United States Office of Research and EPAl600/R-95/136

Environmental Protection Development August 1995

Agency Washington DC 20460 .

,.

I&EPA Short-Term Metho~ds·for

Estimating the Chlronic

To'xicity of Effluel1lt~ and

Receiving Waters. to West

Coast Marine and Estuarine

Organisms

EPA/600jR-95-136

Augu.st 1995

SHORT-TERM METHODS FOR ESTIMATING THE CHRONIC, TOXICITY OF

EFFLUENTS AND RECEIVING WATERS TO WEST COAST MARI:t-lE AND ESTUARINE

ORGANISMS

(First Edition)

Edited'by

Gary A. Chapman1 / Debra L . Denton2/

, ,and James M. Lazorchak3,

lNational Health and Ecological Effects Research

Laboratory/ Newport/ Oregon

2EPA Region IX/ San Francisco/ California.

3National Exposure Research Laboratory/ Cincinnati/ Ohio

NATIONAL EXPOSURE RESEARCH LABORATORY - CINCINNl:\.TI

OFFICE OF RESEARCH AND DEVELOPMENT

u.B. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI/ OHIO 45268

@I Printedon RecycledPaper
DISCLAIMER

This document has been reviewed by the National Exposure

Research Laboratory-Cincinnati (NERL-Cincinnati), U. S".

Environmental Protectiol1 Agency (USEPA) : and approved for·"

publication. The mention of trade names or commercial prQducts

does not constitute endorsement or recommendation for'use.' The

results of data analyses by computer programs described in the

section on data analys:i-s were verifiedusing.data commonly

obtained from effluent toxicity tests. However, these com;puter

programs may not be applicable to all data, (lnd the USEPAassumes

no responsibility for their use.

ii
FOREWORD

Environmental measurements are required t~o determine the

quality ~f ambient waters and the character of: waste effluent.

The National Exposure Research Laboratory-Cincinnati

(NERL-Cincinnati) conducts research to:

• Develop and evaluate analytical methods ,to identify and

measure the concentration of chemical pollutants in'

drinking waters, surface waters, groundwaters,

wastewaters, sediments, sludges, and solid wastes.

• Investigate methods for the identification and

measurement of viruses, bacteria and other

microbiological organisms in aqueous samples and to

determine the responses of aquatic organisms .to water

quality.

• Develop and operate a quality assurance program to

support the achievement of data quality objectives in

measurements of pollutants in drinking water, surface

water, groundwater, wastewater, sedilnent and solid

waste.

• Develqp methods and models to detect and quantify

respons~s in aquatic and terrestrial organisms exposed

to environmental stressors and to correlate the

exposure with effects on chemiqal 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. Thus, the detection of chronically

toxic effluents plays an important role .:in identifying and

controlling toxic discharges to surface waters. This manual is

the first edition of the west coast marine and estuarine chronic

toxicity test manual for effluents. It provides standardized

methods for estimating the chronic toxicity of effluents and

receiving waters to estuarine and marine organisms for use by the

USEPA ,regional programs, the state programs, and t:he National

Pollutant Discharge Elimination System (NPDES) permittees.

iii
PREFACE

This manual contains whole effluent toxicity (WET) test methods

considered by USEPA's Office of Research and Development (ORD) to.

have the necessary characteristics for use in the NPDES program

and other USEPA monitoring activities, in Pacific coastal waters,

for estimating the chronic toxicity of effluents and receiving

waters. All the species included in this report are curren~ly

specified in NPDES permits in one or more of th~ west coas.t

states. The methods will likely be revised to some extent.,

especially if they are proposed in the Federal Register as 304(h)

methods. Revisions would be made based upon comments re'ceived as

a result of the proposed rule public comment period.

,

With one exception, other than changes necessary to identify the

test species used in these methods and corrections of an

editorial nature, the first ten sections of this document are

identical to the first ten sections of the "Short-term Methods

for Estimating the Chronic Toxicity of Effluents and Rece~ving

Waters to Estuarine and Marine Organisms, (Second Edition):."

The exception occurs in chapter 7 where ~he use of synthetic

(standard) dilution water for NPDES permit-related toxicity

testing is not required. Validation and precision tests with

natural seawater and HSB prepared from natural seawater (plus

reagent water as necessary) have been acceptable, and synthetic

waters have shown mixed results in limited testing.

The marine toxicity test procedures in .this manual have been

developed or refined by EPA and the states of California and

Washington over a period of years. A significant number of

organizations and individuals have contributed to this ,effort. A

list of contributors is ,provided in the acknowledgements l?ection.

Among the major efforts that contributed critical data and

critical analysis of the methods in this manual the following

were vital:

1) The California Marine Bioassay Project (MBP). In 1984, the

California State Water Resources Control Board initiated the MBP

to develop sensitive methods for testing t~e toxicity of ;

discharges to California marine waters. The MBP was funded

wholly or in part by the USEPA using Section 205(j) grant funds.

The MBP developed the tests.with abalone (Haliotis rufescens),

iv
topsmelt (Atherinops affinis) , giant kelp (Macrocystis pyrifera) ,

and mysid (Holmesimysis costata) .

2} The EPA West Coast Marine Complex Effluent Program. Started

in 1985, this program provided preliminary 'work for the topsmelt

(Atherinops affi'nis) , revision of methods'for echinoid sperm with

the purple sea urchin (Strongyloceritrotus purpuratus) and the

sand dollar' (Dendraster excentricus) , preparation of all methods

into a standardized format, coordination of' efforts among the

various states and EPA regions 9 and la, -and development of yet'

unadopted test methods with the mysid (Mysidopsj.,s intii) ,and the

kelp (Laminaria saccharina) .

3} The Protocol Review:Committee'(PRC} for the'Triennial Review

of the Marine Toxicity Test Protocols for the California Ocean

Plan. In 1994 this committee reviewed a' number ;of proposed test

methods for inclusion,in the California Ocean Plan. The methods

included in this report are those recommended by: the Protocol

Review Committee. The Mysidopsis intii method developed by EPA

was excluded 'from the recommended procedu'resbecause it was

considered redundant' with the Holmesimysis cos'tata procedure. It

was excluded from this report because its inclusion was also

considered unneccesary by EPA region 10. 'The Laminaria

saccharina test was excluded from the California recommendations

because it was considered redundant wlththe Macrocystis pyrifera

test. It was excluded from this report because the results from

the West Coast Marine Species Chronic Protocol Variabil'ity Study

indicated that more experience with the method was needed to

produce acceptable precision.

4} West Coast Marine Species Chrohic Protocol'Variability Study.

This study was a result of a 1991 settlement agreement among the

Northw'est Pulp and Paper Association, the Washlngtonbept. 'of

Ecology, Puget Sound Water Quality Auth.ority, and Tulal'ip Tribes

of Washington. The year-long study' ini993-94' i:ncluded'monthly

or quarterly interlaboratory toxicity test evaluation of tests

with bivalve molluscs (Crassostrea gigas) and mUissels (Mytilus

sp.), echinoid sperm'tests with purple se:a' urchins'(S'.

purpuratus} and sand dollar (D. exce'tricus) , sexual reproduction'

of kelp (L. saccharina) , and the topsmelt(A. affin'is).'

Following review and recommendations by the PRe 1:0 the State' of"

California for use of the procedures' in this rep<?rt, 'EPA (OR&D'"

v
and Region 9) modified the format for all methods to provide

consistency among the methods as well as consistency with

existing EPA Whole Effluent Toxicity Testing Manuals.

Review of the results from tests using the methods in this report

indicated that they are analogous to, and as sensitive ·as, the

methods previously proposed for estimating the chronic toxicity

of effluents and receiving waters to marine and estuarine

organisms (U.S. EPA 1994). The primary exception is the ,suite of

invertebrate embryo-larval tests contained in this manual. These

tests have been in regulatory and monitoring use on the west

coast, some for many years. They tend to be more sensitive test

organisms to many chemicals and the tests are more robust

statistically. They have no analog in the previous EPA methods

manuals, although a similar test has been proposed by the EPA

laboratory in Narragansett for use in monitoring sedimentassociated contaminants with the bivalve Mulinia lateralis.

vi
ABSTRACT

This manual describes six short-term (forty minutes to seven

days) estuarine andmarine.methods for measuring the chronic

toxicity of effluents'and receiving waters to ei9ht species: the

topsmelt, Atherinops,affinis; the mysid, Holmesimysiscostata;

the sea urchin, Strongylocentrotus purpuratus and sand dollar

Dendraster excentricus;the red abalone Haliotis rufescens; the

bivalves Crassostrea'gigas and mussel Myt;;iltisspp. and the giant

kelp, MC!-crocystis pyrifera. The methods include single 'and

multiple concentration static renewal and static nonrenewal

toxicity tests for effluents and receiving waters. Also included

are guidelines on laboratory safety, quality assurance,

facilities, and equipment 'and supplies; ciilution water; effluent

and receiving .water sample collection, preservation, shipping,

and holding; test conditio~s; toxicity test data analysis; report

preparation; and organism culturing, holding, and handling.

Examples of computer input and output for Dunnett's Procedure,

Probit Analysis, Trimmed Speaman-Karber Method, and the Linear

Interpolation Method are provided in the Appendices.

vii
ACKNOWLEDGEMENTS

The principal authors of this document are: Gary A. Chapman,

OR&D, Newport, Oregon; Debra ~. Denton, Region 9, San Francisco,

California; and James M. Lazorchak, OR&D, Cincinnati, Ohio.

Section 1 through 10 of this manual are only slightly

modified from the same sections in the EPA Manual, "Short-term

Methods for Estimating th~ Chronic Toxicity of Effluents and

Receiving Waters to Marine and Estuarine Organisms" (Second

Edition) and are essentially the work of Klemm, D.J., G.E.

Morrison, T.J. Norberg-King and W.H. Peltier. The numerous

contributors to their manual are acknowledged therein ..

Four of the seven methods in this manual were adapted,from

methods developed by the California State Water Resources Control

Board's Marine Bioassay Project. These methods for red ab~lone,

topsmelt, mysids, and kelp were prepared by the following staff

from the Universit.y of California, Santa Cruz:

Brian A. Anderson

John W. Hunt

Matt Englund

Hilary McNulty

Sheila L. Turpen

The sea urchin embryo/larval development test was modif,ied from a

method prepareq by staff from the Southern California Coastal

Water Research Project:

Steven Bay

Darrin Greenstein

The sea urchin and sand dollar sperm tests and the bivalve

mollusc embryo/larval development tests are ERL-N contributions

287 and 288, respectively, and were prepared by EPA staff:

Gary A. Chapman

Debra L. Denton

viii
The data analysis and statistical sections and appendices

were the work of Technology Applications, Inc. employee:

Laura Gast

Formal Peer-review comments from the follo~~ing persons are

gratefully acknowledged:

Amy Wagner, EPA Region 9 Richmond Lqboratory, who reviewed

all seven methods for technical detail and consistency (any

existing. error$ .sne~ked in after her revie~T) .

Paul Dinnel, Dinnel Marine Research

Suzanne Lussier, EPA Narragansett

Doug Middaugh, EPA Gulf Breeze

George Morrison, EPA Narragansett

Diane Nacci, SAIC

Barry Snyder, Ogden Erivironmental and Energy Services

Glen Thursby, SAIC

Southern California Toxicity Assessment .Group (SCTAG) and

its members, especially chairmen Tim Mikel of ABC Lab$~ and

Tom Dean of Coastal Resources Associates, Inc.

California Protocol Review Committee

.Matthew Reeve, California State Water Resources Control

Board (coordinator)

Business:

Steve Bay, Southern California Coastal Water Research

Project

Tom Dean, Coastal Resources Associates, Inc.

Andrew Glickman, Chevron Research and Technology

Company

Dave Gutoff, City of San Diego Marine IJaboratory

Timothy Hall, National Council of the Paper Industry

for Air and Stream Improvement .

ix
Government:

Gary Chapman, EPA

Debra Denton, EPA

Academia:

Gary Cherr, Bodega Marine Laboratory, University of

California, Davis

Jo Ellen Hose, Occidental College

Donald Reish, California State University, Long Beach

Washington Dept. of Ecology Protocol Variability

Study

Merley McCall, Washington Dept. of Ecology

(coordinator)

Science Advisory Board:

Rick Cardwell, Parametrix, Inc.

Dick Caldwell, Northwest Aquatics

Peter Chapman, EVS Environment Consultants, Ltq.

Gary Cherr (chair), Bodega Marine Lab, UC Davis'

Paul Dinnel, (vice-chair), Dinnel Marine Research

Some people have made continuing contributions to the

development and evaluation of these and related marine toxicity

test procedures. Special recognition need be given to: Paul

Dinnel for extensive work with echinoid sperm and embryo/larval

development tests; Susan Anderson of Lawrence Berkeley

Laboratories (earlier with California State Water Resources

Control Board) for echinoid sperm method modification and whole

effluent testing implementation; Michael Ives, Telonichor Marine

Laboratory, Humboldt State University, for providing his

experience and insight into method miniaturization and

streamlining; Gary Cherr and Jon Shenker at Bodega Mari,ne

Laboratory, UC Dayis, for method development and improvement for

most of these tests, especially for miniaturization of the

bivalve embryo/larval development test; Sally Noack of AScI. who

contributed greatly to testing of the sea urchin sperm cell test;

Robert Smith of EcoAnalysis performed much of the statistical

work of determining the MSD for each of the test methods; Randall

Marshall, Washington Department of Ecology for support and review

x
of the test method development,and implementation; Kevin Brix,

Parametrix, Inc. for providing information on sand-dollar

embryo/larval development tests; Timothy Hall of, NCASI 'for work

with the echinoid sperm test; the Northern California Toxicity

Assessment' Group (NCTAG) for their review of the bivalve mollusc

embryo/larval development test; and Phil Oshida, ,steve Schimmel,

and Steve Bugbee, EPA, for getting the EPA west coast methods

program started and on track.

xi
CONTENTS

Page

Disclaimer . . . . . . . . . . . . . . . . . . .

Foreword . . .

Preface . . .

Abstract

Acknowledgements

Contents . . . .

Section Number

1. Introduction

ii

iii

iv

vii

viii

xii

~age

1

2. Short-Term Methods for Estimating Chronic Toxicity 4

Introduction 4

Types of Tests 8

Static Tests 9

Advantages and Disadvantages of Toxicity Test Types 9

3. Health and Safety

General Precautions

Safety Equipment

General Laboratory and Field

Disease Prevention

Safety Manuals

Waste Disposal

.,

Operations

11

11

11

12

12

13

13

4. Quality Assurance .

Introduction .

Facilities, Equipment, and Test Chambers

Test Organisms .

Laboratory Water Used for Culturing and

and Test Dilution Water .....

Effluent and Receiving Water Sampling and,

Handling . . .

Test Conditions

Quality of Test Organisms

Food Quality ....

Acceptability of Chronic Toxicity Tests

Analytical Methods .

Calibration and Standardization

xii

.'

14

14

14

15

15

16

16

16

17

18

18

19
Replication and Test Sensitivity 19

Variability in Toxicity Test Results 19

Test Precision . 20

Demonstrating Acceptable Laboratory Performance 21

Documenting Ongoing Laboratory Performance 21

Reference Toxicants 24

Record Keeping 24

5 . Facilities, Equipment, and Supplies

General Requirements .

Test Chambers . . . . . .

Cleaning Test Chambers and Laboratory Apparatus

Apparatus and Equipment for Culturing an<l 'I'oxicity

Tests .

Reagents and Consumable Materials

Test Organisms

Supplies

25

25

26

26

27

27

29

29

6. Test Organisms .

Test Species .

Sources of Test Organisms

Life Stage . . . .

Laboratory Culturing

Holding and Handling of Test Organisms

Transportation to the Test Site

Test Organism Disposal ...

30

30

31

32

33

33

34

35

7. Dilution Water .

Types of Dilution Water .

Standard, Synthetic Dilution Water

Use of Receiving Water as Dilution Water

Use of Tap Water as Dilution Water

Dilution Water Holding .....

36

36

36

38

41

42

46

48

Handling,

43

43

43

44

46

Effluent and Receiving Wa.ter Sampling, Sample

and Sample Preparation for Toxicity Tes1:s

Effluent Sampling .

Effluent Sample Types .

Effluent Sampling Recommendations

Receiving Water Sampling ...

Effluent and Receiving Water Sample Handling,'

Preservation, and Shipping

Sample Receiving . . . . . .

$.

xiii
.,

9.

Persistence of Effluent Toxicity During Sample ...<'!?:

Shipment and Holding .

Preparation of Effluent and Receiving Water Samples

for Toxicity Tests .

Preliminary Toxicity Range-finding Tests

Multiconcentration (Definitive) Effluent

Toxicity Tests

Receiving Water Tests

Chronic Toxicity Test Endpoints and Data Analysis

Endpoints .

Relationship between Endpoints Determined by

Hypothesis Testing and Point Estimation Techniques

Precision ...

Data Analysis

Choice of Analysis

Hypothesis Tests

Point Estimation Techniques

48

48

52

53

53

,55

55

56

58

59

61

63

66

10. Report preparation

Introduction

Plant Operations

Source of Effluent, Receiving Water,

Water

Test Methods

Test Organisms

Quality Assurance

Results ...

Conclusions and Recommendations

and Dilution

'68

68

68

68

69

69

70

70

,70

11. Test Method: Topsmelt, Atherinops a££inis, Larval

Survival and Growth Method 1006.0 . . . . . . :71

12. Test Methods: Mysid, Holmesimysis ,costata, Survival

and Growth Test Method 1007.0 . . . . . . 141

13. Test Method: Pacific Oyster, Crassostrea gigas, 'and!

Mussel Mytilus spp. Shell Development Test Method

1005.0 . . . . . ,~. . . 209 ",:' ~'. '''-~;.''

14. Test Methods: Red Abalone, Haliot~s,ru£escens,

Larval Development . . . . . . . . . . . . . 259

xiv
15. Test Method: Sea Urchin, strongylocentrotus' .

purpuratus Embryo-Larval Development Test Method .321

16. 'rest Method: Sea Urchin, Strongylocentrotus

purpuratus and Sand Dollar Dendraster excentricus

Fertilization Test Method 1008.0 .... 389

17. Test Method: Giant Kelp, Macrocystis pyrifera,

Germination and Germ-Tube Length Test Method

100 9 . 0 '. . .' .'. . .' . 466

Cited References

Bibliography

528

549

Appendices . . . . ... . . .. " . 564

. ,.

A. Independence, Randomization, and Outliers '•.

B. Validating Normality and Homogeneity of Variance

Assumptions . . . . . . . . . . . ....

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

D. T test with Bonferroni's Adjustment

E. Steel's Many~one Rank Test .....

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

G. Single Concentration Toxicity Test - Comparis6h

of Control with 100% Effluent or Receiving Water

H. Probit Analysis . . . . . . .

I. Spearman-Karber Method ....

J. Trimmed Spearman-Karber Method

K. Graphical Method .....

L. Linear Interpolation'Method

xv

566

575

587

602

609

615

622

627

631

638

.643

648

SECTION 1

INTRODUCTION

1.1 This manual describes chronic toxicity tests for use in the

National Pollutant Discharge Elimination Systenl (NPDES) Permits

Program to identify effluents and receiving waters containing

toxic materials in chronically toxic concentrations. The test

methods 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 hypothesis testing or point estimate

techniques (see Section 9, Chronic Toxicity Test Endpoints And

Data Analysis) and appropriate dilution, application, and

persistence factors. The tests are performed as a part of

self-monitoring permit requirements, compliance biomonitoring

inspections, toxics sampling inspections, and special

investigations. Data from chronic toxicity tests performed as

part of permit requirements are evaluated during·compliance

evaluation inspections and performance audit inspections.

1.3 Modifications of these tests are also used in toxicity

reduction evaluations and toxicity identification evaluations to

identify the toxic components of an effluent, to aid in the

development and implementation of toxicity reduction plans, and

to compare and control the effectiveness of various treatment

technologies for a given type of industry, irrespective of the

receiving water (USEPA, 1988ci USEPA, 1989bi USEPA, 1989ci USEPA,

1989di USEPA, 198gei USEPA, 1991ai USEPA, 1991bi USEPA, 1992).

1.4 This methods manual serves as a companion to the acute

toxicity test methods for freshwater and marine organisms (USEPA,

1993a), the short-term chronic toxicity test methods for

freshwater organisms (USEPA, 1993b), the short-term chronic

toxicity test methods for east coast organisms ;(USEPA, 1994), and

the'manual for evaluation of laboratories performing aquatic

toxicity tests (199ic). .

1.5 Guidance for the implementation of toxicity tests in the

NPDES program is provided ,in the Technical Suppl:)rt Document for

Water Quality-Based Toxics Control (USEPA, 199113.).

1
similar to those developed for the freshwater organisms and east

coast marine organisms to evaluate the toxicity of effluents

discharged to estuarine and coastal marine' waters under the NPDES

permit program. Methods are presented in this manual for ten

species from six phylogenetic groups. The red abalone larval

development test method, the giant kelp germination and ge~m-tube

length test method, the mysid survival and growth test method and

the topsmelt survival and growth test method were developed and

extensively field tested by University of' California', Santa Cruz

through the California State Water Resources Control Board:! s

Marine Bioassay Project. The purp~e urchin and sand dollar

fertilization test method was developed by U.S. 'Environmental

Research Laboratory-Newport,' Oregon. The purple urchin and sand

dollar development test method was developed by the Southern

California Coastal Water Research Project. The Pacific oy~ter

and mussel survival and larval development test method was

modified from ASTM 1989 by the Washington Department of Ecology

and the USEPA. The methods vary in duration from 40 minutes to

seven days.

1.7 The ten species for which toxicity test methods provided

are: the topsmelt, Atherinops affinis, the red' abalone, Haliotis

rufescens; the Pacific oyster, Crassostrea gigas, mussel Mytilus

spp.; the mysid, Holmesimysis costata; the sea urchin, .

Strongylocentrotus purpuratus, the sand dollar, Dendraster

excentricus; and the giant kelp, Macroystis pyrifera.

