryos will hatch in seven to 14 days, depending

upon the incubation temperature and salinity (Middaugh and Lempesis, 1976).

3.5 FEEDING AND STOCKING DENSITY

3.5.1 Upon hatching, Menidia larvae should be fed immediately. Newly hatched

brine shrimp (Artemia) nauplii (less than eight hours old) are fed to the

larvae twice daily. It is essential to feed M. menidia and M. peninsu7ae

larvae newly-hatched brine shrimp nauplii (USEPA, 1987). Utilization of

older, larger, brine shrimp nauplii will result in starvation of the larvae

since they are unable to ingest the larger food organisms. Three to four days after hatching, the fish are able to consume older (larger) brine shrimp nauplii. Because of their small size M. bery77ina larvae must be fed a

mixohaline rotifer, Branchionus p7icati7us from day of hatch through day five.

Thereafter, they are able to consume newly-hatched and older Artemia nauplii

(USEPA, 1987; USEPA 1988). Methods for culturing brine shrimp are discussed

in the Appendix. A stocking density of about 300 larvae is suitable in an

76-L aquarium.

3.6 CULTURE MAINTENANCE

3.6.1 To avoid excessive build up of algal growths, periodically scrape the

walls of aquaria. Activated charcoal in the aquarium filtration systems

should be changed weekly and detritus (dead brine shrimp nauplii or cysts) siphoned from the bottom of holding aquaria each week. Salinity may be

maintained at the proper level by addition of distilled or deionized water to

compensate for evaporation.

4. VIDEO TRAINING TAPE AVAILABLE FOR CULTURING METHODS

4.1 A video training tape and supplemental report (USEPA, 1990) on culturing

inland silversides are available from the National AudioVisual Center,

Customer Services Section, 8700 Edgeworth Drive, Capitol Heights, MD 20743-

3701 (Phone 301-763-1891), as part of a video package on short-term chronic

toxicity tests for marine organisms (Order No. A18545). The package includes

methods for sheepshead minnows, inland silversides, sea urchins, and Champia, and costs $85.00.

256
50 TEST ORGANISMS

5.1 Fish one to 14 days old are used in acute toxicity tests. Most of the

water in the holding aquarium should be siphoned off before removal of larvae.

Larvae can then be siphoned from the holding tanks into a holding vessel. It

is essential that larvae not be handled with a dip net, because it will result

in very high mortality. A large-bore, fire-polished glass tube, 6 mm 1.0. x

500 mm long (1/4 in. 10 X 18 in. long), equipped with a rubber squeeze bulb

should be used to transfer the larvae from the holding vessel to the test

vessels. It is more convenient to first transfer five fish to each of several

small beakers containing 20 mL of saline dilution water. The appropriate

number of fish (multiples of five) can then be added to test vessels.

257
SELECTED REFERENCES

Anderson, B.S., D.P. Middaugh, J.W. Hunt, and S.L. Turpen. 1991. Copper

toxicity to sperm, embryos, and larvae of topsmelt, Antherinops affinis,

with notes on induced spawning. Mar. Environ. Res. 31:17-35.

Anderson, W.O., J.K. Dias, R.K. Dias, D.M. Cupka, and N.A. Chamberlain. 1977.

The macrofauna of the surf zone off Folly Beach, SC. NOAA Tech. Rept. NMFS

SSRF-704. 23 pp.

ASTM. 1993. Standard practice for using brine shrimp nauplii as food for test

animals in aquatic toxicology. Designation: E 1203-87, Annual Book of ASTM

Standards, Vol. 11.04, American Society for Testing and Materials,

Philadelphia, PA.

Barkman, R.C., and A.D. Beck. 1976. Incubating eggs of the Atlantic

silverside on nylon screen. Prog. Fish-Cult. 38:148-150.

Bayliff, W.H. 1950. The life history of the silverside, Menidia menidia

(Linnaeus). Contr. Chesapeake Biol. Lab., Publ. 90:1-27.

Beck, A.D. 1979. Laboratory culture and feeding of the Atlantic silverside,

Menidia menidia. Conference on aquaculture and cultivation of fish fry and

its live food. Polish Hydrobiological Soc. Syzmbark, Poland, September,

1977. Spec. Publ. No.4, European Mar. Soc. pp. 63-85.

Bengtson, D.A. 1985. Laboratory experiments on mechanisms of competition and

resource partitioning between Menidia menidia (L.) and Menidia bery77ina

(Cope) (Osteichthyes: Atherinidae). J. Exp. Mar. Biol. Ecol. 92(1):1-18.

Bigelow, H.B., and W.C. Schroeder. 1953. Fishes of the Gulf of Maine.

U.S. Fish Wildl. Servo Fish. Bull. 53:1-577.

Briggs, P.T. 1975. Shore-zone fishes in the vicinity of Fire Island Inlet,

Great South Bay, New York. N.Y. Fish Game 22:1-12.

Chernoff, B., J.V. Conner, and C.F. Bryan. 1981. Systematics of the Menidia

bery77ina complex (Pisces:Atherinidae) from the Gulf of Mexico and its

tributaries. Copeia 2:319-335.

Chesmore, A.P., D.J. Brown, and R.D. Anderson. 1973. A study of the marine

resources of Essex Bay. Mass. Div. Mar. Fish. Monogr. Ser. No. 13. 38 pp.

Clark, F.N. 1925. The life history of Leuresthes tenuis, an atherine fish with

tide controlled spawning habits. Calif. Fish. Game Comm. Bull. 10:1-51.