1.7.1 Many of the tests included in this document are based on

the following:

1. "Marine Bioassay Project Seventh Reports (Reports 1-7)"

by Brian S. Anderson, John W. Hunt, and Hilary R.

McNulty, University of California, Santa Cruz; Mark D.

Stephenson, California Department of Fish and Game; and

Francis H. Palmer, ,Debra L. Denton, and Matthew Reeve,

State Water Resources Control Board.

2. "Procedures Manual for Conducting Toxicity Tests

Developed by the Marine Bioassay Project by Brian S.

Anderson, John W. Hunt, Shiela L. Turpen, A.R. Coulon,

University of California, Santa Cruz; Mike Martin,

California of Depart~ent of Fish and Game; Debra L.

Denton and Frank H. Palmer, State Water Resources Control

Board, 90-10WQ, 112 pp.

3. "Standard Practice for Conducting Static Acute Toxicity

Tests with Larvae of Four Species of Bivalve Mollu6cs.

ASTM 1989.

2
1.7.2 Three of the methods incorporate the chronic endpoints of

growth or development (or both) in addition to lethality. The

sea urchin sperm cell test uses fertilization as an endpoint and

has the advantage of an extremely short exposure period (40

minutes) .

1.8 The validity of similar marine/estuarine methods in

predicting adverse ecological impacts of toxic discharges was

demonstrated in field studies (USEPA, 1986d).

1.9 The use of any marine o~ estuarine test species or test

conditions other than those described in the methods summary

tables in this manual or in th~ east coast marine ,manual

(USEPA/600/4-91/003) shall be subject to application and approval

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

1.10 These methods are restricted to use by or under the

supervision of analysts experienced in the use or conduct of

aquatic toxicity testing and the interpretation of data from

aquatic toxicity testing. Each analyst must demon:strate the

ability to generate acceptable test results with these methods

using the procedures described in this methods manual.

1.11 The manual was prepared in the establishedNERL-Cincinnati

format (USEPA, 1983).

3
SECTION 2

SHORT-TERM METHODS FOR ESTIMATING CHRONIC TOXICITY

2.1 INTRODUCTION

2.1.1 The objective of aquatic toxicity tests with effluents or

pure compounds is to estimate the "safe" or "no-effect"

concentration of these substances, which is defined as the

concentration which will permit normal propagation of fisn and

other aquatic life in the receiving waters. The endpoints that

have been considered in tests to determine the adverse effects of

toxicants include death and survival, decreased reproductIon and

growth, locomotor activity, gill ventilation rate, heart rate,

blood chemistry, histopathology, enzyme activity, olfacto~y

function, and terata. Since it is not feasible to detect and/or

measure all of these (and other possible) effects of toxic

substances on a routine basis, observations in toxicity tests

generally have been limited to only a few effects, such a$

mortality, growth, and reproduction.

2.1.2 Acute lethality is an obvious and easily observed effect

which accounts for its wide use in the early period of evaluation

of the toxicity of pure compounds and complex effluents. The·

results of these tests were usually expressed as the

concentration lethal to 50% of the test organisms (LC50) over

relatively short exposure periods (one-to-four days) .

2.1.3 As exposure periods of acute tests were lengthened~ the

LC50 and lethal threshold concentration were observed to decline

for many compounds. By lengthening the tests to include one or

more complete life cycles and observing the more subtle effects

of the toxicants, such as a reduction in growth and reproduction,

more accurate, direct, estimates of the threshold or safe

concentration of the toxicant could be obtained. However,

laboratory life cycle tests may not accurately estimate the

"safe" concentration of toxicants because they are conducted with

a limited number of species under highly controlled, steady state

conditions, and the results do not include the effects of. the

stresses to which the organisms would ordinarily be expos~d in

the natural environment.

2.1.4 An early published account of a full life cycle, fish

toxicity test was that of Mount and Stephan (1967). In this

study, fathead minnows, Pimephales promelas, were exposed to a

graded series of pesticide concentrations throughout their life

cycle, and the eff~cts of the toxicant on survival, growth, .and

4
reproduction were measured and evaluated. This work was soon

followed by full lif~ cycle tests using other toxicants and fish

species.

2.1.5 McKim (1977) evaluated the data from 56 :full life' cycle

tests, 32 of which used the fathead minnow, Piml9phales promelas,

and concluded that the embryo-larval and early :luvenile life .

stages were the most sensitive ptages. He prop6sed the use of

partial life cycle toxicity tests with the early life stages

(ELS) of fish to establish water quality£riteria.

2.1.6 Macek and Sleight (1977) found that exposure of critic~l

life ~tages of fish to toxicants provides estimates of

chronically saf~conceptrations,remarkably similar to ~hose

derived from full life cycle toxicity tests. They reported that

"for a great majority of foxicants, the concentration which will

not be acutely toxic to the most sensitive life stages is the

chronically safe concentration for fish, and that the most

sensitive life stages are the embryos and fry." Critical life

stage exposure was considered to be exposure of the embryos

during most, preferably all, of the embryogenic (incubation)'

period, and exposure of the fry for 30 days post-hatch for warm

water fish with embryogenic periods ranging from one-to-fourteen

days, and for 60 days post-hatch for fish with longer embryogenic

periods. They concluded that in the majority of cases, the

maximum acceptable toxicant concentrafion (MATC) could be

estimated from the results of exposure of the embryos during

incubation, and the larvae for 30 days post:-:hatch.'

2.1.7 Because of the high cost of full life-cycle fish toxicity

tests and the emerging consensus that the ELS test data usually

would be adequate for estimating chronically sa,fe .concentrations,

there was a rapid shift by aquatic toxicologists to 30- to. 90-day

ELS toxicity tests for estimating chronically ,safe concentrations

in the late 1970s. In 1980,USEPA adopted the policy that ELS

test data could be used in establishing water quality criteria if

data from full life-cycle tests were not available (USEPA,

1980a) .

2.1.8 Published reports of the results 6f ELS tests indicate

that the relative sensitivity of. growth and survival as endpoints

may be species dependent, toxicant dependent; or both. Ward and

Parrish (1980) examined the literature on ELS tests th~t used

embryos and juveniles of the sheepshead minnow, (~prinodon

variegatus, and found that growth was not a statistically

sensitive indicator of toxicity in 16 of 18 tests. They

,suggested that the ELS tests be shortened to 14 days posthatc~

and that growth be eliminated as an indicator of toxic effects.

5
2.1.9 In a review of the literatur~ on 173 fish full life-cycle

and ELS tests performed to determine the chronically safe ' concentrations of a wide variety of toxicants, such as metals,

pesticides, organics, inorganics, detergents, and complex

effluents, WOltering (1984) found that at the lowest effect

concentration, significant reductions were observed in fry , survival in 57%, fry growth in 36%, and egg hatchability in 191

of the tests. He also found that fry survival and growth were

very often equally sensitive, and concluded that the growth

response could be' deleted from routine application of the ELS

tests. The net result would be a significant reduction in the

duration and cost of screening tests with no appreciable impact

on estimating MATCs for chemical hazard assessments. Benoit, et

al. (1982), however, found larval growth to be the most

significant measure ~f effect and survival to be equally or 'less

sensitive than growth in early life-stage tests with four organic

chemicals.

2.1.10 Efforts to further reduce the length of partial lifecycle toxicity tests for fish without compromising their

predictive value have resulted in the development of an

eight-day, embryo-larval survival and'teratogenicity test for

fish and other aquatic vertebrates (USEPA, 1981i Birge et al.,

1985), and a seven-day larval survival and growth test (Norberg

and Mount, 1985).

2.1.11 The similarity of estimates of chronically safe

concentrations of toxicants derived from short-term,

embryo-larval survival and teratogenicity tests to those--derived

from full life-cycle tests has been demonstrated by Birge et al.

(1981), Birge and Cassidy (1983), and Birge et al. (1985).

2.1.12 Use of a seven-day, fathead minnow, Pimephales promelas,

larval survival and growth test was first proposed by Norberg and

Mount at the 1983 annual meeting of the Society for Environmental

Toxicology and Chemistry (Norberg and Mount, 1983). This test

was sUbsequently used by Mount and associates in field

demonstrations at Lima, Ohio (USEPA, 1984), and at many other

locations (USEPA, 1985c, USEPA, 1985di USEPA, 198~ei USEPA,

1986ai USEPA, 1986bi USEPA, 1986ci USEPA, 1986d). Growth w~s

frequently found to be more sensitive than survival in

determining the effects of complex effluents.

2.1.13 Norberg and'Mount (1985) performed three single toxicant

fathead minnow larval growth tests with zinc, copper, and

DURSBAN@, using dilution water from Lake Superior. The results

6
were comparable to, 'and had confidence intervals that overlapped

with, chrOnic values r~ported in the liter~ture for both ELS and

full li~e-cycle tests.

2.1.14 USEPA,(1987b) and USEPA (1987c) adapted the fathead

minnow larval growth and survival test for use with the

sheepsh~ad minnow ~tid·the inland silverside,' respectively. When

daily renewal 7-day sheepshead minnow lar~al growth and s~ivival

tests and 28~day ELS tests were pel;"formed with industrial and

municipal effluents, growth was more sensitive than survival in

seven out of 12 larvai grow,th and survival tests, equally

sensitive i~ four tests, and less sensi~ive in only one test. 'Iri

four cases, the ELS test may have been three to 10 ,times more

sensitive to effluents than the larval growth and ~urvival tes~.

In tests using' copper, the No Observabl'e Effect Concentrations

(NOECs) were the same for both types of test, and growth was the

most sensitive endpoint for both. In a four laboratory .

comparison, six of seven tests produced identic:al NOECs for

survival and gro-w:th (USEPA, 1987a). Data indicate that the

inland silverside is at least equally s.ensitive or more sensitive

to effluents' and single compounds than the shee~p$head minnow, and

can be tested over a wider salinity range, 5-30%0 (USEPA,

1987a) "

2.1.15 Lussier et al. (1985) and USEPA (1987e), determined that

survival and growth are often as sensitive as r~production in

28-day life...:.cycle tests with th~ mysid, Mysidopsis bahia.

2.1.16 Nacci and Jackim (1985~ and USEPA; (1987g)'compared th'e

resuits from the sea 'urchin fertilization test, uslngorganic ",

compounds, with results from acute toxicity tests using the

freshwater organisms, fathead minnows, Pimphales promelas, and

Daphnia magna. The test was also compared to acute toxicity

tests using Atlantic silverside, Menidia menidia, and the mysid,

Mysidopsis bahia, and five metals. For six' of 'the eight organic

compounds, the results of the fertilization tes:~ and the acute .

toxicity test correlated well (r2 = 0.85). However, the results

of the fertilization test with the five metals did not cotrelat~

well with the results from the acute tests.

2.1.17 USEPA {1987f) evaluated two industrial effluents

containing heavy metals, five industrial efflueI~ts containing

organic ,chemicals (including dyes and pesticides), and 15

domestic wastewaters using the two-day red macr(~alga, Champia

parvula, sexual reproduction test. Nine single compounds were

used to compare the effects on sexual reproduction using a , ,

7
two-week exposure and a two-day exposure. For six of the :nine

compounds tested, the chronic values were the same for bo~h

tests.

2.1.18 The use of short-term toxicity tests in ,the NPDES Program

is especially attractive because they provide a more direct ,

estimate of the safe concentrations of effluents in receiving

waters than was provided by acute toxicity tests, at an only

slightly increased level of effort, compared to the fish full

life-cycle chronic and 28-day ELS tests and the 28-day mysid

life-cycle test.

2.2 TYPES OF TESTS

2.2.1 The selection of the test type will depend on the NPDES

permit requirements, the objectives of the test, the available

resources, the requirements of the test organisms, and effluent

characteristics such as fluctuations in effluent toxicity.

2.2.2 Effluent chronic toxicity is generally measured using a

multi-concentration, or definitive test, consisting of a control

and a minimum of five effluent concentrations. The tests'are

designed to provide dose-response information, expressed as the

percent effluent concentration that affects the survival, ,

fertilization, growth, and/or development within the prescribed

period of time (40 minutes to seven days). The r~sults of the

tests are expressed in terms of either the highest concentration

that has no statistically significant 'observed effect on those

responses when compared to the controls or the estimated

concentration that causes a specified percent reduction in

responses versus the controls.

2.2.3 Use of pass/fail tests consisting of a single effluent

concentration (e.g., the receiving water concentration or RWC)

and a control is not recommended. If the NPDES permit has a

whole effluent toxicity limit for acute toxicity at the RWC, it

is prudent to use that permit limit as the midpoint of a series

of five effluent concentrations. This will ensure that there is

sufficient information on the dose-response relationship.: For

example, if the RWC is >25% then, 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%. If the RWC is <25%

effluent the concentrations may be: (1) 4 times the RWC, (2) 2

times the RWC, (3) RWC, (4) RWC/2, and (5) RWC/4.

8
two treatments, a control and the undiluted receiving water, but

may also consist of a series of receiving water dilutions.

2.2.5 A negative result from a chronic toxicity' test does not

preclude the presence of toxicity. Also, . because of the

potential temporal variability in the toxicity of effluents, a

negative test result with a particular sample does not preclude

the possibility that samples collected at some other time might.

exhibit chronic toxicity.

2.2.6 The frequency with which chronic toxicity tests are

conducted under a given NPDES permit is determined by the

regulatory agency on the basis of factors such as th~ variability

and degree of toxicity of the waste, production schedules, and

process changes.

2.2.7 Tests recommended for use in this methods manual may be

static non-renewal or static renewal. Individual methods specify

which type of test is to be conducted. .

2.3 STATIC TESTS

2.3.1 static non-renewal tests - The test organisms are exposed

to the same test solution for the duration of the test.

2.3.2 Static-renewal tests - The test organisms are exposed to a

fresh solution of the same concentration of sample every 24 h or

o.ther prescribed interval, . either by transferrin9 the test

organisms from one test chamber to another, or by replacing all

or a portion of solution in the test chambers.

2.4 ADVANTAGES AND DISADVANTAGES OF TOXICITY TEST TYPES

2.4.1 STATIC NON-RENEWAL, SHORT-TERM·TOXICITY TESTS:

Advantages:

1. Simple and inexpensive.

2. More cost effective in determining compliance with ,permit

conditions.

3. Limited resources (space, manpower, equipment) required;

would permit staff to perform more tests in the same

amount of time.

4. Smaller volume of effluent required than for static

renewal or flow-through tests.

9
Disadvantages:

1. Dissolved oxygen (DO) depletion may result from higq

chemical oxygen demand (COD), biological oxygen demand

(BOD), or metabolic wastes.

2. Possible loss of toxicants through volatil{za~ion and/or

adsorption to the exposure vessels. ,

3. Generally less sensitive than renewal because the toxic

substances may degrade or be adsorbed, thereby reducingthe apparent toxicity. Also, there is l,ess chance of

detecting slugs of toxic wastes, or other temporal

variations in waste properties.

2.4.2 STATIC RENEWAL, SHORT-TERM TOXICITY TESTS:

Advantages:

1. Reduced possibility of DO depleti'on from high COD and/or

BOD, or ill effects from metabolic wastes from orga~isms

in the test solutions.

2. Reduced possibility of loss of toxicants through

volatil~zation and/or adsorption to' the exposure ve~sels.

3. Test organisms that rapidly deplete 'energy reserves, are

fed when the test solutions are renewed, and are

maintained in a healthier state.

Disadvantages:

1. Require greater volume of effluent than non-renewal'

tests.

2. Generally less chance 'of temporal variations In waste

properties.

10
SECTION 3

HEALTH AND SAFETY

3.1 GENERAL PRECAUTIONS

3.1.1 Each laboratory should develop and "maintain an effective

health and safety program, requiring an ongoing commitment by the

laboratory management and includes: (1) a safety officer with

the responsibility and authority to develop and maintain a safety

program; (2) the preparation of a formal, written, health and

safety plan, which is provided'to the laboratory staff; (3) an

ongoing training program on laboratory safety; and (4) regularly

scheduled, documented, safety inspections. .

3.1.2 Collection and use of effluents in toxi,city tests may'

involve significant risks to personal safety and health.

Personnel collecting effluent samples and conducting toxicity

tests should take all safety precautions neceE:sary for the

prevention of bodily injury and illpess which might result from

ingestion or invasion of infectious agents, inhalation or

absorption of corrosive or toxic substances through skin contact,

and asphyxiation due to a lack of oxygen or the presence of

noxious gases.

3.1.3 Prior to sample collection and laboratory wor~, personnel

should determine that all necessary safety equipment and

materials have been obtained and are in good condition.

3.1.4 Guidelines for the handling and disposal of hazardous

materials must be strictly followed.

3.2 SAFETY E,QUIPMENT

3.2.1 PERSONAL SAFETY GEAR

3.2.1.1 "Personnel must use safety equipment, as required, such

as rubber aprons, laboratory coats, respirators, gloves, safety

glasses, hard hats, and safety shoes. Plastic netting on glass

beakers, flasks and other glassware minimizes breakage and

subsequent shattering of the glass.

3.2.2 LABORATORY SAFETY EQUIPMENT

3.2.2.1 Each laboratory (including mobile labora.tories) should

be provided with pafety equipment such as first a.id kits, fire

extinguishers, fire blankets, emergency showers, chemical spill

clean-up kits, and eye fountains.

11
3.2.2.2 Mobile laboratories should be equipped with a telephone

to enable personnel to summon help in case of emergency. .

3.3 GENERAL LABORATORY AND FIELD OPERATIONS

3.3.1 Work with effluents should be performed "in compliarice with

accepted rules pertaining to the handling of hazardous materials

(see safety manuals listed in Section 3, Health and Safety,

Subsection 3.5). It is recommended that personnel collecting

samples and performing toxicity tests should not work alone.

3.3.2 Because the chemical composition of effluents is usually

only poorly known, they should be considered as potential health

hazards, and exposure to them should be minimized. Fume and

canopy hoods over the toxicity test areas must be used whenever

possible.

3.3.3 It is advisable to cleanse exposed parts of the body

immediately after collecting effluent samples.

3.3.4 All containers should be adequately labeled to indicate

their contents.

3.3.5 Staff should be familiar with safety guidelines on,

Material Safety Data Sheets for reagents and other chemicals

purchased from suppliers. Incompatible materials should not be

stored together. Good housekeeping contributes to safety and

reliable results.

3.3.6 Strong acids and volatile organic solvents employed in

glassware cleaning must be used in a fume hood or under an

exhaust canopy over the work area.

3.3.7 Electrical equipment or extension cords not bearing the

approval of Underwriter Laboratories must not be used.

Ground-fault interrupters must be installed in all "wet"

laboratories where electrical equipment is used.

3.3.8 Mobile laboratories should be properly grounded to protect

against electrical shock.

3.4 DISEASE PREVENTION

3.4.1 Personnel handling samples which are known or suspected to

contain human wastes should be immunized against tetanus, typhoid

fever, polio, and hepatitis B.

12
3 . 5 SAFETY MANUALS

3.5.1 For further guidance on safe practices when collecting

effluent samples and conducting toxicity tests, check with the

permittee and consult general safety manuals, including USEPA

(1986e), and Walters and Jameson (1984).

3.6 WASTE DISPOSAL

3.6.1 Wastes generated during toxicity testing must be properly

handled and disposed of in an appropriate manner. Each testing

facility will have its own waste disposal requirements based on

local, state and Federal rules and regulations .. It is extremely

important that these rules and regulations be known, understood,

and complied with by all persons responsible for, or. otherwise

involved in, performing toxicity testing activities. Local fire.

officials should be notified of any potentially hazardous

conditions.

13
SECTION 4

QUALITY ASSURANCE

4.1 INTRODUCTION

4.1.1 Development and maintenance of a toxicity test laboratory

quality assurance (QA) program (USEPA, 1991b) requires an ongoing

commitment by laboratory management. Each toxicity test

laboratory should (1) appoint a quality assurance officer with

the responsibility and authority to develop and maintain a QA

program, (2) prepare a quality assurance plan with stated data

quality objectives (DQOs), (3) prepare written.descriptions of

laboratory standard operating procedures (SOPs) for culturing,

toxicity testing, instrument calibration, sample chain-ot-custody

procedures, laboratory sample tracking system, glassware

cleaning, etc., and (4) provide an adequate, qualified technical

staff for culturing and toxicity testing the organisms, and

suitable space and equipment to assure reliable data.

4.1.2 QA practices for toxicity testing labor~tories must

address all activities that affect the quality of the final

effluent toxicity data, such as: (1) effluent· sampling and

handling; (2) the source and condition of the test organisms; (3)

condition of equipment; (4) test conditions; (5) instrum~nt

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 de~ailed

discussion of quality assurance and general guidance on good

laboratory practices and laboratory evaluation related to

toxicity testing, see FDA (1978); USEPA (1979d); USEPA (1980b);

USEPA (1980c); USEPA (1991c); DeWoskin (1984); and Taylor (1987).

4.1.4 Guidelines for the evaluation of laboratory performing

toxicity tests and laboratory evaluation criteria are found in

USEPA (1991c).

4.2 FACILITIES, EQUIPMENT, AND TEST CHAMBERS

4.2.1 Separate test organism CUlturing and toxicity testing

areas should be provided to avoid possible loss of cultures due

to cross-contamination. Ventilation systems should be designed

and operated to prevent recirculation or leakage of air from

chemical analysis laboratories or sample storage and preparation

14
areas into organism culturing or testing areas, and from testing

and sample preparationareas ,into culture room~).

4.2.2 Laboratory and toxicity test temperature control equipment

must be adequate to maintain recommended test Hater temperatures.

Recommended materials must be used in the fabrication of the test

equipment which comes in contact with the effluent (~ee Section

5, Facilities, Equipment* and Supplies;. and specific toxicity

test method) . ..