Conover, D.O. 1979. Density, growth, production and fecundity of the Atlantic

silverside, Menidia menidia (Linnaeus), in a central New England estuary. M.S. Thesis, Univ. Massachusetts, Amherst, MA. 59 pp. .

Conover, D.O. 1984. Adaptative significance of temperature-dependent sex

determination in a fish. Am. Nat. 123(3):297-313.

258
Conover, D.O., and B.E. Kynard. 1981. Environmental sex determination:

Interaction of temperature and genotype in a fish. Science 213:577-579.

Conover, D.O., and B.E. Kynard. 1984. Field and laboratory observations of

spawning periodicity and behavior of a northern population of the Atlantic

si1verside, Menidia menidia (Pisces: Atherinidae). Environm. Bio1. Fish.

11(3}:161-171.

Conover, D.O., and S. Murawski. 1982. Offshore winter migration of the

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Dahlberg, M.D. 1972. An ecological study of Georgia coastal fishes. U.S. Fish

Wildl. Servo Fish. Bull. 70(2):323-353.

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ecological conditions in the shore zone of the Delaware River Estuary, with

notes on other species collected in deeper water. Univ. Delaware Mar.

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Elston, R., and B. Bachen. 1976. Die1 feeding cycle and some effects of light

on feeding intensity of the Mississippi si1verside, Menidia audens, in

Clear Lakes, California. Trans. Amer. Fish. Soc. 105:84-88.

Gettor, C.D. 1981. Ecology and survival of the key si1verside, Menidia

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University of Miami, Coral Gables, FL. 128 pp.

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A new early life-stage toxicity test using the California grunion,

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Safety 10:12-21.

Goodman, L.R., M.J. Hemmer, D.P. Middaugh, and J.C. Moore. 1992. Effects of

fenvalerate on the early life-stages of topsme1t (Atherinops affinis).

Environ. Toxico1. 11(3):409-414.

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Early life-stage toxicity test with tidewater si1versides (Menidia

peninsu7ae) and chlorine-produced oxidants. Environ. Toxicol. Chern.

2:337-342.

Gosline, W.A. 1948. Speciation in the fishes of the genus Menidia. Evo1.

2:306-313.

Hemmer, M.J., D.P. Middaugh, and V. Comparetta. 1992. Comparative acute

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259
Hildebrand, S.F., and W.C. Schroeder. 1928. Fishes of Chesapeake Bay. Bull.

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Johnson, M.S. 1975. Biochemical systematics of the atherinid genus Menidia.

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422.

Loosanoff, V.L. 1937. The spawning run of the Pacific surf smelt, Hypomesus

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Antherinidae). Estuaries 8(1):39-47.

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Environ. Toxicol. Chern. 11(3):393-399.

260
Middaugh, D.P., R.G. Domey, and G.I. Scott. 1984. Reproductive rhythmicity of

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261
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silverside in southern Illinois. North Amer. J. Fish. Manage. 8:127-131.

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262
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Wexler, M. 1983. The fish that spawns on land. Nat. Wildl. 21(3):33-36.

Wurtzbaugh, W., and H. Li. 1985. Diel migration of a zooplanktivorous fish

(Menidia bery77ina) in relation to the distribution of its prey in a large eutrophic lake. Limnol. Oceanogr. 30(3):565-576.

Yan, H-Y. 1984. Occurrence of spermatozoa and eggs in the gonad of a tidewater

silverside, Menidia bery77ina. Copeia 2:544-545.

263
APPENDIX B

SUPPLEMENTAL LIST OF ACUTE TOXICITY TEST SPECIES

TEST ORGANISM

LIFE

STAGE

FRESHWATER SPECIES: VERTEBRATES - WARMWATER

Cyprinella leedsi'

Lepomis macrochirus

Ictalurus punctatus

Bannerfin shiner

Bluegill sunfish

Channel catfish

25

20,25 II

1-14 days II II

II II

FRESHWATER SPECIES: INVERTEBRATES - COLDWATER

Pteronarcys spp. Stonefl ies* 12 1arvae

Pacifastacus

leniusculus Crayfi sh* II juveniles

Baetis spp. Mayfl ies* II nymphs

Ephemere77a spp. " II II

FRESHWATER SPECIES: INVERTEBRATES - WARMWATER

Hyale77a spp. Amphipods 20,25 juveniles Gammarus lacustris II II II

G. fasciatus II II II

G. pseudolimnaeus II II II

Hexagenia limbata Mayfl ies II nymphs

H. bilineata II II II

Chironomus spp. Midges II larvae

*Stoneflies, crayfish, and mayflies may have to be field collected

and acclimated for a period of time to ensure the health of the

organisms and that stress from collection is past. Species , identification must be verified.

Test conditions for Cyprinella leedsi are found in Table 13.

264
SUPPLEMENTAL LIST OF ACUTE TOXICITY TEST SPECIES (CONTINUED)

TEST ORGANISM

TEST

TEMP

( 0C)

SALINITY

(%0)

LIFE

STAGE

MARINE AND ESTUARINE SPECIES: VERTEBRATES - COLDWATER

Parophrys vetulus English sole 12 32-34 1-90 days

Ci thari chys

sitigmaeus Sanddab " " " " Pseudopleuronectes

americanus Winter flounder " " post metamorphosis

MARINE AND ESTUARINE SPECIES: VERTEBRATES - WARMWATER

Paralichthys

dentatus Fl ounder 20,25 32-34 1-90 days

P. lethostigma " " " " " Fundulus simillis Killifish " 20-32 1-30 days Fundulus

heteroc7itus Mummichog " 25-32 " " Lagodon rhomboides Pinfish " 20-32 1-90 days Orthipristis

chrysoptera Pigfish " 15-30 " " Leostomus xanthurus Spot " 10-30 " " Gasterosteus

acu7eatus Threespine

stickleback " 20-32 1-30 days Atherinops affinis Topsmelt 21 10-30 7-15 days

265
SUPPLEMENTAL LIST OF ACUTE TOXICITY TEST SPECIES (CONTINUED)

TEST ORGANISM

TEST

TEMP

( CC)

SALINITY

(%0)

LIFE

STAGE

MARINE AND ESTUARINE SPECIES: INVERTEBRATES - COLDWATER

Panda7us jordani Oceanic shrimp 12 25-32 juvenile

Strongy7ocentrotus

droebachiensis Green sea urchin II 32-34 gametes/embryo Strongy7ocentrotus

purpuratus Purple sea urchin II II 11 II

Dendraster

excentricus Sand doll ar II II II II

Cancer magister Dungeness crab II II juvenil e Ho7mesimy~is

Mysid II 11 costata 1-5 days

MARINE AND ESTUARINE SPECIES: INVERTEBRATES - WARMWATER

Ca77inectes sapidus Blue crab 20,25 10-30 juvenile Pa7aemonetes pugio, Grass shrimp II 10-32 1~10 days

P. vu7garis, 11 II 11 II 11 11

P. intermedius II 11 11 11 II II

Penaeus setiferus White shrimp II 20-32 post-larval Penaeus duorarum Pink shrimp II 11 11 II

Penaeus aztecus Brown shrimp II 11 II II

Crangon

septemspinosa Sand shrimp II 25-32 II II

Mysidopsis a7myra Mysid II 10-32 1-5 days Neomysis americana II II II II 11

Metamysidopsis

e70ngata II II II II 11

Crassostrea

virginica American oyster II 20-32 embryo Crassostrea gigas Pacific oyster II 25-32 11

Arbacia punctu7ata Purple sea urchin II 32-34 gametes/embryo

2Test conditions for Homesimysis costata are found in Table 15.

266
APPENDIX C

DILUTOR SYSTEMS

Two proportional dilutor systems are illustrated: the solenoid valve

system, and the vacuum siphon system.

1. Solenoid and Vacuum Siphon Dilutor Systems

The designs of the solenoid and vacuum siphon dilutor systems incorporate

features from devices developed by many other Federal and state programs, and

have been shown to be very versatile for on-site bioassays in mobile

laboratories, as well as in fixed (central) laboratories. The Solenoid Valve

system is fully controlled by solenoids (Figures 1, 2, and 3), and is

preferred over the vacuum siphon system. The Vacuum Siphon system (Figures 1,

4, and 5), however, is acceptable. The dilution water, effluent, and

pre-mixing chambers for both systems are illustrated in Figures 6, 7, and 8.

Both systems employ the same control panel (Figure 9).

If in the range-finding test, the LC50 of the effluent falls in the

concentration range, 6.25% to 100%, pre-mixing is not required. The

pre-mixing chamber is bypassed by running a TYGON@ tube directly from the

effluent in-flow pipe to chamber E-2 (see Figures 3 and 5), and Chambers E-l

and 0-1 and the pre-mixing chamber are deactivated.

The dilutor systems described here can also be used to conduct tests of

the toxicity of pure compounds by equipping the control panel with an

auxiliary power receptacle to operate a metering pump to deliver an aliquot of

the stock solution of the pure compound directly to the mixing chamber during

each cycle. In this case, chamber E-l is de-activated and chamber 0-1 is

calibrated to deliver a volume of 2000 mL, which is used to dilute the aliquot

to the highest concentration used in the toxicity test.

267
....,. ....,., 

'1

)

I

at'to'.....tt ['! \ ; I - \,_..... I

00 i, . ----'l.lI'JI., .,.

Figure 1. Photographs of the solenoid valve system (left), and the vacuum siphon sy
LIQUID LEVEL

SWITCH

19 mm (3/4 In.) HOLE

12 mm (1/2 In.) WASTE LINE

DILUTION WATER DILUTION WATER OVERFLOW

CHAMBERS CYCLE

COUNTER

LAPSE TIME EFFLUENT

CLOCK CHAMBER

~LPAN~_ PRE·MIXING CHAMBER

MAGENTIC N NORMALLY OPEN SOLENOIOVALVE

STIRRER EFFLUE T '" 7 mm (9/32 In.) 10

CHAMBERS "

u EFFWENT OVERFLOW

19 mm (3/4 In.) HOLE

12 mm (1/2 In.) LINE

EFFWENT

INFLOW

III

TEST CHAMBERS

OJ

1-20 LITERS CAPACITY

D

Figure 2. Solenoid valve dilutor system, general diagram (not to scale).

269
EFFLUENT CHAMBERS

DILUTION WATER

CHAMBERS

::"

..:.

D-1 D-2

"

i

l

EFFLUENT I

CHAMBER- .'