4.3 TEST ORGANISMS

4.3.1 The t~sto~ganisms used .inthe procedures described in

this manual are the red abalone, Haliotis rufescens; the Pacific

oyster, C'rassostrea gigas, and mussel, Mytill,lsspp.; the

topsmelt,Atherinops affini.s; the mysld, Holme.s:imysis costa·ta;

the sea urchin" Strongylocentrptus purpura.tus., ,and the s.and

dollar Denstraster excentricus;and the giant kelp, Macrocystis

pyrifera. The organisms used should bedisease""free and apPear

healthy, behave normally, feed. well, and~. have low mortal i ty in

cultures, during holding, and in test control. Test organisms

should be positively identified to· species ("see Section 6, Test

Organisms). .

4.4 LABORATORY WATER USED FOR CULTURING AND TEST DILUTION WATER

.4.4.1 The quality of water used fo'r test· organism cuIturing and

for dilution water used in toxicity tests is· extremely important.

Water .for these two uses should come from the same source. The

dilution water: used' in effluent toxicity t'ests 'will depend on the

objectives of the study and logistical constraints, as discussed

in Sectioh 7, .Dilution Water. The. dilution water' u'sed in the

toxicity tests may be natural seawater, hypersaline-brine

(100%0) prepared from natural seawater; or artificial seawater

prepared from commercial sea· salts, such as FOR'rY FATHOMS® or HW

MARINEMIX®,. if recorcunended in the method. GP2 synthetic .

seawater, made from reagent grade chemical salts in conjunction

with natural seawater, may also be·used'if reconunended. Types of

water are discussed in Section 5, Facilities, Equipment, ahd

Supplies. Water used for culturing and test dilutibn water

should be analyzed for toxic metals and organics at least

annually or whenever difficul ty is·· encountered' in meeting minimum

acceptability criteria- for control survival and reproduction or

growth. The concentration of the metals; AI, As, Cr, Co, Cu, Fe,

Pb, Ni,. Zn, expressed as total metal, should no+:' exceed 1 pg/L

each, and Cd, Hg, and Ag, expressed as total metal, sbould not

exceed 100 ng/L each. Total organochlorine pesticides plus PCBs

should be less than 50 ng/L (APHA; 1992). Pesticide

15
concentrations should not exceed USEPA's National Ambient Water

Quality chronic criteria values where available.

4.5 EFFLUENT AND RECEIVING WATER SAMPLING AND HANDLING

4.5.1 Sample holding times and temperatures of effluent samples

collected for on-site and off-site testing must conform to

conditions described in Section 8, Effluent and Receiving Water

Sampling, Sample Handling, and Sample Preparation for Toxicity

Tests.

4.6 TEST CONDITIONS

4.6.1 Water temperature and salinity must be maintained within

the limits specified for each test. The temperature of test

solutions must be measured by placing the thermometer or probe'

directly into the test solutions, or by placing the thermometer

in equivalent volumes of water in surrogate vessels positioned at

appropriate locations among the test vessels. Temperature should

be recorded continuously in at least one vessel during the

duration of each test. Test solution temperatures must be

maintained within the limits specified for each test. DO

concentrations and pH should be checked as specified in each test

method.

4.7 QUALITY OF TEST ORGANISMS

4.7.1 If the laboratory performs short-term chronic toxicity

tests routinely but does not have an ongoing test organism

culturing program and must obtain the test organisms from an

outside source, the sensitivity of a batch of test organisms must

be determined with a reference toxicant in a short-term chronic

toxicity test performed monthly (see Section 4, Quality

Assurance, Subsections 4.14, 4.15, 4.16, and 4.17). Where ~cute

or short-term chronic toxicity tests are performed with effluents

or receiving waters using test organisms obtained from outside

the test laboratory, concurrent toxicity tests of the same type

must be performed with a reference toxicant, unless the test

organism supplier provides control chart data from at least the

last five monthly short-term chronic toxicity tests using the

same reference toxicants and test conditions (see Section 6, Test

Organisms) .

4.7.2 The supplier should certify the species identification of

the test organisms, and provide the taxonomic reference (citation

and page) or name(s) of the taxonomic expert(s) consulted.

16
4.7.3 If the laboratory maintains breeding cultures, the

sensitivity of the offspring should be determinE~d in a short-term

chronic toxicity test performed with a referencE~ toxicant at

least once each month (see Section 4, Quality Assurance,

Subsectio~.4.14, 4.15, 4.16, and 4.17). I£ preferred, this

reference toxicant test may be performed concurrently, with _an

effluent toxicity test. However, if a given speci~s of tes~

organism produced by inhouse cultures is used only monthly, orless frequently in toxicity tests, a. reference toxicant test must

be performed concurrently with each short-term chronic effluent

and/or receiving water toxicity test.

4.7.4 If a routine reference toxicant test fails to meet

acceptability criteria, the test must beimmediCltely 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 Section 4,

Quality Assurance, Subsection 4.16.5).

4.8 FOOD QUALITY

.4.8.1 The" nutritional quality of the food used in culturing and

testing fish and invertebrates is an important factor in the

quality of the toxicity test data. This is especially true for

the unsaturated fatty acid content of brine shrimp nauplii,

Artemia. Problems with the nutritional suitability of the food

will be reflected in the survival, growth, and reproduction of

the test organisms in cultures and toxicity tests. Artemia cysts

and other foods must be obtained as described in Section 5,

Facilities, Equipment, and Supplies.

4.8.2 Problems with the nutritional suitabili~y of food will be

reflected in the survival, growth, development 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 ",vi ththe

performance of organisms fed with a food of known quality in

side-by-'side tests. I f the food is used for culturing, its

suitability should be determined using a short-b9rm chronic test

which will determine the affect of food quality on g~owth or

reproduction of each of the relevant test species in culture,

using four replicates with each food source. Where applicable,

foods used only in chronic toxicity tests can be compared with a

food of known quality in side-by-side, multi-concentration

chronic tests, using the reference toxicant regularly employed in

the laboratory QA program. For list of commercial sources of

Artemia cysts, see Table 2 of Section 5, Facilities, Equipment,

and Supplies.

17
4 • 1 7 REFERENCE TOXICANTS

4.17.1 Reference toxicants such as zinc sulfate (ZnS04), cadmium

chloride (CdC12 ), copper sulfate (CUS04), and copper chloripe

(CuC12 ), are suitable for use in the NPDES Program and other

Agency programs requiring aquatic toxicity tests. NERL- ,

Cincinnati plans to releaseUSEPA-certified solutions of cadmium

and copper for use as reference toxicants, through cooperative

research and development agreements with commercial suppli~rs,

and will continue to develop additional reference toxicantp for

future release. Interested parties can determine the

availability of "EPA Certified" reference toxicants by checking

the NERL-Cincinnati electronic bulletin board, using a modem to

access the following telephone number: 513-569-7610. Standard

reference materials also can be obtained from commercial supply

houses, or can be prepared inhouse using reagent grade chemicals.

The regulatory agency should be consulted before reference'

toxicant(s) are selected and used.

4.18 RECORD KEEPING

4.18.1 Proper record keeping is important. A complete file must

be maintained for each individual toxicity test or group of tests

on closely related samples. This file must contain a record of

the sample chain-of-custody; a copy of the sample log she~t; '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 iri the test(s);, such

as species, source, age, date of receipt, and other pertinent

information relating to their history and health; information on

the calibration of equipment and instruments; test conditions

employed; and results of reference toxicant tests. Laboratory

data should be recorded on a real-time basis to prevent the loss

of information or inadvertent introduction of errors into the

record. Original data sheets should be signed and dated by the

laboratory personnel performing the tests.

4.18.2 The regulatory authority should retain records pertaining

to discharge permits. Permittees are required to retain records

pertaining to permit applications and compliance for a mi~imum of

3 years [40 CFR 122.41 (j) (2) ] .

24
SECTION 5

FACILITIES, EQUIPMENT, AND SUPPLIES

5 .1 GENERAL REQUIREMENTS

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

mobile laboratory. Faccilities must include equipment for rearing

and/or holding organisms .. Culturing facilities for test

organisms may be desirable in fixed laboratories which perform

large numbers of tests. Temperature .control can be achieved

using circulating water baths, heat exchangers, or environmental

chambers. Water used for rearing, holding, acclimating, and

testing organisms may be natural seawater or water made up from

hypersaline brine derived from natural seawater, or water made up

from reagent grade chemicals (GP2) or commercial (FORTY FATHOMS®

or HW MARINEMIX®) artificial sea salts when specifically

recommended in the method. Air used for aeration must be free of

oil and toxic vapors. Oil-free air pumps should be used where

possible. Particulates can be removed from the air using

BALSTON® Grade BX or equivalent filters (Balston, Inc.,

Lexington, Massachusetts), and oil and other organic vapors can

be removed using activated carbon filters (BALSTON®, C-1 filter,

or equivalent).

5.1.2 The facilities must be well ventilated_,and free of fumes.

Laboratory ventilation systems should be checked t6 ensure that

return air from chemistry laboratories and/or sample handling

areas is not circulated to test organism culture; rooms or

toxicity test rooms, or that air from toxicity test rooms does

not contaminate culture areas. Sample preparation, culturing,

and toxicity testing areas should be separated to avoid crosscontamination of cultures or. toxicity test solutions with toxic.

fumes. Air pressure differentials between such rooms should. not

result in a net flow of potentially contaminated air to sensitive

areas through open or loosely-fitting doors. Organisms should be

shielded from external disturbances.

5.1.3 Materials used for exposure chambers, tubing, etc., which

come in contact with the effluent and dilution water, should be

carefully chosen. Tempered glass and perfluorocarbon plastics

(TEFLON®) should be used whenever possible to minimize sorption

and leaching of toxic substances. These materials may be reused

following decontamination. Containers made of plastics, such as

polyethylene, polypropylene, polyvinyl chloride, TYGON@, etc.,

may be used as test chambers or to ship, store, and transfer

effluents and receiving waters, but they should not be reused

unless absolutely necessary, because they might carryover

25
1. Sensitive species may not be present in the receiving

water because of previous exposure to the effluent or

other pollutants.

2. It is often difficult to collect organisms of the

required age and quality from the receiving water.

3. Most states require collection permits, which may be

difficult to obtain. Therefore, it is usually mor~ cost

effective to culture the organisms in the laboratory or

obtain them from private, state, or Federal sources.

4. The required QA/QC records, such as the single-laboratory

precision data, would not be available for non

standardized test species.

5. Since it is mandatory that the ident~ty of test organisms

is known to the species level, it would be necessary to

examine each organism caught in the wild to confirm its

identity, which would usually be impractical or, at the

least, very stressful to the organisms.

6. Test organisms obtained from the wild must be observed in

the laboratory for a minimum of one week prior to use, to

ensure that they are free of signs of parasitic or

bacterial infections and other adverse effects. Fish

captured by electroshocking must not be used in toxicity

testing.

6.2.5.2 Guidelines for collection of naturally occurring

organisms-are provided in USEPA, (1973); USEPA, (1990a) and

USEPA, (1993a).

6.2.5.3 Regardless of their source, test organisms and

broodstock 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 s.tages, such

as juvenile mysids and larval fish, is required for all tests."

There may be special cases, however, where the limited '

availability of organisms will require some deviation from the

recommended life stage. In a given test, all organisms should be

approximately the same age and should be taken from the same

source. Since age may affect the results of the tests, it would

enhance the value and comparability of the data if the same

species in the same life stages were used throughout a monitoring

program at a given facility.

32
6.4 LABORATORY CULTURING

6.4.1 Instructions for cUlturini, holding and/6r handling the

reconunended test organisms and broodstock are included in

sp~cified test methods.

6.5 HOLDING AND' HANDLING TEST ORGANISMS

6.5.1 Test organisms should not be subjected to changes of more

than 3°C in water temperature or 3~ in salinity in any 12 h

period.

6.5.2 Organisms should be handled as little as possible. When

handling is necessary, it should be done as gently, carefully,

and quickly as possible to minimize stress. Organisms that are

dropped or touch dry surfaces or are injured during handling must

be discarded. Dipnets are best for handling larger organisms.

These nets are conunercially available or can be made from smallmesh nylon netting, silk bolting cloth, plankton netting, or

similar material. Wide-bore, smooth glass tubes i (4 to 8 nun ID)

with rubber bulbs or pipettors (such ~s a PROPIPETTE@ or other

pipettor) should be used for transferring smaller organisms such

as mysids, and larval fish.

6.5.3 Holding tanks for broodstock are usually supplied with a

good quality water (see Section 5, Facilities, Equipment, and

Supplies) with a flow-through rate of at least two tank-volumes

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

flows through an activated carbon or undergravel filter to remove

dissolved .metabolites Culture water can also bl:; piped through

high intensity ultraviolet light sources for disinfection, and to

photo-degrade dissolved organics. '

6.5.4 Crowding should be avoided because it will stress the

organisms and lower the DO concentrations to unacceptable levels.

The DO must be maintained at a minimum of 4.0 mg/L. 'The

solubility of oxygen depends on temperature, saLLnity, and

altitude. Aerate gently if necessary.

6.5.5 The_organisms should be observed carefully each day for

signs of disease, stress, physical damage, or mortality. Dead

and abnormal organisms should be removed as soon as observed. It

is not unconunon for some larval fish and mysid mortality (5-10%)

to occur during th~ first 48 h in a holding tank because of

individuals that failed to feed and die of starvation.

6.5.6 Organisms in the holding ~anks should generally be fed as

in the cultures (see culturing methods in the respective

methods) .

33
6.5.7 Broodstock and test organisms should be observed carefully

each day for signs of disease, stress, physical damage, and

mortality. Dead and abnormal specimens should be removed as soon

as observed.

6.5.8 A daily record of feeding, behavioral observations, and

mortality should be maintained.

6.6 TRANSPORTATION TO THE TEST SITE

6.6.1 Test organisms and broodstock are transported from the

base or supply laboratory to a remote test site (see the

appropriate test method). Adequate DO is maintained by r~placing

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 fal,l below 4.0 mg/L.

6.6.2 Upon arrival at the test site, organisms are trans,ferred

to receiving water if receiving water is to be used as the test

dilution water. All but a small volume of the holding wa:ter

(approximately 5%) is removed by siphoning, and replaced slowly

over a 10 to 15 minute period with dilution water. If receiving

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

exposing the test organisms to it, because of the possibility

that it might be toxic. For this reason, it is recommended that

only approximately 10% of the test organisms be expos8d 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 transfer~ed to the dilution

water.

?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 b~ 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 The marine organisms may be used at all concentrations of

effluent by adjusting the salinity of the effluent to salinities

specified for the appropriate species test condition or, to the

salinity approximating that of the receiving water, by adding

sufficient dry ocean salts, such as FORTY FATHOMS®, or

equivalent, GP2, .or hypersaline brine.

34
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 accliniated in synthetic

saline water prepared with the dry salts. Caution: addition of

dry ocean salts to dilution water may result iq an increase in

pH. (The pH of estuarine and coastal saline waters is normally

7.5-8.3) .

6.6.6 All effluent concentrations and the control(s) used in a

test should have the same salinity. The change: in salinity upon

acclimation at the desired test dilution should not exceed 6~.

The required salinities for culturing and toxi~ity tests with

estuarine and marine species are listed in the test method

sections.

6.7. TEST ORGANISM DISPOSAL

6.7.1 When the toxicity testes) is concluded, ~ll test organisms

(including controls) should be humanely destroyed and disposed of

in an appropriate manner.

35
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 lar~ely on the objectives of the study.

7.1.1.1 If the objective of the test is to estimate the 'chronic

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

permit-related toxicity testing, a standard dilution water

defined in each test method is used. If the test organisms have

been cultured in water which is different from the test dilution

water, a second set of controls, using culture water, should be

included in the test.

7.1.1.2 If the objective of the test is to estimate the chronic

toxicity of the effluent in uncontaminated natural seawater

(receiving water), or with other uncontaminated natural seawater.

Seasonal variations in the quality of receiving waters may affect

effluent toxicity. Therefore, the salinity of saline receiving

water samples should be determined before each use. If the test

organisms have been cultured in water which is different from the

test dilution water, a second set of controls, using culture

water, should be included in the test.

7.1.1.3 If the objective of the test is to determine the

additive or mitigating effects of the discharge on already

contaminated receiving water, the test is performed using

dilution water consisting of recetving water collected 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

reagent water and reagent grade chemicals (GP2) or commercial sea

salts (FORTY FATHOMS®, HW MARINEMIX®) (Table \3). The source

water for the deionizer can be ground water or tap water. This

synthetic water should be used only if specified in the test

method. These salts may be directly added to' effluents to

achieve appropriate salinities for testing high effluent

concentration (e.g., greater than 60% effluent) where the use of

hypersaline brine is insufficient to obtain test salinities.

7.2.2 REAGENT WATER USED TO PREPARE STANDARD, SYNTHETIC,

DILUTION WATER

36
7.2.2.1 Reagent water is defined as distilled 6r deionized water

that does not contain substances which are toxic to the test

organisms. Deionized water is obtained from a ~1ILLIPORE

MILLI-Q@, MILLIPORE® QPAI{lM2 'or equivalent system. It is

advisable to provide a preconditioned (deionized) feed water by

using a Culligan®, Continental®, or equivalent system in front of

the MILLI-Q® System to extend the life of the MILLI-Q® cartridges

(see Section 5, Facilities, Equipment, and Supplies) .

7.2.2.2 The recommended order of the cartridges in a

four-cartridge deionizer (i.e., MILLI-Q® System 'or equivalent)

is: (1) ion exchange, (2) ion exchange, (3) carbon, and (4)

organic cleanup (such as ORGANEX-Q®, or equivalent), followed by

a final bacteria filter. The QPAI{lM2 water system is a sealed.

system which does not allow for the rearranging of the

cartridges. However, the final cartridge is an .ORGANE'X-Q®

filter, followed'by a final bacteria filter. Commercial

laboratories using this system have not experienced any

difficulty in using the water for culturing or testing.

Reference to the MILLI-Q® systems throughout the remainder of the

manual includes all MILLIPORE® or equivalent systems.

7.2.3 STANDARD, SYNTHETIC SEAWATER

7.2.3.1 To prepare 20 L of a standard, synthetic, reconstituted

seawater (modified GP2), using reagent grade chemicals (Table 2),

with a salinity of 31%0, follow the instructions below. Other

salinities can be prepar~d by making the appropriate dilutions.

Larger or smaller volumes of modified GP2 can be prepared by .

using proportionately larger or smaller amounts of salts and

dilution water.'

1. Place 20 L of MILLI-Q® or equivalent. deionized water in a

properly cleaned plastic carboy.

2. Weigh reagent grade. salts listed in Table: 2 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 1L/h for 24 h.

4. Check the pH and salinity.

7.2.3.2 Synthetic seawater can also be prepared by adding

commercial sea salts, such as FORTY FATHOMS@, HW MARINEMIX®, or

equivalent, to deionized water. For example,. thirty-one parts

per thousand (31%0) FORTY FATHOMS® can be prepared. by dissolving

31 g of sea salts per liter of deionized water~ <The salinity of

the resulting solutions should be checked with a ,refractometer.

37
•

TABLE 2. PREPARATION OF GP2 ARTIFICIAL SEAWATER USING

REAGENT GRADE CHEMICALS 1,2,3

Amount (g)

Compound Concentration Required for

(giL) 20 L

NaCl 21. 03 420.6

Na2S04 3.52 70.4

KCl 0.61 12.2

KBr 0.088 1. 76

Na2B407 . 10 H2O 0.034 0.68

MgC12 • 6 H2O 9.50 190.0

CaC1 2 • 2 H2O 1. 32 26.4

SrCl:! . 6 H2O 0.02 0.400

NaHC03 0.17 3.40

1

3

- ~-

Modified GP2 from Spotte et al. (1984).

The constituent salts and concentrations were taken from

USEPA (1993a). The salinity is 30.89 giL.

GP2 can be diluted with deionized (DI) water to the desired

test salinity.

- 7.2.4 Artificial seawater is to be used only if specified in the

method. The suitability of GP2 as a medium for cUlturing

organisms has not been determined.

7.3 USE OF RECEIVING WATER AS DILUTION WATER

7.3.1 If the objectives of the test require the use of

uncontaminated receiving water as dilution water, and the

receiving water is uncontaminated, it may be possible to collect

a sample of the receiving water close to the outfall, but away

38
from or beyond the influence of the effluent. However, if the

receiving water is contaminated, it may be necessary to collect

the sample in an area "remote" from the dischargE~ site, matching

as closely as possible the physical and chemical ,characteristics

of the receiving water near the' outfall.

7.3.2 The sample should be collected immediately prior to the

test, but never more than 96 h before the test begins. Except

where it is used within 24 h, or .in the case where large volumes.

are required for flow through tests, the sample should be chilled

to 4°C during or immediately following collection, and maintained

at that temperature prior to use in the test.

7.3.3 The investigator should collect uncontaminated water

having a salinity as near as possible to the salinity of the

receiving water at the discharge site. Water should be collected

at slack high tide, or within one hour after high tide. If there

is reason to suspect contamination of the water ih the estuary,

it is advisable to collect uncontaminated water from an adjacent

estuary. At times it may be necessary to collect water at a

location closer to the open sea, where the salinity is relatively

high. In such cases, deionized water or uncontaminated

freshwater is added to the saline water to dilute, it to the

,required test salinity. Where necessary, the salinity of a

surface water can be increased by the addition of artificial sea

salts, such as FORTY FATHOMS@, HW MARINEMIX@, or equivalent, GP2,

a natural seawater of higher salinity, or hypersaline brine.

Instructions for the preparation of hypersaline brine by

concentrating natural seawater are provided below.

7.3.4 Receiving water containing debris or indigenous organisms,

that may be confused with or attack the test organisms, should be

filtered through a sieve having 60 pm mesh openinqs prior to

use.

7.3.5 HYPERSALINE BRINE

7.3.5.1 Most industrial and sewage treatment effluents entering

marine and estuarine systems have little measurable salinity.

Exposur~ of larvae to these effluents Will usually require

increasing the salinity of the test solutions. It is important

to maintain an essentially con_~tant salinity across all

treatments. In some applications it may be desirable to match

the test salinity with that of the receiving water (See Section

7.1). Two salt sources are available to adjust salinities --

artificial sea salts and hypersaline brine (HSB) derived from

natural seawater. Use of artificial sea salts is necessary only

when high effluent concentrations preclude salinity adjustment by

HSB alone.

39
7.3.5.2 Hypersaline brine (HSB) can be made by concentrating

natural seawater by freezing or evaporation. HSB should be made

from high quality, filtered seawater, and can be added to the

effluent or to reagent water to increase salinrty. HSB has

several desirable characteristics for use in effluent toxicity

testing. Brine derived from natural seawater contains the

necessary trace metals, biogenic colloids, and some of the

microbial components necessary for adequate growth, survival,

and/or reproduction of marine and estuarine organisms, and it can

be stored for prolonged periods without any apparent degradation.

However, even if the maximum salinity HSB (100~) is used as a

diluent, the maximum concentration of effluent (O~) that can be

tested is 66% effluent at 34~ salinity. .'

7.3.5.3 High quality (and preferably high salinity) seawater

should be filtered to at least 10 pm before placing into the

freezer or the brine generator. Water should be collected on an

incoming tide to minimize the possibility of contamination.

7.3.5.4 Freeze Preparation of Brine

7.3.5.4.1 A convenient container for making HSB by freezing is

one that has a bottom drain. One liter of brine can be made from

four liters of seawater. Brine may be collected by partially

freezing seawater at -10 to -20 a C until the remaining liquid has

reached the target salinity. Freeze for approximately six hours,

then separate.the ice (composed mainly of fresh water) from the

remaining liq~id (which has now become hypersaline) .

7.3.5.4.2 It is preferable to monitor the water until the target

salinity is achieved rather than allowing total freezing followed

by partial thawing. Brine salinity should never exceed 100~.

It is advisable not to exceed about 70~ brine salinity unless

it is necessary to test effluent concentrations greater than 50%.

7.3.5.4.3 After the required salinity is attained, the HSB

should be filtered through a 1 pm filter and poured directly

into portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and the date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after several months in storage.

7.3.5.5 Heat Preparation of Brine

7.3.5.5.1 The ideal container for making brine using 'heatassisted evaporation of natural seawater is one that (1) ,has a

high surface to volume ratio, (2) is made of anon-corrosive

material, and (3) is easily cleaned (fiberglass containers are

ideal). Special care should be used to prevent any toxic

40
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 is to use a thermostatically controlled heat

exchanger made from fiberglass. If aeration is needed, use only

oil-free air compressors to prevent contaminatic~n.

7.3.5.5.2 Before adding seawater to the brine denerator,

thoroughly clean the generator, aeration supply:tube, heater, and

any other materials that will be in direct cont~ct with the

brine. A good quality biodegradable detergent should be used,

followed by several (at least three) thorough reagent water

rinses.

7.3.5.5.3 Seawater should be filtered to at least 10 pm before

being put into the brine generator. The temperature of the

seawater i~ 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%0 and that the temperature does no": exceed 40°C.

Additional seawater may be added to the brine to:obtain the

volume of brine required.

7.3.5.5.4 After the required salinity is attained, the HSB

should be filtered through a 1 pm filter and poured directly

into portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and the .date the brine

was generated. Containers of HSB should be storE;d in the dark at

4°C (even room temperature has been acceptable). i HSB is usually

of acceptable quality even after several months in storage.

7.3.5.6 Divide the salinity of the HSB by the expected test

salinity to determine the proportion of reagent water to brine.

For example, if the salinity of the brine is 100'~ and the test

is to be conducted at 34%0, 100%0 divided by 34~ = 1.94. Thus,

the proportion is one part brine plus 1.94 reagent water.

7.3.5.8 To make 1 L of seawater at 34%0 salinity from a

hypersaline brine of 100%0, 340 mL of brine and 66':) mL of

reagent water are required.

7.4 USE OF TAP WATER AS DILUTION WATER

7.4.1 The use of tap water in the reconstituting~of synthetic

(artificial) seawater as dilution water is discouraged unless it

is dechlorinated and fully treated. Tap water can be

dechlorinated by deionization, carbon filtration, or the use of

41
sodium thiosulfate. Use of 3.6 mg/L (anhydrous) sodium

thiosulfate will reduce 1.0 mg chlorine/L (APHA,1992).

Following dechlorination, total residual chlorine should not

exceed 0.01 mg/L. Because of the possible toxicity of

thiosulfate to test organisms, a control lacking thiosulfate

should be included in toxicity tests utilizing thiosulfatedechlorinated water.

7.4.2 To be adequate for general laboratory use fpllowing

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, pr algal slime growth and the

problems associated with it. The container should be kept

covered and the contents should be protected from light.

42
SECTION 8

EFFLUENT AND RECEIVING WATER SAMPLING, SAMPLE HANDLING,

AND SAMPLE PREPARATION FOR TOXICITY ,]~ESTS

8 . 1 EFFLUENT SAMPLING.

8.1.1 The effluent sampling point should be the same as that

specified in the NPDES discharge permit (USEPA, 1988b).

Conditions for exception would be: (1) better access to a

sampling point between the final treatment and the discharge

outfall; (2) if the processed waste is chlorinated prior to

discharge, it may also be desirable to take samples prior to

contact with the chlorine to determine toxicity of the

.unchlorinated effluent; or (3) in the event there is a desire to

evaluate the toxicity of the influent to municipal waste

treatment plants or separate wastewater streams in industrial

facilities prior to their being combined with other wastewater

streams or non-contact cooling water, additional sampling points

may be chosen. '

8.1.2 The decision on whether to collect grab or composite

samples is based on the objectives of the test and an

understanding of the short and long-term operations and schedules

of the discharger. If the effluent quality varies considerably

with time, which can occur" where holding times are short, grab

samples may seem preferable because of the ease of collection and

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

the sampling duration of a grab sample is so short that full

characterization of an effluent over a 24-h period would require

a prohibitively large number of separate samples and tests.

Collection of a 24-h composite sample, however, may dilute

toxicity spikes, and average the quality of the E!ffluent over the

sampling period. Sampling recommendations are provided below

(also see USEPA, 1993a). :

I

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 colle~tion should be

recorded.

8.2 EFFLUENT SAMPLE TYPES

8.2.1 The advantages' and disadvantages of effluent grab and

composite samples are listed below:

43
8.2.1.1 GRAB SAMPLES

Advantages:

1. Easy to collect; require a minimum of equipment and

on-site time.

2. Provide a measure of instantaneous toxicity. Toxicity

spikes are not masked by dilution.

Disadvantages:

1. Samples are collected over a very short period of time

and on a relatively infrequent basis. The chances of

detecting a spike in toxicity would depend on the

frequency of sampling, and the probability of missing

spikes is high.

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: I~

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 pamples.

8.3.2 When 7-day tests are conducted off-site, a minimum of

three samples are collected. If these samples are collected on

Test Days 1, 3, and 5, the first sample would be used for'test

initiation, and for test solution renewal on Day 2. The second

sample would be used for test solution renewal on Days 3 and 4.

The third sample would be used for test solution renewal on Days

5, 6, and 7.

8.3.3 Sufficient sample must be collected to perform the

required toxicity and chemical tests. A 4-L (l-gal) CUBITAINER®

will provide sufficient sample volume for most tests.

44
8.3.4 THE FOLLOWING EFFLUENT SAMPLING METHODS ~~E 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 cbntinuously

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.

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 ~ecause 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 dischaige 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

successive8-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) .

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

the discharge of a slug of toxic waste (orie sample is

collected) .

45
8.4 RECEIVING WATER SAMPLING

8.4.1 Logistical problems and difficulty in securing sampling

equipment generally preclude the collection of composite

receiving water samples for toxicity tests. Therefore, based on

the requirements of the test, a single grab sample .or series of

daily grab samples of receiving water is collected for use in the

test.

8.4.2 The sampling point is determined by the objectives of the

test. At estuarine and marine sites, samples should be collected

at mid-depth.

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

receiving water at estuarine and marine effluent sites, receiving

water samples are collected at several distances away from the

discharge. The time required for ~he effluent-receiving-water

mixture to travel to sampling points away from the point of

discharge, and the rate and degree of mixing, may be difficult to

ascertain. Therefore, it may not be possible to correlate

receiving water toxicity with effluent toxicity at the discharge

point unless a dye study is performed. The toxicity of receiving

water samples from five stations in the discharge plume can be

evaluated using the same number of test vessels and test

organisms as used in one effluent toxicity test with five

effluent dilutions.

8.5 EFFLUENT AND RECEIVING WATER SAMPLE HANDLING, PRESERVATION,

AND SHIPPING

8.5.1 Unless the samples are used in an on-site toxicity ~est

the day of collection, it is recommended that they be held -at

approximately 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 residu~l

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 elapsed time

(holding time) from sample collection to first use of the sample

in test initiation must not exceed 36 h. EPA believes that 36 h

46
is adequate time to deTiver the sample to the laboratories

performing the test in most cases. In the isolated cases, where

the permittee can document that this delivery time cannot be met,

the permitting authority can allow an option foi on-site testipg

or a variance for an extension of shipped sample holding time.

The request for a variance in sample holding time, directed to

the USEPA Regional Administrator under 40 CFR 136.3(e), must

include supportive data which show that the toxicity of the

effluent sample is not reduced (e.g., because of volatilization

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

extending the holding time beyond 36 h. However, .in no case

should more than 72 h elapse between collection and first use of

the sample. In static-renewal tests, the original sample may

also be used to prepare test solutions for renewal at 24 hand 48

h after test initiation, if stored at 4°C, with minimum head

space, as described in Paragraph 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 content~ 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 thesa:mple arrives at

the laboratory and is unpacked. Insulating materlal 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 4-1, (I-gal)

CUBITAINERS® or new plastic "milk" jugs. All sample containers

should be rinsed with dilution 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 carriE;rs vary with

the carrier.

47
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

approximately 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 unless a variance has

been granted by the NPDES permitting authority.

8.7 PERSISTENCE OF EFFLUENT TOXICITY DURING SAMPLE SHIPMENT AND

HOLDING

8.7.1 The persistence of the toxicity of an effluent prior to

its use in a toxicity test is of interest in assessing the

validity of toxicity test data, and in determining the possible

effects of allowing an extension of the holding time. Where a

variance in holding time (>36 h, but ~72 h) is requested by a

permittee (See subsection 8.5.4), information on the effects of

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

be obtained by comparing the results of multi-concentration

chronic toxicity tests performed on effluent samples held 36 h

with toxicity test results using the same samples after they were

held for the requested, longer period. The portion of the sample

set aside for the second test must be held under the same

conditions as during shipment and holding.

8.8 PREPARATION OF EFFLUENT AND RECE IVING WATER SAMPLES FOR

TOXICITY TESTS

8.8.1 Adjust the sample salinity to the level appropriate' for

objectives of the study using hypersaline brine or artificial sea

salts.

8.8.2 When aliquots are removed from the sample container, the

head space above the remaining sample should be held to a

minimum. Air which enters a container upon removal of sample

should be expelled by compressing the container before re~losing,

if possible (i.e., where a CUBITAINER® used), or by using an

appropriate discharge valve (spigot).

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

NYLON® sieve having 2 to 4 rom 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 pm

mesh openings. Since filtering may increase the dissolved oxygen

(DO) in an effluent, the DO should be determined prior to

48
filtering. Low dissolved oxygen concentrations wi.ll indicate a

potential problem in performing the test. Caution: filtration may

remove some toxicity.

I

8.8.4 If the samples must be warmed to bring them to the

prescribed test temperature, supersaturation of the dissolved

oxygen and nitrogen may become a problem. To avoid this problem,

the effluent and dilution water are checked with DO probe after

reaching test temperature and, if the DO is greater than 100%

saturation or lower than 4.0 mg/L, based on temperature and

salinity, the solutions are aerated moderately (approximately 500

mL/min) for a few minutes, using an airstone, unti,l the DO is

lowered to 100% saturation (Table 3) or until the DO is within the

prescribed range (~4.0 mg/L). Caution: avoid excessive aeration. I

8.8.4.1 Aeration during the test may alter the results and should

be used only as a last resort' to maintain the required DO.

Aeration can reduce the apparent toxicity of the test solutions by

stripping them of highly volatile toxic substances, or change the

toxicity by altering the pH. However, the DO in the test solution

must not be permitted to fall below 4 .. 0 mg/L.

8.8.4.2 In static tests (non-renewal or renewal) low DOs may

commonly occur in the higher concentrations of wastewater.

Aeration is accomplished by bubbling air through a pipet at the

rate of lqO 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 I is initiated

during the first 8 h of the test, the DO may be exhausted during an

unattended period, thereby invalidating the test.

8.8.5 At a minimum, pH, or salinity, and total residual chlorine

are measured in the undiluted effluent or receiving water, and pH

and salinity are measured in the dilution water. '

8.8.6 Total ammonia is measured in effluent and rE~ceiving 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 valqe obtained from

Table 4,

49
•

TABLE 3. OXYGEN SOLUBILITY (MG/L) IN WATER AT EQUILIBRIUM

WITH AIR AT 760 MM HG· (AFTER RICHARDS AND CORWIN,

1956)

TEMP SALINITY (to)

o 5 10 15 20 25 30 35 40 43

0 14.2 13.8 13.4 12.9 12.5 12.1 11. 7 11.4 10.8 10.6

1 13.8 13.4 13.0 12.6 12.2 11. 8 11. 4 11. 0 10.6 10.3

2 13.4 13.0 12.6 12.2 11. 9 11.5 11.1 10.7 10.3 10.0

3 13.1 12.7 12.3 11. 9 11. 6 11.2 10.8 10.4 10.0 9.8

4 12.7 12.3 12.0 11. 6 11.3 10.9 10.5 10.1 9.8 9.5

5 12.4 12.0 11. 7 11. 3 11. 0 10.6 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 11.5 11.2 10.8 10.5 10.2 9.8 9.5 9.2 8.9 8.7

10 10.9 10.7 10.3 10.0 9.7 9.4 9.1 8.8 8.5 8.3

12 10.5 10.2 9.9 9.6 9.3 9.0 8.7 8.4 8.1 7.9

14 10.0 9.7 9.5 9.2 8.9 8.6 8.3 8.1 7.? 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

'M

50
_______________________1 _

TABLE 4.

pH

PERCENT UNIONIZED NH3 IN AQUEOUS AMMONIA SOLUTIONS:

TEMPERATURE 15-26°C AND pH 6.0-8.91

TEMPERATURE (OC)

15 16 17 18 19 . 20 21 22 23 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 2.0,7 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.:n 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 4.71; 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.% 9.56 10.::: 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 (1985a) .

------_..._---------------------- 51
under the appropriate pH and temperature, and multiplying it by the

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

8.8.7 Effluents and receiving waters can be dechlorinated using

6.7 mg/L anhydrous sodium thiosulfate to reduce 1 mg/L chlorine

(APRA, 1992). Note that the amount of thiosulfate required to

dechlorinate effluents is greater than the amount needed to

dechlorinate tap water, (see Section 7, Dilution Water). Since

thiosulfate may contribute to sample toxicity, a thiosulfate,

control should be used in the test in addition to the normal

dilution water control.

8.8.8 The DO concentration in the samples should be near

saturation prior to use. Aeration will bring the DO and other

gases into equilibrium with air, minimize oxygen demand, and

stabilize the pH. However, aeration during collection, transfer,

and preparation of samples should be 'minimized to reduce the loss

of volatile chemicals.

8.8.9 Mortality or impairment of growth or reproduction due to pH

alone may occur if the pH of the receiving water sample falls

outside the range of 7.5 - 8.5 for marine. Thus, the presence of

other forms of toxicity (metals and organics) in the sample may be

masked by the toxic effects of low or high pH. The question about

the presence of other toxicants can be answered only by performing

two parallel tests, one with an adjusted pH, and one without an

adjusted pH. Freshwater samples are adjusted to pH 7.0, and marine

samples are adjusted to pH 8.0, by adding 1N NaOH or 1N Hel

dropwise, as required, being careful to avoid overadjustment.

8.9 PRELIMINARY TOXICITY RANGE-FINDING TESTS

8.9.1 USEPA Regional and State personnel generally have observed

that it is not necessary to conduct a toxicity range-finding test

prior to initiating a static, chronic, definitive toxicity test.

However, when preparing to perform a static test with a sample of

completely unknown quality, ,or before initiating a flow-through

test, it is advisable to conduct a preliminary toxicity rangefinding test. '

8.9.2 A toxicity range-finding test ordinarily consists of a downscaled, abbreviated static acute test in which groups of five

organisms are exposed to several widely-spaced sample dilutions in

a logarithmic series, such as 100%, 10.0%, 1.00%, and 0.100%, and a

control, for 8-24 h. Caution: if the sample must also be used for

the full-scale definitive test, the 36-h limit on holding time (see

Subsection 8.5.4) must not be exceeded before the definitive test

is initiated.

52
8.9.3 It should be noted that the toxicity of a sample observed in

a range-finding test may be significantly different from the

toxicity observed in the follow-up, chronic, definitive test

because: (1) the definitive test may be longer; 'and (2) the test

may be performed with a sample collected at a different time, and

possibly differing significantly in the level of toxicity.

8.10 MOLTICONCENTRA'1'ION (DEFINITIVE) EFFLUENT 'J?OXICITY TESTS

8.10.1 The tests recommended for use in determining discharge

permit compliance in the NPDES program are multiconcentration or

definitive tests. These tests provide a statistical measure of

effluent toxicity, defined as mortality, fertilization, growth,

and/or development. The tests may be static-renewal or static nonrenewal.

8.10.2 The tests consist of a control and a minimum of five

effluent concentrations commonly selected to approximate a

geometric series, s.uch as 60%, 30%, 15%, 7.5%, and 3.75%, using a

~O. 5 dilution series. '.

8.10.3 These tests are also to be used in determining compliance

with permit limits on the mortality of the receiving water

concentration (RWC) of effluents by bracketing the RWC with

effluent concentrations in the following manner. For example, if

the RWC is >25% then, 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%. If the RWC is <25% ef:fluentthe

concentrations may be: (1) 4 times the RWC, (2) 2 times the RWC,

(3) RWC/2, and (4) RWC/4.

8.10.4 If acute/chronic ratios are to be determined by

simultaneous acute and short-term chronic tests with a single

species, using the same sample, . both types of tests must use the

same test conditions, i.e., pH, temperature, salinity, etc.

8.11 RECEIVING WATER TESTS

8.11.1 Receiving water toxicity.tests generally consist of 100%

receiving water and a control. The salinity of the control should

be comparable to the receiving water.

8.11.2 The data from the two treatments are analyzed by hypothesis

testing to determine if test organism survival, fertilization,

growth or development in the receiving water differs significantly

from the control. Four replicates and 10'organisms per replicate

are required for each treatment (see Summary of Test Conditions and

Test Acceptability Criteria in the specific test method) .

53
8.11.3 In cases where the objective of the test is to estimate the

degree of toxicity of the receiving water, a definitive,

multiconcentration test is performed by preparing dilutions of the'

receiving water, using a 0.5 dilution series, with a suitable

control water.

54
-SECTION 9

CHRONIC TOXICITY TEST ENDPOINTS ANDDA~~ ANALYSIS

9.1 ENDPOINTS

9.1.1 The objective of'chronic aquatic toxicity tests with

effluents and pure compounds is to estimate the highest" "safe" or

"no-effect concentration" of these substances. 'E'or practical

reasons, the responses observed in these tests are usually iimited

to survival, fertilization, germination, growth and larval

development and the results of the tests are usually expressed in

terms of the highest toxicant concentration that"has-no . .

statistically significant observed effect on these responses, when

compared to the controls. The terms ~urrently used to define the

endpoints employed in the rapid, chronic andsub--chronic toxicity

tests have been derived from the terms previously used for full

life-cycle tests. As shorter chronic tests were developed, it

became common practice to apply the same terminology to 'the "

endpoints. The terms used in this manual are as follows:

9.1.1.1 Safe Concentration - The highest concentration·,of toxicant

that will ermit normal propagation of fish and other aquatic life

in receiving waters. The concept of a "safe concentration" is a

biological concept, whereas the "no-observed-effEict concentration"

(below) is a statistically defined concentration. 1._ .07

9.1.1.2 No-Observed-Effect-Concentration (NOEC) - The highest

concentration of toxicant to which organisms.are exposed~ina full

life-cycle or partial life-cycle (short-term) test, that dau~e~ no

observable adverse effects on the test organisms (i.e., the high.est

concentration of toxicant in which the values for,the observed

responses are not statistically significantly dif'feren't from the'

controls). This value is used, along with -other factors, to

determine toxicity ~imits in permits.

9.1.1.3 Lowest-Observed-Effect-Concentration (LOEC) _. The lowest

concentration of toxicant to which organisms are·exposed in a lifecycle or .

partial life-cycle (short-term) test, which causes adverse effects

on the test organisms' (i. e., where the values for the observed

responses are statistically significantly different from the

controls).

9.1.1.4 Effective Concentratlon (EC) - A point estimate of the

toxicant concentration that would cause an observable adverse

affect on a quantal, "'111 or nothing," response (such as death,

fertilization, germination or, development) in a given percent of

the test organisms, calculated by point estimation techniques. If

55
analysis alone, unless (1) the assumptions of a strict threshold

model are accepted, and (2) it is assumed that the amount of

adverse effect present at the threshold is statistically detectable

by hypothesis testing. In this case, estimates obtained from a

statistical analysis are indeed estimates of a "no-effect"

concentration. If the assumptions are not deemed tenable, then

estimates from a statistical analysis can only be used in

conjunction with an assessment from a biological standpoint of, what

magnitude of adverse effect constitutes a "safe" concentration. In

this instance, a "safe" concentration is not necessarily a truly'

"no-effect" concentration, but rather a concentration at which the

effects are judged to be of no biological significance.