NORMALLY CLOSED ()o---- SOLENOID VALVE

7 mm (9132 In.) 10

I ~---- 6 mm 00 DEUVERY TUBE

NORMALLY CLOSED

SOLENOID VALVES

E1. 02· D6 -7 mm (9/32 In.) 10

01 • 9.5 mm (3(8 In.) 10

6 rnm 00 DEUVERY TUBE

NORMALLY OPEN SOLENOID VALVE

PRE-MIXING _ 7 mm ~/32 In.) 10

CHAMBER

MAGNETIC _ t4--+,Io--- 6 mm OD DEUVERY TUBE

STIRRER ;;;!===dT"lr~=--'E--3---;--

10mmOD /"

ADJUSTABLE

STANDPIPE DRAIN

!+- MIXING CHAMBER

1200 ml CAPACITY

1---- 10 mm 00 DEUVERY TUBE

:rEST CHAMBER

1-20 UTER CAPACITY

NOTE: WHEN 100% EFflUENT IS USED AS THE HIGHEST EFFLUENT CONCENTRATION,

E-1, 0-1, AND THE PRE·MIXING CHAMBER ARE BYPASSED BY CONNECTING A

TYGON TUBE TO THE EFFLUENT INFLOW, AND RUNNING rr DIRECTLY TO E-2.

IN THIS CASE. SOLENOIDS FOR E-' AND 0-1, AND THE PRE·MIXING CHAMBER ARE DISCONNECTED. 0-2 + E-3 • 50% EFFLUENT; D-3 + E-4· 25% EFFLUENT.

ETC.

Figure 3. Solenoid valve dilutor system, detailed diagram (not to scale).

270
SOLENOID SYSTEM EQUIPMENT LIST

1. Dilator Glass.

2, Stainless Steel Solenoid Valves

a. 3, normally open, two-way, 55 psi, water, 1/4" pipe size, 9/32"

orifice size, ASCO 8262152, for incoming effluent and dilution water

pipes and mixing chamber pipe. b. 1, normally closed, two-way, 15 psi, water, 3/8" pipe size, 3/8"

orifice size, ASCO 8030B65, for 0-1 chamber evacuation pipe.

c. 12, normally closed, two-way, 36 psi, water, 1/4" pipe size, 9/32"

orifice size. ASCO 8262C38, for remaining dilution chambers (02-06)

and effluent chamber (EI-E6) evacuation pipes. 3. Stainless steel tubing, seamless, austenitic, 304 grade for freshwater

and 316 grade for saline water.

a. 10 ft of 3/8" 00, 0.035" wall thickness, for dilution water and

effluent pipes.

b. 60 ft of 1/4" 00, 0.035" wall thickness, for dilution water and

effluent pipes.

c. 1 ft of 3/4 11 00, 0.035" wall thickness, for standpipe in 01 chamber.

4. Swagelok tube connectors, stainless steel.

a. 4, male tube connectors, male pipe size 1/4", tube 00 3/8".

b. 2, male tube connectors, male pipe size 1/2", tube 00 3/8".

c. 26, male tube connectors, male pipe size 1/4", tube 00 1/4".

d. 2, male tube connectors, male pipe size 3/8", tube 00 3/8".

e. 2, male adaptor, tube to pipe, male size 1/2", tube 00 3/8".

5. 7, 1200 mL stainless steel beakers.

6. Several lbs each of Neoprene stoppers, sizes 00, 0, and 1; 1 lb of

size 5.

7. 14 - aquarium (1-20 liters).

8. Magnetic stirrer.

9. 2 - PVC ball valves, 1/2" pipe size.

10. Dilutor control panel - see Fig. 32 and equipment list.

11. Plywood sheeting, exterior grade: one - 4' x 8' x 3/4", one - 4' x 8' x

1/2".

12. Pine or redwood board, 1" x 8", 20 ft.

13. Epoxy paint, 1 gal.

14. Assorted wood screws, nails, etc.

15. 25 ft - 14" 10, TEFLON® tubing, to connect the mixing chambers to the

test chambers.

271
L1aUID LEVEL SWITCH

NORMALLY CLOSED

SOLENOID VALVE

DILUTION WATER 9.5 mm (3/8 In.) 10

CHAMBERS

NORMALLY OPEN

SOLENOID VALVES

7 m 9/32 In.) 10

EFFLUENT CHAMBER

PRE-MIXING

CHAMBER

CONTROL

PANEL VACUUM LINE

MAGNETIC ./'

STIRRER EFFLUENT

CHAMBERS

EFFLUENT

INFL0F-W,:",:-_..J.U.....L..,

CYCLE _-+-I~

COUNTER

LAPSE

TIME CLOCK

FLOW CONTROL VALVES

DILUTiON

INFLOW

MIXING CHAMBERS

I I I I I I I I

TEST CHAMBERS 1·20 LITERS CAPACITY

Figure 4. Vacuum siphon dilutor system, general diagram (not to scale).

272
VACUUM SIPHON SYSTEM EQUIPMENT LIST

1. Dilutor Glass.

2. Stainless steel solenoid valves.

a. 2, normally open, two-way, 55 psi, water, 1/4" pipe size, 9/32"

orifice size, ASCO 8262152, for incoming effluent and dilution water

pipes. b. 2, normally closed, two-way, 15 psi, water, 3/8" pipe size, 3/8"

orifice size, ASCO 8030B65, for dilution water chamber 0-6 and

effluent chamber E-2.

3. Stainless steel tubing, seamless, austenitic, 304 grade for freshwater

and 316 grade for saline water.

a. 60 ft of 3/8" 00, 0.035" wall thickness, for dilution water and

effluent pipes. b. 20 ft of 5/16" 00, 0.035" wall thickness, for standpipes in mixing

chambers.

c. 1 ft of 3/4" 00, 0.035" wall thickness, for standpipe in 01 chamber.