9.2.5 A better understanding of the relationship between endpoints

derived by hypothesis testing (NOECs) and point estimation

techniques (LCs, ICs, and ECs) would be very helpful in choosing

methods of data analysis. Norberg-King (1991) reported that the

IC25s were comparable to the NOECs for 23 effluent and reference

toxicant data sets analyzed. The d~ta sets' included short-term

chronic toxicity tests for the sea urchin; Arbacia punctulata, the

sheepshead minnow, Cyprinodon variegatus, and the red macroalga,

Champia parvula. Birge et al. (1985) reported that LCls derived

from Probit Analyses of data from short-term embryo-larval t'ests

with reference toxicants were comparable to NOECs for several

organisms. Similarly, USEPA (1988d) reported that the IC25s were

comparable to the NOECs for a set of daphnia,' Ceriodaphnia dilbia

chronic tests with a single reference toxicant. However, the scope

of these comparisons was very limited, and sufficient information

is not yet available to establish an overall relationship between

these two types of endpoints, especially when derived from effluent

toxicity test data.

9.3 PRECISION

9.3.1 HYPOTHESIS TESTS

9.3.1.1 When hypothesis tests are used to analyze toxicity test

data, it is not possible to express precision in terms of a

commonly used statistic. The results of the test are given in

terms of two endpoints, the No-Observed-Effect Concentration (NOEC)

and the Lowest-Observed-Effect Concentration (LOEC). The NOEC and

LOEC are limited to the concentrations selected for the test. The

width of the NOEC-LOEC interval 'is a function of the dilution

series, and differs greatly depending on whether a dilution factor

of 0.3 or 0.5 is used in the test design. Therefore, USEPA

recommends the use of the ~0.5 dilution factor (see Section '4,

Quality Assurance). It is not possible to place confidence limits

on the NOEC and LOEC derived from a given test, and it is difficult

to quantify the precision of the ~OEC-LOEC endpoints between tests.

If the data from a series of tests performed with the-same

58
toxicant, toxicant concentrations, and test species, were analyzed

with hypothesis tests, precision could only be assessed by a

qualitative comparison"of the NOEC-LOEC intervals, with the

understanding that maximum precision would be attained if all tests

yielded the same NOEC-LOEC interval. In practice, the precision of

results of repetitive chronic tests is considered acceptable if the

NOECs vary by no more than one concentration interval above or

below a central tendency. Using-these guidelines, the "normal"

range of NOECs from toxicity tests using a 0.5 dilution factor

(two-fold difference between adjacent concentrations), would be·

four-fold.

9.3.2 POINT ESTIMATION TECHNIQUES

9.3.2.1 Point estimation techniques have the advantage of

providing a point estimate of the toxicant concentration causing a

given amount of adverse (inhibiting) effect, the precision of which

can be quantitatively assessed (1) within tests by calculation of

95% confidence limits, and (2) across tests by calculating a

standard deviation and coefficient of variation.

9.4 DATA ANALYSIS

9.4.1 ROLE OF THE STATISTICIAN

9.4.1.1 The use of the statistical methods described in this

manual for routine data analysis does not requirE~ the assistance of

a statistician. However, the int~rpretation of the results of the

analysis of the data from any of the toxicity tests described in

this manual can become problematic because of the inherent

variability and sometimes unavoidable anomalies in biological data.

If the data appear unusual in any way, or fail to meet the

necessary assumptions, a statistician should be consulted.

Analysts who are not proficient in statistics are strongly advised

to seek the assistance of a statistician before selecting the

method of analysis and using any of the results.

9.4.1.2 The statistical methods recommended in this manual are not

the only possible methods of statistical analysis. Many other

methods have been proposed and considered. Certainly there are

other reasonable and defensible methods of statistical analysis for

this kind of toxicity data. Among alternative hypothesis tests

some, like Williams' Test, require additional assumptions, while

others, like the bootstrap methods, require computer~intensive

computations. Alternative point estimation approaches most

probably would require the services of a statistician to determine

the appropriateness of the model (goodness of fit), higher order

linear or nonlinear models, confidence intervals for estimates

generated by inverse regression, etc. In addition, point

estimation or regression approaches would requirE! the specification

59
by biologists or toxicologists of some low level of adverse effect

that would be deemed acceptable or safe. The statistical methods

contained in this manual have been chosen because they are (1)

applicable to most of the different toxicity test data sets for

which they are recommended, (2) powerful statistical tests, (3)

hopefully "easily" understood by nonstatisticians, and (4) amenable

to use without a computer, if necessary.

9.4.2 PLOTTING THE DATA

9.4.2.1 The data should be plotted, both as a preliminary step to

help detect problems and unsuspected trends or patterns in the

responses, and as an aid in interpretation of the results. Further

discussion and plotted sets of data are included in the methods and

the Appendices.

9.4.3 DATA TRANSFORMATIONS

9.4.3.1 Transformations of the data, (e.g., arc sine square root

and logs), are used where necessary to meet assumptions of the

proposed analyses, such as the requirement for normally distributed

data.

9.4.4 INDEPENDENCE, RANDOMIZATION, AND OUTLIERS

9.4.4.1 Statistical independence among observations is a critical

assumption in all statistical analysis of toxicity data. One of

the best ways to ensure independence is to properly follow rigorous

randomization procedures. Randomization techniques should be

employed at the start of the test, including the randomization of

the placement of test organisms in the test chambers and

randomization of the test chamber location within the array of

chambers. Discussions of statistical independence, outliers and

randomization, and a sample randomization scheme, are included in

Appendix A.

9.4.5 REPLICATION AND SENSITIVITY

9.4.5.1 The number of replicates employed for each toxicant

concentration is an important factor in determining the sensitivity

of chronic toxicity tests. Test sensitivity generally increases as

the number of replicates is increased, but the point of diminishing

returns in sensitivity maybe reached rather quickly. The level of

sensitivity required by a hypothesis test or the confidence

interval for a point estimate will determine the number of

replicates, and should be based on the objectives for obtaining the

toxicity data.

9.4.5.2 In a statistical analysi~ of toxicity data, the choice of

a particular analysis and the ability to detect departures from the

assumptions of the analysis, such as the normal distribution of the

60
data and homogeneity of variance, is also dependent on the number

of replicates. More than the minimum number of replicates may be

required in situations where it is imperative to obtain optimal

statistical results, such as with tests used in E~nforcement cases

or when it is not possible to repeat the tests. For example, when

the 'data are analyzed by hypothesis testing, thenonparametric

alternatives cannot be used unless there are at least four

replicates at ~achtoxicant concentration.

9.4.6 RECOMMENDED ALPHA LEVELS

9.4.6.1 The data analysis examples included in the manual specify

an alpha level of 0.01 for testing the assumptions of hypothesis

tests and an alpha level of 0.05 for the hypothesis tests

themselves. These levels are common and well accepted levels for

this type of analysis and are presented as a recommended minimum

,significance level for toxicity data analysis. .'

9.5 CHOICE OF ANALYSIS

9.5.1 The recommended statistical analysis of most data from

chronic toxicity tests with aquatic organisms follows a decision

process illustrated in the flowchart in Figure 2. An initial

decision is made to use point estimation techniques (Probit

Analysis, the Spearman-Karber Method, the Trimmed' Spearman-Karber,

the Graphical Method or Linear Interpolation Method) and/or to use

hypothesis testing (Dunnett's Test, the t test with the Bonferroni

adjustment, Steel's Many-one Rank Test, or Wilcoxbn Rank Sum Test).

If hypothesis testing is chosen, subsequent decisions are made on

the appropriate procedure for a given set of data, depending on the

results of tests of assumptions, as illustrated in the flowchart.

A specific flow chart is included in the analysis section for each

test",

9.5.2 Since a single chronic toxicity test might; yield information

on more than one parameter (such as survival, growth, and

development), the lowest estimate of a "no-observ~::ld-effect

concentration" from any of the responses would be used as the

"no-observed-effect concentration" for each test., It follows

logically that in the statistical analysis of the data,

concentrations that had a significant toxic effect on one of the

observed responses would not be subsequently tested for an effect

on some other response. This is one reason for excluding

concentrations that have shown a statistically siqnificant

reduction in survival from a subsequent hypothesis test for effects

on another parameter such as growth. A second reason is that the

exclusion of such concentrations usually results :~n a more powerful

and appropriate statistical analysis. In performJcngthe point

estimation techniques recommended in this manual, ,i an all-data

61
I DATA (SURVIVAL, GROWTH, REPRODUCTION, ETC.) I

I

, I HYPOTHESIS TESTING' I POINT

ESTIMATION t

TRANSFORMATION? I "

+ ENDPOINT ESTIMATE

LC, EC,le I SHAPIRO-WILK'S TEST I NON-NORMAL DISTRIBUTION

I

NORMAL DISTRIBUTION

+ HETEROGENEOUS

VARIANCE

BARTLETI'S TEST ..

- HOMOGENEOUS

" VARIANCE

NO STATISTICAL ANALYSIS NO 40R MORE

RECOMMENDED REPLICATES?

-- YES

, , .-- EQUAL NUMBER OF EQUAL NUMBER OF

REPLICATES? REPLICATES?

NO t YES YES t

NO

, " T-TESTWITH DUNNETI'S STEEL'S MANY-ONE WILCOXON RANK SUM

BONFERRONI TEST WITH

ADJUSTMENT TEST RANK TEST BONFERRONIADJUSTMENT

I 'I

ENDPOINT

•

ESTIMATES

NOEC, LOEC .

Figure 2. Flowchart for statistlcal analysis of test data.

62
approach is used. For example, data from concentrations above the

NOEC for survival are included in determining ICp estimates

usingthe Linear Interpolation Method.

9.5.3 ANALYSIS OF GROWTH DATA

9.5.3.1 Growth data from the topsmelt, Atherin~ps affinis, mysid,

Holmesimysis costata, survival and growth tests, and the giant

kelp, Macrocystis pyriferia, germination and germ-tube length test,

are analyzed using hypothesis testing according to the flowchart in

Figure 2. The above mentioned growth data may also be analyzed by

generating a point estimate with the Linear Interpolation Method.

Data from effluent concentrations that have tested significantly

different from the control for survival are excluded from further

hypothesis tests concerning growth effects. Grm"th is defined as

the change in dry weight of the orginal number of test organisms

when group weights are obtained. When analyzing the data using

point estimating techniques, data from all concentrations are

included in the analysis.

9.5.4 AN~YSIS OF FERTILIZATION, GERMINATION ANp DEVELOPMENT DATA

9.5.4.1 Data from the purple urchin, Strongylooentrotus purpuratus

'and the sand dollar, Denstraster excentricus, fe:ttilization test

and development test; the red abalone Haliotis rufescens, the

Pacific oyster, Crassostrea gigas, and mussel, ~vtilus spp., larval

development tests; and the giant kelp, Macrocyst.is pyrifera,

germination test may be analyzed by hypothesis b~sting after an arc

sine transformation according to the flowchart in Figure 2. The

fertilization, larval development or germination data may also be

analyzed by generating a point estimate with the Linear

Interpolation Method.

9.5.5 ANALYSIS OF MORTALITY DATA

9.5.5.1 Mortality data are analyzed by Probit ~lalysisi 'if

appropriate, or other point estimation techniques, (i.e!, the

Spearman-Karber Method, the Trimmed Spearman-Karber Method, or the . . I ' • - "

Graphical Method) (see Appendices G-I) (see discussion below). The

mortality data can also be analyzed by hypothesis testing, after an

arc sine square root transformation (see Appendices B-F), according

to the flowchart in Figure 2.

9.6 HYPOTHESIS TESTS

9.6.1 DUNNETT'S PROCEDURE

9.6.1.1 Dunnett's Procedure is used to determinE~ the NOEC. The

procedure consists of an analysis of variahce (~rO~A) to determine

the error term, which is then used in a multiple comparison

63
procedure for comparing each of the treatment means with the

control mean, in a series of paired tests (see Appendix C). Use of

Dunnett's Procedure requires at least three ,replicates per

treatment to check the assumptions of the test. In cases where the

numbers of data points (replicates) for each concentration are not

equal, a t test may be performed with Bonferroni's adjustment for

multiple comparisons (see Appendix D), instead of using Dunnett's

Procedure.

9.6.1.2 The assumptions upon which the use of Dunnett's Procedure

is contingent are that the observations within treatments are

normally distributed, with homogeneity of variance. Before

analyzing the data, these assumptions must be tested using the

procedures provided in Appendix B.

9.6.1.3 If, after suitable transformations have been carried out,

the normality assumptions have not been met, Steel's Many-one Rank

Test should be used if there are four or more data points

(replicates) per toxicant concentration. If the numbers of,data

points for each toxicant concentration are not equal, the Wilcoxon

Rank Sum Test with Bonferroni's adjustment should be used (see

Appendix F). .

9.6.1.4 Some indication of the sensitivity of the analysis should

be provided by calculating (1) the minimum difference between means

that can be detected as statistically significant, and (2) the

percent change from the control mean that this minimum difference

represents for a given test.

9.6.1.5 A step-by-step example of the 'use of Dunnett's Procedure

is provided in Appendix C.

9.6.2 T TEST WITH THE BONFERRONI ADJUSTMENT

9.6.2.1 The t test with the Bonferroni adjustment is used as an

alternative to Dunnett's Procedure when the number of replicates is

not the same for all concentrations. This test sets an upper bound

of alpha on the overall error rate, in contr~~t to Dunnett's

Procedure, for which the overall error rate i,s fixed at alpha.

Thus, Dunnett's Procedure is a more powerful test.

9.6.2.2 The assumptions upon which the use of the t test with the

Bonferroni adjustment is contingent are that the observations

within treatments are normally distributed, with homogen~~ty of

variance. These assumptions must be tested using the procedures

provided in Appendix B.

9.6.2.3 The estimate of the safe concentration derived from this

test is reported in terms of the NOEC. A step-by-step example of

the use of a t-test with the Bonferroni adjustment is provided in

Appendix D.

64
9.6.3 STEEL'S MANY-ONE RANK TEST

9.6.3.1 'Steel's Many-one Rank Test is a multiple comparison

procedure for comparing several treatments with a control. This

method is similar to Dunnett's procedure, except that it is not

necessary to meet the assumption of normality. The data are

ranked, and the 'analysis is performed 'on the ranks rather than on

the data themselves. If the data are normally or nearly normally

distributed, Dunnett's Procedure would be more sensitive (would

detect smaller differences between the treatments and control) . For data that are not normally distributed, Steel's Many-one Rank

Test can be much more efficient ,(Hodges and Lehmann, 1956).

9.6.3.2 It is necessary to have at least four replicates per

toxicant concentration to use Steel's test. Unlike Dunnett's

procedure, the sensitivity of this test cannot be stated in terms

of the minimum difference between treatment means and the control

mean that can be detected as statistically significant.

9.6.3.3 The estimate of the safe concentration i~ reported as the

NOEC. A step-by-step example of the use of Steel's Many":'One Rank

Test is provided in Appendix E.

9.6.4 WILCOXON RANK SUM TEST

9.6.4.1 The Wilcoxon Rank Sum 'rest is a noriparametric test for

comparing a treatment with a control. The data are ranked and the

analysis proceeds exactly as in Steel's Test except that

Bonferroni's adjustment for multiple comparisons is used instead of

Steel's tables. ,When Steel's test can be used (i.e., when there

are equal numbers of data points per toxicant concentration), it

will' be more powerful (able to detect smaller dif:ferences as

statistically significant) than the Wilcoxon Rank Sum Test with

Bonferroni's adjustment.

9.6.4.,2 The estimate of the safe concentration i:3 reported as the

NOEC. A step-by-step example of the use of the Wilcoxon Rank Sum

Test is provided in Appendix F.

9.6.5 A CAUTION IN THE USE OF HYPOTHESIS TESTING

9~6.5.1 If in the calculation of an NOEC by hypothesis testing,

two tested concentrations cause statistically significant adverse

effects, but an intermediate concentration did not cause

statistically significant effects, the results should be used with

extreme caution.

65
9.7 POINT ESTIMATION TECHNIQUES

9.7.1 PROBIT ANALYSIS

9.7.1.1 Probit Analysis is used to estimate an LC or EC value and

the associated 95% confidence interval. The ~nalysis consi~ts of

adjusting the data for mortality in the control, and then using a

maximum likelihood technique to estimate the parameters of the

underlying log tolerance distribution, which is assumed to have a

particular shape.

9.7.1.2 The assumption upon which the use of Probit Analysis is

contingent is a normal distribution of log tolerances. If the

normality assumption is not met, and at least two partial

mortalities are not obtained, Probit Analysis should not be used.

It is important to check the results of Probit Analysis to

determine if use of the analysis is appropriate. The chi-square

test for heterogeneity provides a good test of appropriateness of

the analysis. The computer program (see discussion, Appendix H)

checks the chi-square statistic calculated for the data set against

the tabular value, and provides an error message if the calculated

value exceeds the tabular value.

9.7.1.3 A discussion of Probit Analysis, and examples of computer

program input and output, are found in Appendix H.

9.7.1.4 In cases where Probit Analysis is not appropriate, the

LeSO and confidence interval may be estimated by the

Spearman-Karber Method (Appendix I) or the trimmed Spearman-Karber

Method (Appendix J). If a test results in 100% survival and 100%

mortality in adjacent treatments (all or nothing effect), the LC50

may be estimated using the Graphical Method (Appendix K) .

9.7.2 LINEAR INTERPOLATION METHOD

9.7.2.1 The Linear Interpolation Method (see Appendix L) is a

procedure to calculate a point estimate of the effluent or other

toxicant concentration [Inhibition Concentration, (IC)] that causes

a given percent reduction (e.g., 25%, 50%, e~c.) in the

reproduction or growth of the test organisms. The procedure was

designed for general applicability in the analysis of data from

short-term chronic toxicity tests.

9.7.2.2 Use of the Linear Interpolation Method is based on the .. assumptions that the responses (1) are monotonically non-increasing

(the mean response for each higher concentration is less than or

equal to the mean response for the previous concentration), (2)

follow a piece-wise linear response function, and (3) are from a

random, independent, and representative sample of test data. The

assumption for piece-wise linear response cannot be tested

66
statistically, and no defined statistical procedure is provided to

test the assumption for monotonicity. Where the observed means are

not strictly monotonic by examination, they are adjusted by

smoothing. In cases where the responses at the low toxicant

concentrations are much higher than in the controls, the smoothing

process may result in a large upward adjustment in the control

mean.

9~7.2.3 The inability to test the monotonicity an'd piece wise

linear response assumptions for this method makes .it difficult to

assess when the method is, or is not, producing_reliable results.

Therefore, the method should be used with caution when the results

of a toxicity test approach an "all or nothing" response from one

concentration to the next in the conce.ntration series, and when it

appears that there is a large deviation from monotonicity. See

Appendix L for a more detailed discussion of the use of this method

and a computer program available for performing calculations.

67
SECTION 10

REPORT PREPARATION

The toxicity data are reported, together with other appropriate

data. The following general format and content are recommended for

the report:

10.1 INTRODUCTION

1. Permit number

2. Toxicity testing requirements of permit

3. Plant location

4. Name of receiving water body

5. Contract Laboratory (if the test was performed

under contract)

a. Name of firm

b. Phone number

c. Address

10.2 PLANT OPERATIONS

1. Product (s)

2. Raw materials

3. Operating schedule

4. Description of waste'treatment

5. Schematic of waste treatment

6. Retention time (if applicable)

7. Volume of waste flow (MGD, CFS, GPM)

8. Design flow of treatment facility at time of sampling

10.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION WATER

l.

a.

b.

c.

d.

e.

f.

g.

Effluent Samples

Sampling point

Collection dates and times

Sample collection method

Physical and chemical data

Mean daily discharge on sample collection date

Elapsed time from sample collection to delivery

Sample temperature when received at the laboratory

68
Dilution Water Samples

Source

Collection date and time

Pretreatment

Physical and chemical characteristics,

2.

a.

b.

c.

d.

e.

f.

g.

3.

a.

b.

c.

d.

Receiving Water Samples

Sampling point

Collection dates and times

Sample collection method

Physical and chemical data

Tide stages

Sample, temperature when received at

Elapsed time from sample collection

'I

the ,laboratory

to delivery

10.4 TEST METHODS

1. Toxicity test method used (title, number, source)

2. Endpoint(s) of test

3. Deviation (s) from reference method, if any" and the

reason(s)

4. Date and time test started

5. Date and time test terminated

6. Type of volume and test chambers

7. Volume of solution used per chamber

8. Number of organisms used per test chamber

9. Number of replicate test chambers per treatment

10. Acclimation of, test organisms (temperature ,land salinity

mean and range)

11. Test temperature (mean and rahge)

12. Specify if aeration was needed

13. Feeding frequency, and amount and type of food

14. Test salinity (mean and range)

10.5 TEST ORGANISMS

1. Scientific name and how determined

2. Age

3. Life stage

4. Mean length and weight (where applicable)

5. Source

6. Diseases and treatment (where applicable)

7. Taxonomic key used for species identificatHm

69
10.6 QUALITY ASSURANCE

1. Reference toxicant used routinely; source

2. Date and time of most recent reference toxicant test; test

results and current control (cusum) chart

3. Dilution water used in reference toxicant test

4. Results (NOEC or, where applicable, LOEC, LC50, IC or EC

value)

5. Physical and chemical methods used

10 • 7 RESULTS

1. Provide raw toxicity data in tabular form, including daily

records of affected organisms in each concentration

(including controls), and plots of toxicity data

2. Provide table of the statistical endpoints; LC50s,NOECs,

Ee or IC value, etc.

3. Indicate statistical methods used to calculate endpoints

4. Provide summary table of physical and chemical data

5. Tabulate QA data

10.8 CONCLUSIONS AND RECOMMENDATIONS

1. Relationship between test endpoints and permit limits.

2. Action to be taken.

70
SECTION 11

TOPSMELT, Atherinops affinis

7-DAY LARVAL GROWTH AND SURVIVAL TEST :METHOD

Adapted from a method developed by

Brian S. Anderson, John W. Hunt, Sheila Turpen,

Hilary R. McNulty, and Matt A. Engl-Lmd

Institute of Marine Sciences, University of California

Santa Cruz, California

(in association with)

California Department of Fish and G,ame

Marine Pollution Studies Laboratory

34500 Coast 'Route I, Monterey, CA 93940

TABLE OF CONTENTS

Appendix I

11.1

11.2

11.3

11.4

11.5

11.6

11. 7

11. 8

11. 9

11.10

11.11

11.12

11.13

11.14

Scope and Application

Summary of Method

Interferences

Safety

Apparatus and Equipment

Reagents and Supplies

Effluents and Receiving Water Collection,

Preservation, and Storage

Calibration and Standardization

Quality Control

Test Procedures

Summary of Test Conditions and Test

Acceptability Criteria

Acceptability of Test Results

Data Analysis

Precision and Accuracy

Step~by Step Summary

71
SECTION 11

TOPSMELT, ATHERINOPS AFFINIS

LARVAL SURVIVAL AND GROWTH TEST

11.1 SCOPE AND APPLICATION

11.1.1 This method estimates the chronic toxicity of effluents

and receiving waters to the topsmelt, Atherinops affinis, using

nine-to-fifteen day old larvae in a seven-day, static-renewal

exposure test. The effects include the synergistic,

antagonistic, and additive effects of all chemical, physical, and

biological components which adversely affect the physiological an

biochemical functions of the test organisms.

11.1.2 Daily observations of mortality "make it possible to also

calculate acute toxicity for desired exposure periods (i.e., 24-

h, 48-h, 96-h LC50s) .

11.1.3 Detection limits of the toxicity of an effluent or

chemical substance are organism dependent.

11.1.4 Brief excursions in toxicity may "not be detected using

24-h composite samples. Also, because of the long sample

collection period involved in composite sampling and because the

test chambers are not sealed, highly volatile and highly

degradable toxicants in the source may not be detected in the

test.

11.1.5 This method is commonly used in one of two forms: (1) a

definitive test, consisting of a minimum of five effluent

concentrations and a control, and (2) a receiving water test(s),

consisting of one or more receiving water concentrations and a

control.

11.1.6 This method should be restricted to use by, or under the

supervision of, professionals experienced in aquatic toxicity

testing. Specific experience with any toxicity test is usually

needed before acceptable results become routine.

11.2 SUMMARY OF METHOD

72
11.2.1 This method provides step-by-step instructions for

performing a 7-day static-renewal toxicity test using survival

and growth of topsmelt larval fish to determine the toxicity of

substances in marine and estuarine waters. The test endpoints

are survival and growth.

1.3 INTERFERENCES

11.3.1 Toxic substances may be introduced by contaminants in

dilution water, glassware, sample hardware, and testing equipment

(see Section 5/ Facilities, Equipment, and Supplies) .

,

11.3.2 Improper effluent sampling and handling may adversely

affect test results (see Section 8/ Effluent and Receiving Water

Sampling and Sample Handling, and Sample Preparation for Toxic~ty

Tests) .

11.3.3 Pathogenic 'and/or predatory organisms in the dilution

water and effluent may affect test organism survival, and

confound test results.

11.3.4 Food added during the test may sequester metals and other

toxic substances and confound test results.

11.4 SAFETY

11.4.1 See Section 3/ Health and Safety.

11.5 APPARATUS AND EQUIPMENT

11.5.1 Tanks, trays, or aquaria -- for holding and acclimating

topsmelt, e.g., standard salt water aquarium or Instant Ocean

Aquarium (capable of maintaining seawater at 10-:WOC) / with

appropriate filtration and aeration system. (See Anderson et

al., 1994/ Middaugh and Anderson, 1993).

11.5.2 Air pump, air lines, and air stones -- for aerating water

containing broodstock or for supplying air to test solutions with

low dissolved oxygen.

73
11.5.3 Constant temperature chambers or water baths -- for

maintaining test solution temperature and keeping dilution ~ater

supply, and larvae at test temperature (20°C) prior to the test.

11.5.4 Water purification system -- Millipore Super-Q, Deionized

water (DI) or equivalent.

11.5.5 Refractometer

11.5.6 Hydrometer(s)

for determining salinity.

for calibrating refractometer.

11.5.7 Thermometers, glass or electronic, laboratory grade

for measuring water temperatures.

11.5.8 Thermometer, National Bureau of Standards eertified (see

USEPA METHOD 170.1, USEPA, 1979) -- to calibrate laboratory

thermometers.

11.5.9 pH and DO meters -- for routine physical and chemical

measurements.

11.5.10 Standard or micro-Winkler apparatus -- for determining

DO (optional) and calibrating the DO meter.

11.5.11 Win~ler bottles -- for .dissolved oxygen determinations.

11.5.12 Balance -- Analytical, capable of accurately weighing to

0.00001 g.

11.5.13 Fume hood -- to protect the analyst from effluent or

formaldehyde fumes.

11.5.14 Glass stirring rods for mixing test solutions.

11.5.15 Graduated cylinders Class A, borosilicate slass or

non-toxic plastic labware, 50-1000 mL for making test solutions.

(Note: not to be used interchangeably for gametes or embryos and

test solutions).

11.5.16 Volumetric flasks -- Class A, borosilicate glass or nontoxic plastic labware, 10-1000 mL for making test solutions.

74
11.5.17 Pipets, automatic -- adjustable, to COVE!r a range of

delivery volumes from 0.010 to 1.000 mL.

11.5.18 Pipet bulbs and fillers -- PROPIPET@ or :equivalent.

11.5.19 Wash bottles - - for reagent water, for t,opping off

graduated cylinders, for rinsing small glassware and instrument

electrodes and probes.

11.5.20 Wash bottles -- for dilution water.

11.5.21 20-liter cubitainers or polycarbonate wa.ter cooler jugs

-- for making hypersaline brine.

11.5.22 Cubitainers, beakers, or similar chambe~s of non-toxic I

composition for holding, mixing, and dispensing dilution water

and other general non-effluent, non-toxicant contact uses.' These

should be clearly labeled and not used for other purposes.

11.5.23 Beakers - - six Class A, borosilicate gla1ss or non-toxic

plasticware, 1000 mL for making test solutions.

11.5.24 Brine shrimp, Artemia, culture'unit

11.6.25 and Section 4, Quality Assurance.

see Subsection

11. 5.25 Separatory funnels, 2 -L - - two-four for ~ulturing

Artemia.

11.5.26 Siphon tubes (fire polished glass) -- fo~ solution

renewals and handling larval fish.

11.5.27 Droppers, and glass tubing with fire polished edges, 4

mm ID -- for transferring larvae.

11.5.28 Siphon with bulb and clamp -- for cleaning test

chambers.

11.5.29 Light box -- for counting and observing larvae.

11.5.30 White plastic tray -- for collecting larvae during

cleaning of the test chambers.

75
11.5.31 Forceps -- for transferring dried larvae to weighing

pans.

11.5.32 Desiccator ~- for holding dried larvae~

11.5.33 Drying oven -- 50-105°C range, for drying larvae.

11.5.34 NITEX@ mesh screen tubes - (~150 ~m, 500 ~m, 3 to '5 mm)

-- for collecting Artemia nauplii and fish larvae. (NITEX® is

available from Sterling Marine Products, 18 Label Street,

Montclair, NJ 07042; 201-783-9800).

11.5.35 60 ~m Nitex® filter -- for filtering receiving water.

11.6 REAGENTS AND SUPPLIES

11.6.1 Sample containers -- for sample shipment and storage (see

Section 8, Effluent and Receiving Water Sampling, and Sample

Handling, and Sample Preparation for Toxicity Tests) .

11.6.2 Data sheets (one set per test) -- for data recording

(Figures 1 and 2) .

11.6.3 Tape, colored -- for labelling test chambers and

containers.

11.6.4 Markers, water-proof -- for mark~ng containers, etc.

11.6.5 Parafilm -- to cover graduated cylinders and vessels.

11.6.6 Gloves, disposable -- for personal protection from

contamination.

11.6.7 Pipets, serological -- 1-10 mL, graduated.

11.6.8 Pipet tips

11.6.9 Coverslips

for automatic pipets.

for microscope slides.

11.6.10 Lens paper -- for cleaning microscope optics.

11.6.11 Laboratory tissue wipes for cleaning and drying

electrodes, microscope slides, etc.

76
,

11.6.12 Disposable countertop covering for <protection of work • • I

surfaces and minimizing spills and contamlnatlon.:

11.6.13 pH buffers 4, 7, and 10 (or as per instr~ctions of

instrument manufacturer) -- for standards and cal:Lbration check I '

(see USEPA Method 150.1, USEPA, 1979).

11.6.14 Membranes and filling solutions -- for dissolved oxygen

probe (see USEPA Method 360.1, USEPA, 1979), or rE~agents for

modified Winkler analysis.

11.6.15 Laboratory quality assurance samples and standards

for the above methods.

11.6.16 Test chambers -- 600 mL, five chambers per

concentration. The chambers should be borosilicate glass (for

effluents) or nontoxic disposable plastic labware! (for 'reference

toxicants). To avoid contamination from the air ctnd excessive

evaporation of test solutions during the test, the chambers

should be covered during the test with safety glaf~s _plates or a

plastic sheet (6 mm thick) .

11. 6 .17 Ethanol (70%) or formalin (4%) -- for prErserving the

larvae.

11.6.18 Artemia nauplii -- for feeding test organisms.

11.6.19 Weigh boats or weighing paper -- for wei~rhing reference

toxicants.

11.6.20 Reference toxicant solutions (see Subsec~ion 11.10.2.4

and see Section 4, Quality Assurance).

11.6.~1 Reagent water -- defined as distilled or deionized water

that does not contain substances which are toxic to -the' test

organisms (see Section 5, Facilities, Equipment, a:nd Supplies and

Section 7, Dilution Water).

11.6.22 Effluent and receiving water -- see Section 8, Effluent

and Surface Water Sampling, and Sample Handling, and Sample

Preparation for Toxicity Tests.

77
11.6.23 Dilution water and hypersaline brine,-- see Section 7,

Dilution Water and Section 11.6;24, Hypersaline Brines. The

dilution water should be uncontaminated l-~m-filtered natural

seawater. Hypersaline brine should be prepared from di'lution

water.

11.6.24 HYPERSALINE BR~NES

11.6.24.1 Most industrial and sewage treatment effluents

entering marine and estuarine systems have little measurable

salinity. Exposure of larvae to these effluents will usually

require increasing the salinity of the test solutions. It ,is

important to maintain an essentially constant salinity across all

treatments. In some applications it may be desirable to match

the test salinity with that of the recei~ing water (See Section

7.1). Two salt sources are available to adjust salinities --

artificial sea salts and hypersaline brine (HSB) derived from

natural seawater. Use of artificial sea salts is necessary only

when high effluent concentrations preclude salinity adjustment by

HSB alone.

11.6.24.2 Hypersaline brine (HSB) can, be made by concentrating

natural seawater by freezing or evaporation. HSB, should be made

from high quality, filtered seawater, and can be added to the

effluent or to reagent water to increase salinity. HSB has

several desirable characteristics for use in effluent toxicity

testing. Brine derived from natural seawater contains the

necessary trace metals, biogenic colloids, and some of the

microbial components necessary for adequate growth, survival,

and/or reproduction of marine and estuarine organisms~ and it can

be stored for prolonged periods without any apparent degradation.

However, even if the maximum salinity HSB (100~) is used as a

diluent, the maximum concentration of effluent (O~) that can be

tested is 66% effluent at 34~ salinity (see Table 1) .

11.6.24.3 High quality (and preferably high salinity) seawater

should be filtered to at least 10 ~m befqre placing into t~e

freezer or the brine generator. Water should be collected on an

incoming tide to minimize the possibility of 'contamination.

11.6.24.4 Freeze Preparation of Brine

78
11.6.24.4.1 A convenient container for making USB by freezing is

one that has a bottom drain. One liter qf brine can be made from

four liters of seawater. Brine .may be collected by partially

freezing seawater at -10 to -20°C until the rema.ining liquid has

reached the target salinity. Freeze for approximately six hours,

then separate the ice (composed mainly of fresh water) from the

remaining liquid (which has now become hypersaline) .

11. 6.24.4.2 It is preferable to monitor the wat:er until the

target salinity is achieved rather than allowinsr total freezing

followed by partial thawing. Brine salinity should never exceed

100%'0. It is advisable not to exceed about 70%0 brine salinity

unless it is necessary to test effluent concentl~ations greater

than 50%.

11.6.24.4.3 After th,e required salinity is attcllined, the HSB

should be filtered through a 1 Jim filter 'and poure,d directly into

portable containers (20-L cubitainers or polycarbon~te water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and the: date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been acceptable).: HSB is usually

of acceptable quality even after several months 'in storage.

11.6.24.5 Heat Preparation of Brine

11.6.24.5.1 The ideal container for making brine using heatassisted evaporation of 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 ~ontainers are

ideal). Special care should be used to ~revent any toxic

materials from coming in contact with the seawater being used to

generate the brine. If a heater is immersed direc1:ly into the

seawater, ensure that the heater materials do not corrode or

leach any substances that would contaminate the biine. One

successful method is to use a thermostatically cpntrolled hea~

exchanger made from fiberglass. If aeration is heeded, use only

oil-free air compressors to prevent contamination.

11.6.24.5.2 Before adding seawater to the brine generator,

thoroughly clean the generator, aeration supply tube, ~eater, and

any other materials that will be in direct contact with the

brine. A good quality biodegradable detergent should be used,

79
followed by several (at least three) thorough reagent water

rinses.

11.6.24.5.3 Seawater should be filtered to at least 10 ~m before

being put into the brine generator. 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 (dep~:nding

on the volume being generated) to ensure that the salinity does

not exceed 100%0 and that the temperature does not exceed 40,oC"

Additional seawater may be added to the brine to obtain the

volume of brine required.

TABLE 1. MAXIMUM EFFLUENT CONCENTRATION (~) THAT CAN BE TESTED

AT 34%0 WITHOUT THE ADDITION OF DRY SALTS GIVEN THE

INDICATED EFFLUENT AND BRINE SALINITIES.

,

Effluent Brine Brine Brine Brine Brine

i Salinity 60 70 80 90 100

%0 %0 ~o ~o %"0 %"0

i 0 43.33 51.43 57.50 62.22 66.00

1 44.07 52.17 58.23 62.92 66.67

2 44.83 52.94 58.97 63.64 67.35

I 3 45.61 53.73 59.74 64.37 68.04

I

4 46.43 54.55 60.53 65.12 68.75

5 47.27 55.38 61. 33 65.88 69.47

10 52.00 60.00 65.71 70.00 73.33 i

, 15 57.78 65.45 70.77 74.67 77.65 I

I

I 20 65.00 72.00 76.67 80.00 82.50 I

I

, 25 74.29 80.00 83.64 86.15 88.00

11.6.24.5.4 After the required salinity is attained, the HSB

should be filtered through a 1 ~m filter and poured directly into

portable containers (20-L cubitainers or polycarbonate water

80
cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and'the date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after'several months in storage.

11.6.24.6 Artificial Sea Salts

11.6.24.6 ..1 No data fromtopsmelt larval tests using sea salts

or artificial seawater (e.g., GP2) are available for evaluation

at this time, and thei.r use must be considered provisional.

11.6.24.7 Dilution Water Preparation from Brine

11.6.24.7.1 Although salinity adjustment with brine is the .. preferred method, the use of high salinity brines and/or reagent

water has sometimes been associated with discernible adverse

effects on test organisms. For·this reason, it is recommended

that only the. mini~um necessary volume of brine and reagent water

be used to offset the low salinity of' the effluent, and that

brine control~ be included in the test. The remaining ~ilution

water should be natural seawater. Salinity may be adjusted in

one of two ways. First, the salinity of the highest effluent

test concentration may be adjusted to an acceptable salinity, and

then serially diluted. Alternatively, each effluent

concentration can be prepared individually withiappropriate

volumes of effluent and brine.

11.6.24.7.2 When HSB and reagent water'are used, thoroughly mix

together the reagent water and HSB before mixinEj in the effluent.

Divide the salinity of the HSB by the expected -test salinity to

determine the proportion of reagent water to brine. For example,

if the salinity of the brine is' 100%'0 and ,the test is to be

conducted at 34%'0, 100%'0 divided' by 34%'0 = 2.94. The proportion

of brine is 1 part plus 1.94 reagent water. To, make 1 L of

dilution water at 34~ ~alinity from a HSB of 100~, 340 ~L of

brine and 660 mL of reagent water are required. Verify the

salinity of ' the resulting mixture using refracto~eter.

11.6.,24.8 Test Solution Salinity Adjustn:tent

,

11.6.24.8.1 Table 2 illustrates the preparation of test

solutions (up to 50% effluent) at 34%'0 by combining effl-uent,

81
HSB, and dilution water. Note: if the highest effluent

concentration does not exceed 50% effluent, it is conveniertt to

prepare brine so that the sum of the effluent salinity and brine

salinity equals 68~; the required brine volume is then always

equal to the effluent volume needed for ~ach effluent

concentration as in the example in Table 2.

11.6.24.8.2 Check the pH of all brine mixtures and adj·ust to

within 0.2 units of dilution water pH by adding, dropwise, dilute

hydrochloric acid or sodium hydroxide (see subsection 8.8.'9,

Effluent and Receiving Water Sampling, Sampling Handling, and

Sample Preparation for Toxicity Tests) .

11.6.24.8.3 To calculate the amount of brine to add to each

effluent dilution, determine the following quantities: .sal'inity

of the brine (SB, in~), the salinity of the effluent (SE, in

~), and volume of the effluent to be added (VE, in mL). Then

use the following formula to calculate the volume of brine (VB,

in mL) to be added:

VB = VE x (34 - SE)/(S~ - 34)

11.6.24.8.4 This calculation assumes that dilution water

salinity 'is 34 ± 2~.

11.6.24.9 Preparing Test Solutions

11.6.24.9.1 Two hundred mL of test solution are needed for each

test chamber. To prepa~e test solutions at low effluent

concentrations «6%), effluents may be added directly to dilution

water. For example, to prepare 1% effluent, add 10 mL of

effluent to a I-liter volumetric flask using a volumetric pipet

or calibrated automatic pipet. Fill the volumetric flask to the

l-liter mark with dilution water, stopper it, and shake.to mix.

Distribute equal volumes into the replicate test chambers.

11.6.24.9.2 To prepare a test solution at higher effluent

concentrations, hypersaline brine must usually be used. For

example, to prepare 40% effluent, add 400 mL of effluent to a

82
TABLE 2. EXAMPLES OF EFFLUENT DILUTION SHOWING VOLUMES OF

EFFLUENT (x~), BRINE, AND DILUTION WA~rER NEEpEDFOR

ONE LITER OF EACH; TEST SOLUTION.

FIRST STEP;

to achieve a

for dilution

Combine brine with reagent water OJI:' natural

brine of 68-x%"0 and, unless natural seawater

water, also a brine-based dilution water of

seawater

is used

34%"0.

SERIAL DILUTION; I

Step 1. Prepare the highest effluent concentration to be tested

by adding equal volumes of effluent and brine to the appropriate

volume of dilution water. An example using 40% is shown.

Effluent Cone. Effluent Brine : Dilution , (%) x§o (68-x)§o Water* 34§o

40 800 mL 800 mL 400 mL

Step 2. Use elther serlally prepared dllutlons'of the hlghest

test concentration or individual dilutions of 100% effluent.

Effluent Cone. (%) Effluent Source Dilution Water*

(34§o)

20 J.OOO mL of 40% J.OO'O mL

J.O J.OOO mL of 20% J.00l0 mL

5 J.OOO mL of J.O% J.OOO mL

"

2.5 J.OOO mL of 5% J.OOO mL

Control none J.OOO mL .'

INDIVIDUAL PREPARATION

Effluent Cone. Effluent x§o Brine (68-x)§o Dilution Water*

(%) 34§o L

40 400 mL 400 mL : 200 mL "

20 200 mL 200 mL' 600 mL

J.O J.OO mL J.OO mL ! 800 mL

5 SO mL SO mL 900 mL

2.5 25 mL 25 mL 950 mL

I

Control none none J.OOO mL

*May be natural seawater or brlne-reagent water:equlvalent.

83
l-liter volumetric flask. Then, assuming an effluent salinity of

2~ and a brine salinity of 66~, add 400 mL of brine (see

equation above and Table 2) and top off the flask with dilution

water. Stopper the flask and shake well. Pour into a ('100-250

mL) beaker and stir. Distribute equal volumes into the replicate

test chambers. The remaining test solution can be used for

chemistry.

11.6.24.10 Brine Controls

11.6.24.10.1 Use brine controls in all tests where brine is

used. Brine controls contain the same volume of brine as does

the highest effluent concentration using brine, plus the volume

of reagent water needed to reproduce the hyposalinity of the

effluent in the highest concentration, plus dilution water.

Calculate the amount of reagent water to "add to brine controls by

rearranging the above equation, (See SubSection, 11.6.24.8.3)

setting SE = 0, and solving for VE.

VE = VB x (SB - 34)/(34 - SE)

11.6.25 BRINE SHRIMP, ARTEMIA SP.,NAUPLII

cultures and test organisms.

for feeding

11.6.25.1 Newly hatched Artemia sp. nauplii are used for food

for the test organisms. Although there are many commercial

sources of brine shrimp cysts, the Brazilian or Colombian strains

are preferred because the supplies examined have had low

concentrations of chemical residues and produce nauplii of

suitably small size. (One source that hqs been found to be

acceptable is Aquarium Products, 180L Penrod Ct., Glen Burnie,

Maryland 21061). For commercial sources of brine shrimp,

Artemia, cysts, see Table 2 of Section 5, Facilities, Equipment,

and Supplies); and Section 4, Quality Assurance.