4. Swagelok tube connectors, stainless steel.

a. 4, male tube connectors, male pipe size 1/4", tube 00 3/8".

b. 2, male tube connectors, male pipe size 3/8", tube 00 3/8".

c. 2, male adaptor, tube to pipe, male pipe size 1/2", tube 00 3/8".

d. 2, male tube connectors, male pipe size 1/2", tube 00 3/8".

5. 7, 1,200 mL stainless steel beakers.

6. Several lbs each of Neoprene stoppers, sizes 00, 0, and 1; 1 lb of

size 5.

7. 14 - aquarium (1-20 liters).

8. Magnetic stirrer.

9. 2, PVC Ball valves, 1/2" pipe size.

10. Dilutor control panel equipment - see Fig. 32 and equipment list.

11. 7, 120 ml NALGENE® bottles.

12. 3 ft, l-in-2 aluminum bar, for siphon support brackets.

13. Stainless steel set screws, box of 50, for securing S5 tubing in siphon

support brackets.

14. Stainless steel hose clamps, box of 10, size #4 or 5, (need 3 boxes).

15. 6, NALGENE® T's, 5/16" 00.

16. 12, TYGON®Y connectors, 3/8" 1.0.

17. TYGON® tubing, 3/8" 00, 10 ft.

18. Plywood sheeting, exterior grade: one - 4' x 8' x 3/4", one - 4' x 8' X

1/2".

19. Pine or redwood board, 1" x 8", 20 ft.

20. Epoxy paint, 1 gal.

21. Assorted wood screws, nails, etc.

22. 25 ft of 5/16" 10, TEFLON® tubing, to connect the mixing chambers to the

test chambers.