11.6.25.2 Each new batch of Artemia cysts must be evaluated for

size (Vanhaecke and Sorgeloos, 1980, and Vanhaecke et al., 1980)

and nutritional suitability (Leger, et ai., 1985, Leger, et al.,

1986) against known suitable reference cysts by performing a

side-by-side larval growth test using the "new"· and "reference"

cysts. The "reference" cysts used in the suitability te~t may be

a previously tested and acceptable batch of cysts, or may be

obtained from the Quality Assurance Research Division, EMSL,

84
Cincinnati, OR 45268, 513-569-7325. A sample of newly-hatched

Artemia nauplii from each new batch of cysts should be chemically

analyzed. The Artemia cysts should not be. use,d if, the

concentration of total organochlorine pesticid~s 0.15 ug/g w~t

weight or that the total concentration of organochlorine

pesticides plus PCBs exceeds 0.30 ~g/g wet weight (For analytical

methods see USEPA, 1982).

11.6.2S.4

organisms.

11.6.25.3

1.

2.

3.

4.

Artemia nauplii are obtained as follows:

'I. •

Add 1 L of seawater, or an aqueous u:hlonlzed salt ;

(NaCl) solution prepared with 35, g salt or artificial

sea salts per liter, to ,a 2~L separa~ory funnel, or

equivalent. "

Add 10 mL Artemia cysts to the separ,atory funnel and

aerate for 24 h at 27°C. ,Ratchingtime varies with

incubation temperature and the 'geographic strain Of

Artemia used (see USEPA, 1985ai USEPA, 1993ai A$TM,

1993) .

After 24 h, cut off the air supply i:o. the separatory

funnel. Artemia nauplii are phototactic, and will

concentrate at the bottom of the funnel if it is

covered for S-10 minutes with 9- dark: cloth or paper·

towel. To prevent mortality, do not: leave the

concentrated nauplii at the bottom of ~he funnel more

than 10 min without aeration. -.

Drain the nauplii into a funnel fitt,=d with a s:lS0 ~m

NITEX® or stainless steel screen, and rin~e with

seawater or equivalent before use.

I '<,

Testing Artemia nauplii as food for,toxicity test

,

11.6.25.4.1 The primary criteria for acceptabtlityof ~ach neW

supply of brine shrimp cysts is adequate survi'~al, and growth of

the larvae. The larvae used to evaluate the abceptability of the

brine shrimp nauplii must be the same geogr~phical origin ang....

stage of development (9 to 1S days old) ~s, those, used routiIlely

in the toxicity tests. Two 7-day chronic testl3 are performed

side-by-side, each consisting of five replicate tes~ vessel~ .~

containing, five larvae (25 organisms per test,total :of·'SO

organisms). The juveniles in OIJ.e set of test (~hambers is~ed,

85
reference (acceptable) nauplii and the other set is fed nauplii

from the "new" source of Artemia cysts..

11.6.25.4.2 The feeding rate and frequency, test vessels, volume

of control water, duration of the tests, and age of the Artemia

nauplii at the start of the test, should be the same as used for

the routine toxicity tests.

11.6.25.4.3 Results of the brine shrimp, Artemia, nauplii

nutrition assay, where there are only two treatments, can be

evaluated statistically by use of a t test. The "new" food is

acceptable if there are no statistically significant differences

in the survival or growth of the mysids fed the two sources of

nauplii.

11.6.26 TEST ORGANISMS

11.6.26.1 The test organisms for test method are larvaepf the

topsmelt, Atherinops affinis. Topsmelt, occur from the Gulf of

California to Vancouver Island, British Columbia (Miller and Lea,

1972). It is often among the most abundant fish specfes in

central and southern 'California e,stuaries (Allen and Horn, 1975;

Horn, 1979; Allen, 1982). Topsmelt reproduce from May through

August, depositing eggs on benthic algae in the upper ends of

estuaries and bays (Croaker, 1934; Fronk,. 1969).' Off-season

spawning of Atherinops affinis has been successful in a

laboratory-held population (Anderson et al., 1994). Their

embryonic development is similar to that of other atherinids used

widely in toxicity testing (eg, Menidia species, Borthwipk et,

al.,1985; Middaugh et al., 1987; Middaugh and Shenker, 1988), and

methods to assess sublethal effects with these species have

proven to be adaptable for topsmelt (Ande:rson et al.,· 1991,

Middaugh and Anderson, 1993, McNulty et al., 1994).

11.6.26.2 Species Identification

11.6.26.2.1 Topsmelt often co-occur with jacksmelt, Atherinopsis

californiensis. The two species can be distinguished based on

several key characteristics. Jacksmelt have 10-12 scales between

their two dorsal fins; topsmelt have 5-8 'scales between the two

fins. Jacksmelt teeth are arranged in several bands on each jaw

and the teeth are not forked; topsmelt teeth are arranged in one

band and the teeth are forked. . In j acksmelt, the insertion of

86
the first dorsal fin occurs well in advance of the origin of the

anal fin. In topsmelt, the origin of the anal fin is under the

. insertion of the first dorsal fin. Constilt Miller and Lea (1972)

for a guide to the taxonomy of these two fishes.

11.6.26.3 Obtaining Broodstock

11.6.26.3.1 In California, adult topsmelt can be seined from

sandy beaches in sloughs and estuaries from April through August.

The size of the seine used depends on the number of people

deploying it and the habitat being sampled. Larger seines can be

used in open sandy areas, smaller seines are used in smaller

areas with rocky outcroppings. Five or six people are an

adequate number to set and haul a 100-ft beach seine. The seine

is set on an ebbing tide using a small motor skiff with one

person driving and a second deploying the net from the bow. The

net is set parallel to shore then hauled in evenly from the

wings. The net mesh diameter should be small enough to prevent

the fish from damaging themselves; a one-centimeter diameter mesh

in the middle panel and one-and-a-half-centimeter diameter mesh

in the wing panel is adequate. As the net is pulled onto_the

shore, the adult topsmelt are sorted into five-liter plastic

buckets, then immediately transferred to 100-liter transport

tanks.

11.6.26.3.2 State collection permits are usually required for'

collection of topsmelt. Collection is prohibited or restricted

in some areas. Collection of topsmelt is regulated by California

law. Collectors must obtain a scientific collector's ~ermit from

the 'California Department of Fish and Game and observe any

regulations regarding collection, transfer, and maintenance of

fish broodstock.

11.6.26.3.3 Various containers can be used to ~ransport fish;

100-liter covered plastic trash cans have been ·~sed.successfully

to transport topsmelt. New plastic containers should be leached

in seawater for 96 hours prior to transporting fish. Each

container can maintain approximately 20 adult fish for six to

eight hours if adequate aeration is provided. 'Use compressed

oxygen or air to supply aeration to the tanks during transport.

11.6.26.4 Broodstock Culture and Handling

87
11.6.26.4.1 Once in the laboratory the fish should be treated

for 2 days with a general antibiotic in a separate tank (eg.,

Prefuran@ as per label instructions), then divided among 1000-

liter holding tanks. No more than 30 adult fish should be placed

in each tank. Tank temperature should be maintained at l8 0 e

using a 1500-watt immersion heater. To conserve heated seawater,

the seawater in the tanks can be recirculated using the system

similar to that described by Middaugh and Hemmer (1984). A onethirtieth (1/30)-hp electric pump is used to circulate water (10

liters/minute) from the tanks through vertical, biologically

activated nylon filter elements located in a separate reservoir,

then back into the tanks. Fresh seawater should be constantly

provided to the system at 0.5 liters/minute to supplement the

recirculated seawater. The tanks are insulated with one inch

thick closed cell foam to conserve heat. Dissolved oxygen levels

should be maintained at greater than 6.0 mg/liter using aeration.

Salinity should be checked periodically using a refractometer

accurate to the nearest 0.5~i tank salinity should be 34 ± 2~.

11.6.26.4.2 Adult topsmelt in each tank are fed twice daily (at

0900 and 1500 hrs) approximately 0.3g of ·TetraminTM flake food.

Supplemental feedings of krill or chopped squid are recommended.

Tanks are siphoned clean once weekly.

11.6.26.4.3 Dyeless yarn spawning substrates are attached to the

surface of plastic grids cut from light diffuser panel (7 cm x

10 cm x 1 cm) and weighted'to the bottom of each tank.

Substrates are checked daily for the pres.ence of eggs.

11.6.26.4.4 Spawning is induced by a combination of three

environmental cues: lighting, 'tidal' cycle, and temperature.

The photoperiod is 14 hours of light followed by 10 hours ·of

darkness (14L: 10D) with lights on at 0600 and off at 2000 :hours.

Use two cool white 40-watt fluorescent lamps suspended 1.25

meters above the surface of each tank to provide illumination.

Light levels at the surface of the tanks 'should be 12 to 21

p.E/m2 /s.

11.6.26.4.5 A 'tidal signal' of reduced current velocity is

produced once daily in each tank, from 2400 to 0200 hrs, by

turning off the circulating pump (Middaugh and Hemmer, 1984).

A 1500-watt immersion heater is used to maintain constant

temperature at 18°e and to provide temperature spikes. For

88
spiking, the temperature is raised from i8°e to 21°C over a 12 h

period, then allowed to return to 18°C overnight. :The temperature

should be checked to the nearest O.l°e at 1to 4 hour intervals

on days when the temperature spikes are introduced. It is common

for the fish to appear stressed during the temperature increase

and one or two fish may die. If significant mort~lity begins to

. occur, the temperature should be lowered immediately.

Significant egg production usually begins within five days of the

temperature spike (Middaugh, et al., 1992).

11.6.26.5 Culture Materials

11.6.26.5.1 See Section 5, Facilities and Equipment, for a

discussion of suitable materials to be used in laboratory culture

of topsmelt. Be sure all new materials are properly leached in

seawater before use. After use, all culture materials should be

washed in soap and water, then rinsed with seawater before reuse.

11.6.26.6 Test Organisms

11.6.26.6.1. Newly fertilized embryos s~ould be placed in screen

tubes set in aquaria and equipped with gently flowing seawater at

20 ± 1.oC. The embryos' can be left attached to the spawning

substrates but care should be taken to ensure the substrates are

relatively clean and free of food; strands of embryos should not

overlap each other on the substrates, and gentle aeration must be

provided. Beginning about day 9, check the screen tubes daily

for the presence of larvae. Isolate newly-hatched larvae into a

separate screen-tube at 21°C by slow siphoning. Provide larvae

with newly-hatched Artemia nauplii (in excess) at 24-h posthatch; supply gently flowing seawater, and aeration. .Larvae

aged 9 to 15 days are used in toxicity tests '(Mc~~lty et al.,

1994). For information regarding topsmelt larva isuppliers call

the Marine Polluti?n Studies Laboratory (408) 624-0947.

11.6.26.6.2 Larvae can be transported in l-liter ziplock plastic

bags (double-bagged). No more than approximately 100 larvae

should be transported in anyone bag; do not include food. The

seawater in the bags should be aerated with pure oxygen for 30

seconds prior to introduction of the larvae. The bag should be

packed in an ice chest with one or two blue ice blocks (insulated

by newspaper) for transport. The temperature during transport

89
should be held between 15 and 18 De. Larvae should be shipped via

air-express overnight couriers.

11.6.26.6.3 Topsmelt larvae can tolerate a relatively wide range

of salinities (5 to ~35~) if adequate acclimation is provided

(Anderson, et al., In Press). In situations where the test

salinity is significantly lower than the salinity at which the

larvae were cultured, it may be necessary to acclimate the larvae

to the test salinity.

11.7 EFFLUENTS AND RECEIVING WATER COLLECTION, PRESERVATION, AND

STORAGE

11.7.1 See Section 8, Effluent and Receiving Water Sampling,

Sample Handling, and Sample 'Preparation for Toxicity Tests.

11.8 CALIBRATION AND STANDARDIZATION

11.8.1 See Section 4, Quality Assurance

11.9 QUALITY CONTROL

11.9.1 See Section 4, Quality Assurance

11.10 TEST PROCEDURES

11.10.1 TEST DESIGN

11.10.1.1 The test consists of at least five effluent

concentrations plus a dilution water control. Tests that use

brine to adjust salinity must also contain five replicates of a

brine control.

11.10.1.2 Effluent concentrations are expressed as percent

effluent.

11.10.2 TEST SOLUTIONS

11.10.2.1 Receiving waters

11.10.2.1.1 The sampling point is determined by the objectives

of the test. At estuarine and marine sites, samples are usually

collected at mid-depth. Receiving water toxicity is determined

90
with samples used directly as collected or with samples passed

through a 60 J.Lm NITEX® filter and compared without dilution,

against a control. Using five replicate chambers per test, each

containing 200 mL would require approximately 1 J.J of sample per

test per day.

11.10.2.2 Effluents

11.10.2.2.1 'The selection of the efflue~t test concentrations

should be based on the objectives of the study; A dilution

factor of at least 0.5 is commonly used. A. dilution factor of

0.5 provides hypothesis test discrimination of ± 100%, 'and

testing of a 16 fold range of concentrations. Hypothesis test

discrimination shows little improvement as dilution factors are

increased beyond 0.5 and declines rapidly if smaller dilution

factors are used. USEPA recommends that one of the five effluent

treatments must be a concentration of effluent mixed with

dilution water which corresponds to the permitte~'s instream

waste concentration (IWC). At least two of the ,effluent

treatments must be of lesser effluent concentration than the lWC,

with one being at least one-half the concentration of the IWC.

If 100%'0 HSB is used as a diluent, the maximum concentration of

effluent that can be tested will be 66% at 34~ salinity.

11.10.2.2.2 If the effluent is known or suspect,ed to be highly

toxic, a lower range of effluent concentrations should be used

(such as 25%, 12.5%, 6.25%, 3.12% and 1.56%).

11.10.2.2.3 The volume in each test chamber is 200 mL.

11.10.2.2.4 Effluent dilutions should be prepared for all

replicates in each treatment in one container to minimize

variability among the replicates. Dispense into the appropriate

effluent test chambers.

11.10.2.3 Dilution Water

11.10.2.3.1 Dilution water shou~~ be un~ontaminated I-J.Lmfiltered natural seawater or hypersaline brine prepared from

uncontaminated natural seawater plus reagent watler (see Section

7, Dilution Water). Natural seawater may be uncbntaminated

receiving water. This water is used in all dilu'tion steps and as

the control water.

91
11.10.2.4 Reference Toxicant Test

11.10.2.4.1 Reference toxicant tests should be conducted as

described in Quality Assurance (see Section4.7).

11.10.2.4.2 The preferred reference toxicant for topsmelt is

copper chloride (CuC1202H20). Reference toxicant tests provide

an indication of the sensitivity of the test organisms and the

suitability of the testing laboratory (see Section 4 Quality

Assurance). Another toxicant may be specified ~y the appropriate

regulatory agency. Prepare a 10,000 ~g/L copper stock solution

by adding 0.0268 g of copper chloride (CuC12 02H20) to one liter

of reagent water in a polyethylene volumetric flask.

Alternatively, certified standard solutions can be ordered from

commercial companies.

11.10.2.4.3 Reference toxicant solutions should be five

replicates each of°(control) i 56, 100, '180, and 320 ~g/~ total

copper. Prepare one liter of each concentration by adding 0,

5.6, 10.0, 18.0, and 32.0 mL of stock solution., respectively, to

one-liter volumetric flasks and fill with dilution water. Start

with control solutions and progress to the highest concentration

to minimize contamination.

11.10.2.4.4 If the effluent and reference toxicant tests are to

be run concurrently, then the tests must use embryos from the

same spawn. The tests must be handled in the same way and test

solutions delivered to the test chambers at the same time.

Reference toxicant tests must be conducted at 34 ± 2~.

11.10.3 START OF THE TEST

11.10.3.1 Prior to Beginning the Test

11.10.3.1.1 The test should begin as soon as possible,'

preferably within 24 h of sample collection. The maximum holding

time following retrieval of the sample from the sampling device

should not exceed 36 h for off-site toxicity tests unless

permission is granted by the permitting authority. In no case

should the sample be used in a test more 'than 72 h after sample

collection (see Section, 8 Effluent and Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity T~st) .

92
11.10.3.1.2 Just prior to test initiation (approximately 1 h),

the temperature of a sufficient quantity of the'sample to make

the test solutions should be adjusted to the te/3ttemperature (20

± 1°C) and maintained at that temperature durinsr the addition of

dilution water.

11.10.3.1.3 Increase the temperature of the water bath, room, or

incubator to the required test temperature (20 :1: 1°C) .

11.10.3.1.4 Randomize the'placement of test chambers in the

temperature-controlled water bath, room, or incubator at the

beginning of the test, using a position chart. Assign numbers

for the position of each test chamber using a rcindom numbers or

similar process (see Appendix A, for an example'of

randomization). Maintain the chambers in this configuration

throughout the test, using a position chart. Record these

numbers on a separate data sheet together wi'th t:he concentration

and replicate numbers to which they correspond. Identify this

sheet with the date, test organism, test ,number, laboratory, and

investigator's name, and safely store it away uqtil after the

larvae have been examined at the end 6f the test.

11.10.3.1.5 Note: Loss of the randomization sl:1eet would

invalidate the test by making it impossible to analyze the data

afterwards. Make a copy of the randomization shee't and store

separately. Take care to follow the num:qering Eiystem exactly

while filling chambers with the test solutions.

11.10.3.1.6 ~rrangethe test chambers randomly'in the wafer bath

or controlled temperatl.1re room. Once chambers have been labeled

randomly, they can be arranged in numerical order for

convenience, since this will also ensure random placement of

treatments.

11.10.3.2 Randomized Placement of, Larvae into Test Chambers

11.10.3.2.1 Larvae must be randomized before placing them into

the test chambers. Pool all of the test larvae into a l-liter

beaker by si'cw siphoning from the screen-tube. The larvae in the

screen-tube can be concentrated into the bottom by lifting the

tube during siphoning. Using a fire-polished glass tube, place

one larva into as many plastic cups as there ar~test chambers

(including reference toxicant chambers). These cups should

93
contain enough reference seawater to maintain water quality and

temperature during the transfer,process (approx. 50 mL). When

each of the cups contains one larva, repeat the process, adding

one larva at a time until each cup contains 5 animals.

11.10.3.2.2 Carefully pour or pipet off excess water in the

cups, leaving less than 5 mL with the test larvae. If more than

5 mLs of water are added to the test solution with the juveniles,

report the amount on the data sheet. Carefully transfer the

larvae into the test chambers immediately after reducing the

water volume. Again, make note of any excess dilution of the

test solution. Because of the small volumes involved in the

transfer process, this is best accomplished in a constant

temperature room. Be sure that all water used in culture,

transfer, and test solutions is within 1°C of the test

temperature.

11.10.3.2.3 Verify that all five animals are transferred by

counting the number in each chamber after transfer. This initial

count is important because larvae unaccounted for at the end of

the test are assumed to be dead.

11.10.4 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE

11.10.4.1 The light quality and intensity should be at ambient

laboratory conditions are generally adequate. Light intensity

should be 10-20 ~E/m2/s, or 50 to 100 foot candles (ft-c), with a

16 h light and 8 h dark cycle.

11.10.4.2 The water temperature in the test chambers ~hould be

maintained at 20 ± 1°C. If a water bath 'is used to maintain the

test temperature, the water depth surrounding the test cups

should be as deep as possible without floating the chambers.

15.10.4.3 The test salinity should be in the range of 5 to 34~,

and the salinity should not vary by more than ± 2~ among the

chambers on a given day. The salinity should vary by no more

than ±2~ among the chambers on a given day. If 'effluent and

receiving water tests are conducted concurrently, ,the salinities

of these tests should be similar.

15.10.4.4 Rooms or incubators with highyolume ventilation

should be used with caution because the volatilization of the

94
test solutions and evaporation of dilution water may cause wide

fluctuations in salinity. Covering the test chanillers with clean

polyethylene plastic may help prevent· volatilizat:ion and

evaporation of the test solutions.

11.10.5 DISSOLVED OXYGEN (DO) CONCENTRATION

11.10.5.1 Aeration may affect the toxicity of effluent and

should be used only as a last resort to. maintain a satisfactory

DO. The DO concentration should be measured on new solutions at

the start of the test (Day 0). The DO should not. fall below 4.0

mg/L (see Section 8, Effluent and Receiving Water' Sampling,

. Sample Handling~ and Sample Preparation for Toxicity Tests). If

it is necessary to aerate, .all treatments and the. control should

be aerated. The aeration rate should not exceed that necessary

to maintain a minimum acceptable DO and under no circumstances

should it exceed 100 bubbles/minute, using a pipet with a 1-2 mm

orifice, ~uch as a 1 mL KIMAX® serological pipet No. 37033, or

equivalent. Care should be taken to ensure that ~urbulence

resulting from aeration does not cause undue stress to the fish.

11.10.6 FEEDING

11.10.6.1 Artemia nauplii are prepared as de'scribed below.

11.10.6.2 The test larvae are fed newly-hqtched .(less than 24-h~

old) Artemia nauplii once a day from Day 0 through Day 6; larvae

are not fed on Day 7. Equal amounts of Artemia nauplii ~ust be

fed to each replicate test chamber to miriimize the variability of

larval weight. Add 40 newly hatched Artemia naupiii per;larva.

twice daily: once in the morning and once in the afternoon. The

density of Artemia may be determined by pipetting a known volume

of nauplii onto a piece of filter paper and counting the number

using a dissecting microscope. Feeding excessive amounts of

Artemia nauplii will result in a depletion in DO to below an

acceptable level. Siphon as much of the 'uneaten J~rtemia nauplii

as possible from each chamber daily to ensure that the larvae

principally eat newly hatched nauplii.

11.10.7 DAILY CLEANING OF TEST CHAMBERS

11.10.7.1 Before the daiiy renewal of test solutions, uneaten

and dead brine shrimp', dead larvae, and other debris are removed

95
from the bottom of the test chambers with a siphon hose. Because

of their small size during the first few days of the test, larvae

are easily drawn into a siphon tube when cleaning the test

chambers. By placing the test chambers on a light box,

inadvertent removal of larvae can be greatly reduced because they

can be more easily seen. If the water siphoned from the test

chambers is collected in a white plastic tray, the live larv~e

caught up in the siphon can be retrieved, and returned by ,pipette

to the appropriate test chamber and noted on the data sheet.