273
E·1 0·1 0-2

EFFWENT CHAMBERS

10 mm 0.0. U SHAPE SYPHON TUBE

......---- 10 mm 0.0. OEUVERY TUBE

\ J"Ii(----- 10 mm 1.0. CONNECTING TUBE Y FORM

....--- 10 mm 0.0. OEUVERY TUBE

_H----- 10 mm 0.0. AUTOMATIC SYPHON TUBE

~~~:;;~~~~~~5;:::= DILUTION WATER CHAMBERS

:; II -- VACUUM UNE

: : 6 mm 0.0. CONNECTING TUBE T FORM

";! 10 mm 0.0. U SHAPE SYPHON TUBE

"I : 6 mm 0.0. VACUUM UNE TUBE

I, STAINLESS STEEL HOSE CLAMP

,...---- 10 mm 1.0. CONNECTING TUBES Y FORM

m~---- 10 mm 0.0. OEUVERY TUBE

120 ml BOrnE VACUUM BLOCK

~---++---- 10 mm 0.0. OEUVERY TUBE

NORMALLY CLOSED

SOLENOID VALVE - 9.5 mm (318 in.) 10

PRE·MIXING

CHAMBER

EFFWENT

CHAMBER

~--- MIXING CHAMBER 1200 ml CAPACITY

1+---- 10 mm 0.0. OEUVERY TUBE

TEST CHAMBERS CAPACITY 1·20 UTERS

Figure 5. Vacuum siphon dilutor system, detailed diagram (not to scale).

274
EFFLUENT CHAMBER

F1

DILUTION WATER CHAMBERS

F2

OVERFLOW

19 mm (3/4 In.) DIAMETER HOLE

12 mm (1/2 In.) WASTE LINE

19 mm (3/4 In.) HOLE

12 mm (1/2 in.) LINE

(FOR VACUUM SIPHON SYSTEM ONLY)

C1

1__'01 _1

fB1·33_74~1

C2

447 nun ·1

0 0 0 0

o If

Figure 6.

e~~IJr~~~~:'~~~~~DSl~~~~H3,ID DIWN HOU; IS REOUIAED OIlLVFOR ClW,IBER El.

INDMDUAL PART SIZE AND NUr.lBER OF PIECES USING 'nvn (11410.) PlATE GlASS. NOTE: 1.8 nvn.&r' ...).... 304 GAADE (FOR FRESH

::J'i~ ~:nW= glASS. SALINE WATER) STAINUSS STEEL

INSIDE CELL r.lEAlIUREMENTS AND APPROlOlIATE '>'OlUIlE8

WIllII! WiGllI IlEIGI1I l/llI.UIIE

El: I5mm.ll5 ... • 281 .... ·2016 IlL

D1: 125 mm .. 95 mM X 200 INn - 237e: mL 02: 40 mm l iii rmI Jl 200 ma1 • 7eO nL o:t: &ommx t6rm1 x 2((Imrn- _ nt. D4: 8(Jmmx85mm x200mm-'1040 rN.. De: 80mmx 95mm x 2OOrmn- 11«)

Da: 70 mrn x t6mm x 200mm - 1330 mL

Effluent and dilution water chambers (not to scale).

275
A

E2

B

E3 E4' E5 E6

EFFLUENT OVERFLOW

19 mm (3/4 in.) HuLE

12 mm (1/2 in.) LINE

"

c

BOTTOM PLATE (C)

1....-------387 mm ~I

o o o o

T I.....----.. 61 -----..~ 152 --.. 203 -"237 -.270..

92t 0

DRAIN HOLES IN BOTTOM PLATE (C) SHOWN FOR SOLENOID VALVE

DILUTOR SYSTEM ONLY. FOR VACUUM SIPHON DILUTOR SYSTEM, A

DRAIN HOLE IS REQUIRED ONLY FOR CHAMBER E2.

INDIVIDUAL PART SIZE AND NUMBER OF PIECES USING A 6 mm (1/4 in.)

PLATE GLASS ARE SHOWN BELOW. NOTE: 1/16 in. No. 304 (FOR FRESH

WATER) OR No. 316 STAINLESS STEEL (FOR SALINE WATER) MAY BE

SUBSTITUTED FOR GLASS.

LENGTH WIDTH t-KL PIECES (9)

A 180 mm x 80 mm .. 2 (END PLATES)

B 155 mm x 80 mm .. 4 (PARTITIONS)

C 296 mm x 92 mm = 1 (BOTTOM PLATE)

D 296 mm x 180 mm .. 2 (FRONT AND BACK PLATES)

INSIDE CHAMBER MEASURMENTS AND APPROXIMATE VOLUMES.

WIDTH LElliHH HEIGJ:H: YOLUME

E2: 110 mm x 80 mm x 155 mm = 1364 mL

E3: 60 mm x 80mm x 155 mm .. 744 mL

E4: 30 mm x 80 mm x 155 mm = 372 mL

E5: 30 mm x 80 mm x 155 mm 372 mL

t:a. "J!n rnl"'W\ v an n""tn"ll L....,. vv 111111 " ,""v 111111 X 155 mm = 372 mL

Figure 7. Effluent chambers (not to scale).

276
M-2

I'"

~M-4~

M-3

240mm

SIDE VIEW

END VIEW

-1

125mm

M -1

19 mm (3/4 in.) HOLE

O~--+-- 12 mm (1/2 In.) LINE

l4+--15mm

'-------'----'

I... 165mm ~I

INDIVIDUAL PART SIZE AND NUMBER OF PIECES USING

6 mm (1{4 in.) PLATE GLASS. APPROXIMATE CAPACITY

4360mL.

M-1

M-2

M-3

M-4

125 mm x 153 mm

125 mm x 153 mm

240 mm x 165 mm

240 mm x 125 mm

• 1 (END PLATE, WITH HOLE)

- 1 (END PLATE)

- 1 (BOTTOM PLATE)

• 2 (SIDE PLATES)

Figure 8. Pre-mixing chamber (not to scale).

277
TOR-I 

II ...

AI

5

DIET

5t-+----..:.Ii..---

I

• •

-'"

:3

v,.C

60tU FI S, ! r:- '

0- f

." V',C

"'EUT

OND

TDR-2

-----, 

2

Illl VAC NEUT >

" iY

7

115 VAC HOT 1 S2

NOTE: I.'" DEHons CHASSIS GROUND 

PI JI

I

BLUE 2)2 I I

il S.

I

SJI

N

.......

co

Figure 9. Dilutor control panel wiring diagram.
Designation

DILUTOR CONTROL PANEL EQUIPMENT LIST*

CKT Description Manufacturer

A1

CTR-1

ET

L.S.

P1

S1

S2

SJ 1

Encapsulated amplifier

Cycle counter

Elapsed time indicator

Input power fuse

Receptacle

Aux A.C. output jack

Main input power cord

Fill indicator light

Emptying indicator light

Liquid level sensor

(Dual Sensing Probe)

Plug

On-off main power switch (spst)

On-off aux power switch (spst)

Solenoid

Cutler Hammer 13535H98C

Redington #P2-1006

Con rae #636W-AA H&T

Little fuse 342038

Amphenol 91PC4F

Stand. 3-prong AC Rcpt.

Stand. 3-prong AC male

plug

Dialco 95-0408-09-141

Dialco 95-0408-09-141

Cutler Hammer 13653H2

Amphenol 91MC4M

Cutler Hammer 7580 K7

Cutler Hammer 7580 K7

(See Solenoid and Vacuum

System equipment lists)

TDR-1

TDR-2

II

II

Additional Solenoids for

Solenoid Valve System

Time delay relay

Aux time delay relay

II

II

II

Dayton 5x829

Dayton 5x829

II II

fI II

II II

*Consult local electric supply house.

279
APPENDIX D

PLANS FOR MOBILE TOXICITY TEST LABORATORY

D.1. TANDEM-AXLE TRAILER

AIR CONDITIONER

REGION IV

ATHE;IIS GEORGIA

EXTERNAL SIDE VIEW

WINDOW

1....·~ lB.S· ~·~1

CARINETS

7.5'

DOOR

/

WALL CABINETS

TOP ViEW

DILUTERS

HOLDING TANKS

OVER DRAWERS

Figure 1. Mobile bioassay laboratory, tandem axle trailer. Above - external

side view; below - internal view from above.

280
o

REAR INSIDE

SWlTCH"S

OUTSIDE SPOTLIGHT

CEILING LIGHTS

WEATHERPROOF SPOTLIGHT

o

o

o

LICENSE PLATE REAR OUTSIDE

~AIR CONDITIONER,-

r-;:::====:=::======::::;::2~:-l -~-------- 0/ 'xo

EJ D ~DRAWERS+

FRONT INSIDE

o

FRONT OUTSIDE

Figure 2. Mobile bioassay laboratory, tandem-axle trailer, external and internal

end views.

281
LEFT SIDE

CABINETS WITH

SLIDING DOORS

RIGHT SIDE

6" OPENING

WITH

SPRING COVER

ON OUTSIDE

ELECTRIC OUTLETS

DILUTER BOARD

STAINLESS STEEL TROUGH

WHEEL WELL

DUAL WHEEL WELL

CABINETS 18" X 12"

o

AIR CONDITIONER/"

FRONT!/

STAINLESS STEEL TROUGH

-"""--24"

SLIDING DOORS

I I I I I I

aw"" ou"m~H.S,u., UN"," "N' oFRONT \ l rri;::5T LIGHTS

2 DRAWERS - 18" X 18"

- I I I I I I

I - I - I - II

3 DRAWERS

24" X 16"

DUAL WHEEL WELL 36" HIGH CABINETS

SLIDING DOORS

11-.....----------18·-----------....;·~1

Figure 3. Mobile bioassay laboratory, tandem-axle trailer, internal views of

side wall s.

282
APPENDIX D

PLANS FOR MOBILE TOXICITY TEST LABORATORY

0.2. FIFTH WHEEL TRAILER

UPPER CABINETS SINK

DILUTOR SY STEM COUNTER TOP .. DRAWERSII' WATER TANK/

....- >-

I AIR CONDITIONER

'"-

DILUTOR SYSTEM

DOUBLE SHELVES

FOR STATIC TESTS

32" DOOR lPLAN A

CABINET

DOOR

UPPER CABINETS

COUNTER TOP

DRAWERS

DOUBLE SHELVES

FOR STATIC T£Sl

SINK

WATER TANK

UPPER CABINETS

COUN1ER lOP

AIR CONDITIONER

DILUTER SYSTEMS

PLAN B

1-"~;"------_-__--125 FEET .,........... ~-5.5FEET~

Figure 4. Mobile bioassay trailer, fifth-wheel trailer, internal view from

above.

283
APPENDIX E

CHECK LISTS AND INFORMATION SHEETS

E.l. TOXICITY TEST FIELD EQUIPMENT LIST

__Shovels

__Spare tires (trailer, generator)

__Brine shri~~ eggs __Broom

__Brushes (wash)

__Buckets

__Camera

__Chlorine kit (w/chern)

__Cleanser

__Clip board (lg, sm)

__Cork borer set

____Culture dishes (200 mL, Daphnia)

__Daphni a food

__Data sheets: __Bioassay (static)

__Bioassay (flow-thru)

__Dilutor volume delivery

__Calibrator delivery sheet

__Daily events log

nalgene

metal - diesel fuel

(2) Homel i te

Hoses &couplings

blocks

__500 gal

__ 55 gal

__ 22 gal

5 gal

__Boards

__Cinder

__Drl.ll1s:

__Gas can

__Jacks

__Jumper cables

__Oil

__P~s:

__Acetone

__Aerators (battery operated)

__Air line: Clamps

___Aerators (battery operated)

___Air line: __Clamps

__Stones

__Tubing

__Valves

__Dish pan

__Dish rack

__Dissolved oxygen:

__KCL membrane solution

__Membranes

__Meter (YSI)

__Probes

__Reagent: __MnS04

__Alkaline azide

__H2S04

__0.0375 Na thiosulfate

__Extension cords

__Fire extinguisher

First aid kit

__Fish nets, (lg, sm)

__Flash light

__Generator: __Oil

Filter - fuel __Funnel

__Grease gun (wheels)

__Credit card

__Lock/key

__Siphon hose

__Alcohol

__All.ll1inl.ll1 foi l

__Alkalinity analysis (0.02 N H2S04 )

__Boots: __safety

__wading

__Batteries __D cell

__Beakers: __150 mL nalgene

__200 mL glass (3 boxes)

__Bottles: __D.O.

__wash

__Sample

__VOA vials

__500 mL plastic

__Glass organic

__Qt. w/teflon liner

__Starch

__Distilled H20

__Emergency road kit

__Enamel pans (lg, sm)

__Erlenmeyer flasks: 500 mL (2)

__1000 mL

__2000 mL

284
E.l. TOXICITY TEST FIELD EQUIPMENT LIST (CONTINUED)

____Mop

__Jars: 750 mL (4 boxes)

____3 gal (glass) (1)

____5 gal (glass) (1)

____Sample jugs (2)

__Kimwipes (lg, sm)

____Lab coats (2)

__Level

____Light 110 V

__Log book

____Magnetic stirrers: __Lighted

__Other

____Paper towels

____Parachute cord

____Parafi lm

____Pencils, pens

____pH: __Meters, Orion

____Meters, corning

__Buffers, 4,7,10

____Probes (extras)

____Pipets: __Bulbs

__Eyedroppers

____Volumetric (1 mL, 5 mL, 10 mL)

Plastic bags

____Quality assurance - SPCP

____Rain gear

____Reconstituted hard water

____Refractometer

____Respirators (cartridges)

__Super ice

__Tablets (paper)

____Tape: __Cellophane

__Color coded

____Electri ci an

__Masking

__Nylon

____Thermometers: Dial

__Glass

__Tools (lock/key)

__Tygon tubing, 1/8", 1/4", 3/8"

__Volumetric flasks (1000 mL, 2000 mL) __WD40

____Weigh boats

__Wire tags

____Rubber bands

____Ruler

____Safety glasses

____Safety manual

__Sample labels

__Scissors

__Screen bioassay cups

__Sea salts

____Separatory funnels &racks

____Silent giants

__Silicon sealant

____Solenoids (spare)

__Stainless steel tubing pieces

__Standard Methods Hand Book

____Stirring bars

____Stoppers (assorted)

____Submersible pumps: lg, sm.

__screens

2000 mL

____Buffer

____EDTA

indicator

__Aquarium

__Space

__Clamps

____Connectors

Ice chests

____Hose:

____Glass cutter

____Gloves (plastic)

____Graduated cylinders:

25 mL, 50 mL, 100 mL

250 mL, 500 mL, 1000 mL,

____Ground wire &rod

____Hand soap

____Hard hats

____Hardness analysis:

____HCl (20%)

____Heaters:

285
APPENDIX E

CHECK LISTS AND INFORMATION SHEETS

E.2. INFORMATION CHECK LIST FOR ON-SITE INDUSTRIAL

OR MUNICIPAL WASTE TOXICITY TEST

1. PRE-TRIP INFORMATION

Faci 1i ty Name: _

Address:

Phone number:

Plant Representative(s):

Names, Titles, Addresses of Company Personnel:

A. To Receive Correspondence:

B. To Receive Carbons:

Date of Notification Letter:

State Making Notification and Arrangements: __

Special Plant Safety/Security Requirements for EPA Personnel to Observe:

Local Accommodation Recommendations:

Directions to Plant:

Availability of Power Hookups (three 20-amp, 110-V Circuits):

286
Distance from Power Source to Trailer:

Trailer Location:

Possible Source of Dilution Water:

Major Products:

Raw Materi a1s :

(Feet)

Name of Receiving Water: __

Schedule of Plant Operation (continuous, weekdays only, etc.):

Treatment Steps:

Treatment Level (BPT, BAT, etc.): __

Wastewater Retention Time by Lagoon or Treatment Step:

Lagoon Designation

Retention

Hours

Time

Days

______ Calculated;

Total Wastewater Retention Time:

Retention Time Determination:

_____ Hours; _____ Days

____ Actual

Calculation method:

287
Description of Wastewater Tap Point:

Description of Outfall (surface, submerged diffuser, etc.):

Description of Other Waste Disposal Alternatives in Use (spray irrigation,

deepwell, municipal discharge, etc.):

2. ON-SITE INFORMATION

Wastewater General Characteristics:

Color:

Odor:

Solids:

Other:

Serial Number(s) of Discharge(s) to be Tested:

Description of Receiving Water: Uniflow; Tidal; ____ Approximate amplitude, feet

Color:

Odor:

Solids:

Salinity: High tide _

Other:

7QI0:

288

Low tide _

Ave. flow _
Description of Receiving Water Zone of Dilution:

Location and Description of Water Sampling Point(s):

Fresh:

Salt:

Dilution Waste General Characteristics:

Color:

Odor:

Solids:

Other:

Description of Toxicity Test Anomalies (plant production changes, power failure, rain events, etc.):

Duration

Time &Date Time &Date

Description of Plant maintenance:

Anomaly

Attach: DIAGRAM OF WASTEWATER TREATMENT FACILITIES.

289
3. FOLLOW-UP INFORMATION

Date of follow-up letter:

Wastewater Flow (data supplied by discharger):

Week Prior to Testing Week of Testing

Date Discharge (MGD) Date Discharge (MGD)

Average Discharge (MGD):

Organisms Tested On-site or In-Lab:

Species

Flow-thru

test

duration

(h)

Static

test

duration

(h)

Test

Location Dates Results

Possible Recommended Action as a Result of These Findings:

290
APPENDIX E

CHECK LISTS AND INFORMATION SHEETS

E.3. DAILY EVENTS LOG

Page of Pages

Day # of Study

Day # of Flow-through Test

Notes:

Date: _

Site: _

Initials: _

Time:

291
APPENDIX E

CHECK LISTS AND INFORMATION SHEETS

E.4. DILUTOR CALIBRATION FORM

Calibration Site:

Dilutor Number: _ Calibrator:

Date:

Effl uent

Concentration (%) 100.0 50.0 25.0 12.5 6.25 3.12 1. 56

Dilution Water (mL) 0 500 750 876 938 969 984

Trial 1

2

3

Average

Effluent (mL) 1000 500 250 125 62 31 16

Tri al 1

2

3

Average

Mixing Chamber (%):

Wastewater (mL):

Dilution Water (mL):

Dilution Effl uent

Water

Vol (mL)

Tri al 1

2

3

Average

Remarks:

292
Volume Delivery to

Metering Boxes

Each 10 Seconds (mL) Measured Control Box

Observation Cycle Time

Number Date Time Dilution Water Wastewater (min.) Hour Cycles Comments

Ave 4·day turnover =

Final Min/turnover

o

C

ff>

8

;;j

;:l

H

Z

rl

.....

Z

o

o

..,

:l

n

'"

-0

-0

W

I

--J

'"'

o

I

o

C'

,

w

Q

w

o

N

N

1.0

W

USEPA

Athens, GA

DILUTOR VOLUME DELIVERY INFORMATION

Industry:

Date:

Test start'Date:

STANDARD DILUTOR FLOW RATES

Flow rate (mL110 secondsl

Cycle Time (minI Dilution Water Wastewater Turnover Time (min)

2.5 532 246 43

5.0 199 92 85

7.5 123 57 128

10.0 89 41 170

12.5 69 32 213

15.0' 67 22 300

• This turnover rate provides approximately five 90% turnovers per day.

Time:

_