11.10.8 OBSERVATIONS DURING THE TEST

11.10.8.1 Routine Chemical and Physical Observations

11.10.8.1.1 DO is measured at the beginning of the exposure

period in one test chamber at each test concentration and in the

control.

11.10.8.1.2 Temperature, pH, and salinity are measured at the

beginning of the exposure period in one test chamber at each

concentration and in the control. Temperature should also be

monitored continuously or observed and r~corded daily for at

least two locations in the environmental control system or the

samples. Temperature should be measured in a sufficient number

of test chambers at the end of the test to determine temperature

variation in the environmental chamber.

11.10.8.1.3 Record all the measurements on the data sheet.

11.10.8.2 Routine Biological Observatiorts

11.10.8.2.1 The number of live larvae in each test chamber are

recorded daily and the dead larvae are discarded. These data

provide daily mortality rates which may be used to calculate 24,

48, and 96-h LCSOs.

11.10.8.2.2 Protect the larvae from unnecessary disturbances

during th~ test by carrying out the daily test observations,

solution renewals, and removal of dead larvae, carefully., Make

sure the larvae remain immersed at all times during the

performance of the above operations.

96
11.10.9 TEST SOLUTION RENEWAL

11.10.9.1 The test solutions are renewed daily 'using freshly

prepared solutions, immediately after cleaning t.he test chambers.

The qld solution is carefuily sip~oned out, leaving enough water

so that all of the larvae can still swim freely (approximately 50

mL). Siphon from the bottom of' the test chambers: so that dead

Artemia nauplii are removed with the old test solution. It is

convenient to siphon old solutions into a small (-500 mL)

container in order to ensure that no larvae have been

inadvertently removed during solution renewals. If a larva is

siphoned, 'return it to the test chamber and note it on the data

sheet.

11.10.9.2 New solution is siphoned into the test chambers using

a U-shaped glass tube attached to plastic tubing to minimize

disturbance to the larvae.

11.10.9.3 The effluent or receiving water used in the 'test is

stored Jnan ':L~gubator or refrigerator at 4'OC. Plastic

containers such as 8-20 L cubitainers have proven suitable for

effluent collection and storage. For on-site to}cJicity studies no

more than 24 h should elaps~ between col~ection of the effluent

and use in a toxicity test (see Section 8, Effluent and Receiving

Water Sampling, Sample Handling, and Sample Prep,aration for

Toxicity Testsf.

11.10.9.4 Approximately 1 h before test initiation, a sufficient

quantity of effluent or receiving water sample is wa.rmed to 20 ±

1°C to. prepare the test solutions. A sufficient quantity of

effluent should be warmed to make daily test solutions.

11.10.10 TERMINATION OF THE TEST

11.10.10.1 Ending the Test

11.10.10.1.1 Record the time the test is terminated.

11.10.10.1.2 Temperature, pH, dissolved oxygen, and salinity are

measured at the end of the exposure period in one test chamber at

each concentration and in the control.

11.10.10.2 Sample Preservation

97
11.10.10.2.1 The surviving larvae in each test chamber-

(replicate) are counted, and immediately.prepared as a group for

dry weight determination, or are preserved in 4% formalin then

70% ethanol. Preserved organisms are dried and weighed within 7

d. For safety, formalin should be used under a hood. -Ndte:

Death is defined as lack of response to stimulus such as prodding

with a glass rod;' dead larvae are generally opaque and curled.

11.10.10.3 Weighing

11.10.10.3.1 For immediate drying and weighing, siphon or pour

live larvae onto a 500 ~m mesh screen in a large beake~ to retain

the larvae and allow Artemia to be rinsed away. Rinse the larvae

with reagent water to remove salts that might contribute to the

dry weight. Sacrifice the larvae in an ice bath of reagent

water.

11.10.10.3.2 Small aluminum weighing pans can be used to dry and

weigh larvae. An appropriate number of aluminum weigh pans (one

per replicate) are marked for identification and weighed to 0.01

mg, and the weights are recorded on the data sheets.

11.10.10.3.3 Immediately prior to drying, the preserved larvae

are in reagent water. The rinsed larvae.from each test chamber

are transferred, using for~eps, toa tared weighing pans and

dried at 60°C for 24 h, or at 105°C for a minimum of 6 h.

Immediately upon removal from the drying .oven, the weighing pans

are placed in a desiccator to cool and to prevent' the adsorption

of moisture from the air until weighed. Weigh all weighing pans

containing the dried larvae to 0.01 mg, subtract the tare weight

to determine dry weight of larvae in each replicate. Record the

weights.

11.10.10.4 Endpoints

11.10.10.4.1 Divide the dry weight by the number of origin~l

larvae (5) per replicate to determine the average dry weight, and

record on the data sheets. For the controls, also calculate the

mean weight per surviving fish in the test chamber to evaluate. if,

weights met test acceptability criteria (see Subsection 11.11) .

Complete the summary data sheet after calculating the average

measurements and statistically analyzing the dry weights and

98
percent survival for the entire test. Average wE~ights should be

expressed to the nearest 0.01 mg.

11.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEP~~ABILITY CRITERIA

11.11.1 A summary of test conditions and test acceptability

criteria is listed in Table 3.

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY

CRITERIA FOR THE TOPSMELT, ATHERINOPS l.lFFINIS, LARVAL

SURVIVAL AND GROWTH TEST WITH EFFLUENTS AND RECEIVING

WATERS r "

1- Test type: Static-renewal

2. Salinity: 5 to 34%"0 (± 2%0 of: the

selected test salinity)

3. Temperature: 20 ± l°C

4. Light quality: Ambient laboratory illumination

5. Light intensity: 10-20 fJ-E/m2 / s (Ambient

laboratory levels)

6 . Photoperiod: 16 h light, 8 h darkness

7. Test chamber size: 600 mL

solution volume: 200 mL/replicate I

8 . Test

9 . Renewal.of test Daily

solutions:

10. Age of test organisms: 9-15 days post-hatch

11. No. larvae per test 5

chamber:

12. No. replicate chambers 5

per concentration:

13. Source of food: Newly hatched Artemia nauplii

14. :Feeding regime: Feed 40 nauplii per larvae

twice daily (morning arid night)

99
1.5. Cleaning: Siphon daily, immediately

before test solution renewal

and feeding

I

1.6. Aeration: None, unless DO concentration

I falls below 4.0 mg/L, then

aerate all chambers. Rate

should be less than 100

I bubbles/min.

I 1.7. Dilution water: Uncontaminated 1-Jlm-filtered

natural seawater or hyper~aline

brine prepared from natural

seawater

1.8. Test concentrations: Effluent: Minimum of 5' arid a

I control

Receiving waters: 100%

I receiving water and a control

,

19. Dilution factor: Effluents: ~0.5

Receiving waters: None, or ~0.5

i

I

20. Test duration: 7 days [

: 21.. Endpoints: Survival and growth (weight)

,

22. Test acceptability ~80% survival in controls, 0.85

criteria: mg average weight of cont,~ol

larvae (9 day old) , Le50 with

I copper must be ~205 Jlg/L, <25%

I MSD for survival and <50% MSD

! for growth

100
23. Sampling requirement:

24. Sample volume

required:

For on-site tests, samples

collected daily, and used

within 24 h of the time they

are removed from the sampling

device. For off-site tests, a

minimum of threl= samples are'

collected on days one, ,three,

and five with a maximum ,holding

time of 36 h before first use

(see Section 8, Effluent and

Receiving Water· Sampling,

Sample Handling" and Sample

Preparation for Toxicity Tests)

2 L per day

11.12 ACCEPTABILITY OF TEST RESULTS

11.12.1 Tests results are accep~able only if all the following,

requirements are met:

(1) The mean survival of larvae must be at least 80%" in the

,controls.

(2) If the test starts with 9 day old larvae, the mean

weight per larva must exceed 0.85 mg in the reference

and brine controlsi the mean weight of preserved larvae

must exceed 0.72 mg.

(3) The LC50 for survival must be within two standard

deviations of the control chart mean for the

laboratory. The LC50 for survival. with copper must be

<205 f.Lg/L.

(4) The minimum significant difference (%"MBD) of <25%"

relative to the control for survival for the reference

toxicant test. The (%"MSD) of <50%" rel,tive to the

control for growth for the reference toxicant test.

11.13 DATAANALYSIS

101
11.13.1 GENERAL

11.13.1.1 Tabulate and summarize the data. A·sample set of

survival and growth response data is listed in Table 4.

11.13.1.2 The endpoints of toxicity tests using the topsmelt

larvae are based on the adverse effects on survival and growth.

The LC50 and the IC25 are calculated using point estim~tion

techniques (see Section 9, Chronic Toxicity Test Endpoints and

Data Analysis). LOEC and NOEC values, for survival and growth,

are obtained using a hypothesis testing approach such as

Dunnett's Procedure (Dunnett, 1955) or Steel's ,Many-one Rank Test

(Steel, 1959; Miller, 1981) (see Section 9). Separate analyses

are performed for the estimation of the LOEC and NOEC endpoints

and for the estimation of the LCSO and IC2S. Concentrations at

which there is no survival in any of the test chambers are

excluded from the statistical analysis of the NOEC and LOEC for

survival and growth, but included in'the estimation of the LCSO

and IC25. See the Appendices for examples of the manual

computations and examples of data input ~nd program output.

11.13.1.3 The statistical tests described here must be used with

a knowledge of the assumptions upon which the tests are

contingent. Tests for normality and homogeneity of variance are

included in Appendix B. The assistance of a statistician is

recommended for analysts who are not proficient in statistics.

11.13.2 EXAMPLE OF ANALYSIS OF TOPSMELT, ATHERINOPS AFFINIS

SURVIVAL DATA

11.13.2.1 Formal statistical analysis of the survival data is

outlined in Figures 1 and 2. The response used in the analysis is

the proportion of animals surviving in each test or control

chamber. Separate analyses are performed for the estimation of

the NOEC and LOEC endpoints and for the estimation of the LCSO

endpoint. Concentrations at which there is no survival in any of

the test chambers are excluded from statistical analysis of the

NOEC and LOEC, but included in the estimation of the Ie, EC, and

LC endpoints.

11.13.2.2 For the case of equal numbers of replicates across all

concentrations and the control, the eval~ation of the NOEC and

LOEC endpoints is made via a parametric test, Dunnett's

102
Procedure/ or a nonparametric test, Steel's Man)'-one Rank Test,

on the arc sine square root transformed data. lTnderlying

assumptions of Dunnett's Procedure/ normality and homogeneity of

variance/ are formally tested. The test fo'r normality is the

Shapiro-Wilk's Test, and Bartlett's Test is used to test for

homogeneity of variance. If either of these tests fails/ the

nonparametric test, Steel's Many-one Rank Test, is used to

determine the NOEC and LOEC endpoints. If the assumptions of

Dunnett's Procedure are met/ the endpoints are e:stimated by the

parametric procedure.

11.13.2.3 If unequal numbers of replicate,s occur among the

concentration levels tested/ there are parametric and

nonparametric alternative analyses. The parametric analysis is a

t test with the Bonferroni adjustment (see Appendix D). The

Wilcoxon Rank Sum Test with the Bonferroni adjustment is the

nonparametric alternative.

11.13.2.4 Probit Analysis (Finney, 1971; see Appendix H) is used

to estimate the concentratibn that causes a specified percent

decrease in survival from the control. In this analysis/ the

total mortality data from all test replicates at' a given

concentration are combined. If the data do not tit the Probit

Analysis, the Spearman-Karber Method, the Trimme~ Spearman-Karber

Method, or the Graphical Method may be used to estimate the LC50

(see Appendices H-K) .

11.13.2.5 Example of Analysis of Surviv~l Data

11.13.2.5.1 This example uses the survival data from the

Topsmelt Larval Survival and Growth Test. The proportion

surviving in each replicate must first be transf6rmed by the arc

sine square root transformation procedure described in Appendix .

The raw and transformed data, means and variances of the

transformed observations at each copper concentrc~tion and control

are listed in Table 5. A plot of the survival p:roportions is

provided in Figure 5. Since there was 100% mortality in all five

replicates for the 100 ~g/L and 180 ~g/L concentration~, they are

not included in the statistical analysis and are considered

qualitative mortality effects.

103
TABLE 4. SUMMARY OF SURVIVAL AND GROWTH DATA FOR TOPSMELT,

ATHERINOPS AFFINIS, LARVAE EXPOSED TO COPPER FOR

SEVEN DAYSl

Copper Mean

Cone. Replicate Survival Proportions Proportion

(/Lg/L) A B C D E Survival

0.0 LO 0.8 LO LO LO 0.,96

32.0 LO LO LO LO LO LOO

56.0 0.0 0.6 0.2 LO 0.6 0.48

1.00.0 0.0 0.0 0.0 0.0 0.0 0.00

1.80.0 0.0 0.0 0.0 0.0 0.0 0.00

Cone. Replicate Average Dry Weights (mg) ,Mean Dry

(/Lg/L) A B C D E Wgt (mg)

0.0 0.001.34 0.00153 0.00134 0.00146 0.00144 0.00142

32.0 0.00146 0.00142 0.00150 0.00138 0.00128 0.00141

56.0 0.00147 0.00170 0.00124 0.00130 0.00114

1.00.0

180.0

lFive replicates of 5 larvae each.

104
Figure 1. Flowchart ;or stat'istical analysis of the topsmelt,

Atherinis affinis, la~val survival data by hypothesis testing.

105
MORTALITY DATA

.,DEAD

TWO OR MORE

PARTIAL MORTALITIES?

YES

IS PROBIT MODEL

APPROPRrATE?

. (SIGNIFICANT X2 TEST)

YES

PROBITMETHOD

Figure 2. Flowchart for statistical analysis.of the topsmelt,

Atherinis affinis, larval survival data by point estimation.

106
TABLE 5. TOPSMELT, ATHERINOPS AFFINIS, SURVIVAL DATA I

Copper Concentration

(/lg/L)

Replicate Control 32.0 56.0

A 1;0 1.0 0.0

RAW B 0.8 1.0 0.6

C 1.0 1.0 0.2

D 1.0 1.0 1.0

E 1.0 1.0 0.6

A 1.345 1.345 0.225

ARC SINE B 1.107 1. 345 0.886

SQUARE C 1 ..345 01.345 0.464

ROOT D 1.345 1.345 1. 345

TRANSFORM E 1. 345 1. 345 0.886

ED

11.13.2.6 Test fo~ Normality

1.297

0.0113

1

1. 345

00.000

2

0.761

0.187

3

11.13.2.6.1 The first step of the test for normality is to

center the observations by subtracting the mean of all

observations within a concentration from each observation in that

concentration. The centered observations are summarized in Table

6.

107
1.0 r * * * * ........ ......... --- ~M_'oIiUCI'OlUCH~

0.9

0.8 ,:

z

07 j 0

a::

0 0.6 '\ * D..

0

a::

0- 0.5 ..,

-' 1 ..... 1

0 <

0.4 i > CD -

>

a::

=> 0.31 (/J

0.2

4 *

0.1 j

0.0

0 32 56 100 180

COPPER CONCENTRATION (ugll)

Figure 3 Plot of mean survival proportion data in Table 5.

-
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-,wILK'S EXAMPLE

Copper ConcE~ntration

( flg/]~)

Replicate Control 32.0 56.0

A 0.048 0.000 -0.536

B ...,0.190 0.000 0.125

C 0.048 0.000 -0.297

D 0.048 0.000 0.584

E 0.048 0.000 0.125

11.13.2.6.2 Calculate the denominator, D, of the statistic:

n - 2

D = E (X.-X)

i.l

Where:

11.13.2.6.3

11.13.2.6.4

largest

Xi = the ith centered observation

X = the overall mean of t4e centE~red observations

n the total number of centered observations

For this set of data,

n = 15

x = (0.003) = 0.000

15

D 0.793

Order the centered observations from smallest to

109
where Xli) denotes the ith ordered observation. The ordered

observations for this example are listed in Table 7.

TABLE 7. ORDERED CENTERED OBSERVATI,ONS. FOR THE SHAPIRO-WILKIS

EXAMPLE

i Xli) i Xli)

1 -0.536 9 0.048

2 -0.297 10 0.048

3 -0.190 11 0.048

4 0.000 12 0.048

5 0.000 13 0.125

6 0.000 14 0.125

7 0.000 15 0.584

8 0.000

11.13 .2.6 .5 From Table 4, Appendix B, ·for the number of

observations, n, obtain the coefficients a1, a 2 , ••• a k where k is

n/2 if n is even and (n-1)/2 if n is odd. For the data in this

example, n = 15 and k = 7. The ai values' are listed in Table 8.

11.13.2.6.6 Compute the test statistic, W, as follows:

k 2

W = [Ea (X (n-i.1) -X (i») ]

D i.1 i

The'differences xln-i+1)

in this example,

Xli) are listed in Table 7. For the data

1 2

W = --(0.817) = 0.842

0.793

110
11.13.2.6.7 'The decision rule for this test is: to compare W as

calculated in Subsection 11.13.2.6.6 to a critical value found in

Table 6, Appendix B. If the computed W is less than the critical

value, conclude that the data are not normally distributed. For

the data in this example, the critical value at a significance·'

level of 0.01 and n = 15 observations is 0.835. Since W ='0.842

is greater than the critical value, conclude ,that the dat,a are

normally distributed.

11.13.2.6.8 Since the variance of the lowest, copper

concentration group is zero, Bartlett's test statistic can not be

calculated. Therefore, the survival data variances are

considered to be heterogeneous.

11.13.2.6.9 Since the data do not meet the assumption of

homogeneity of variance,' Steel's Many-one Rank Test will be used

TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S

EXAMPLE

i X (n-i+ll - X (il

1 0.5150 1.12.0 X(lSJ X(l)'

2 0.'3306 0.422 X(l4) - X(2)

3 0.2495 0.315 X(l3) - X(3l

4 0.1878 0.048 X(12) X(4)

5 0.1353 0.048 X(l1.J - Xes)

6 0.0880 0.048 X(lO) - X(6)

7 0.0433 0.048 X(9) - XC?)

to analyze the survival data.

11.13.2.7 Steel's Many-one Rank Test

11.13.2.7.1 For each control and concentration combination,

combine the data and arrange the observations il~ order of size

111
from smallest to largest. Assign the ranks (1, 2, ... , 10) to

the ordered observations with a rank of 1 assigned to the

smallest observation, rank of 2 assigned to the next larger

opservation, etc. If ties occur when ranking, assign the average

rank to each tied observation.

11.13.2.7.2 An example of assigning ranks to the combined data

for the control and 32.0 ~g/L copper concentration is,given in

Table 9. This ranking procedure is repeated for each

control/concentration combination. The complete set of rankings

is summarized in Table 10. The ranks are next summed ~or ~ach

copper concentration, as shown in Table 11.

11.13.2.7.3 For this example, 'determine if the survival in any

of the copper concentrations is significantly lower than the

survival in the control. If this occurs, the rank sUm at that

concentration would be significantly lower than the ,rank sum of

the control. Thus, compare the rank sums for the survival at

each of the various copper concentrations with some "minimum" or

critical rank sum, at or below which the survival would be

considered significantly lower than the control. At a

significance level of 0.05, the minimum rank sum in a test with

two concentrations (excluding 'the control) and five replicates is

18 (see Table 5; Appendix E) .

11.13.2.7.4 Since the rank sum for the 56.0 ~g/L copper

concentration is equal to the critical value, the proportion

surviving in the 56.0 ~g/L concentration is considered

significantly less than that in the cont~ol. Since the other

rank sum is not less than or equal to the critical value, it is

not considered to have a significantly lower proportion surviving

than the control. Hence, the NOEC and the LOEC are the 32.0 ~g/L

and 56.0 f1.g/L concentrations, respec.tively.

11.13.2.8 Calculation of the LC50

11.13.2.8.1 The data used for the calculation of the LC50 is

summarized in Table 12. For estimating the LC50, the data for

the 100 f1.g/L and 180 f1.g/L copper concentrations with 100%

mortality are included.

112
TABLE 9. ASSIGNING RANKS TO THE CONTROL AND 32.0 ~g/L,

COPPER CONCENTRATION FOR STEEL'S MANY-ONE RANK

TEST

Rank

1

6

6

6

6

6

6

Transformed

Proportion

Surviving

1.107

1.345

1.345

1.345

1.345

, 1.345

1.345

Copper

Conc1entration

(~g/L)

Control

32.0

32.0

32.0

3:2.0

32.0

Control

;

I '

TABLE 10. TABLE OF RANKS

Copper Concentration <,ug/L)

Replicate Control 32.0 " 56.0

A 1.345 (6, 8) 1.345 (6) , 0.225 (1)

B 1.107 (1, 5) '1.345 (6) 1 0 . 886 "(3.5) I

C 1.345 (6, 8) 1.345 (6) 0.464 (2)

D 1.345 (6, 8) 1.'345 (6) . 1.345 (8)

E 1.345 (6, 8) 1.345 (6) :0.886 ' (3 .5)

113
TABLE 11. RANK SUMS

Copper Concentration Rank Sum

(p.g/L)

32.0 30

56.0 18

11.13.2.8.2 Because there are is only one partial mortality in

the set of copper concentration responses, Probit Analysis is not

appropriate to calculate the LC50 and 95% confidence interval for

this set of test data. Inspection of the data reveals that, once

the data is smoothed and adjust~d, the proportion mortality in

the lowest effluent concentration will be zero and the proportion

mortality in the highest effluent concentration will be one,

Therefore, the Spearman-Karber Method is appropriate for this

data.

11.13.2.8.3 Before the LC50 can be calculated the data must be

smoothed and adjusted. For the data in this example, because the

observed proportion mortality for the 32.0 ~g/L copper

concentration is less than the observed response proportion for

the control, the observed responses for the control and this

group must be averaged:

s s

Po = P1 =

0.040+0.000

2

0.020

Where: pj = the smo~thed observed mortality proportion" for

effluent concentration i.

11.13.2.8.3.1 Because the rest of the responses are monotonic,

additional smoothing is not necessary. ~he smoothed observed

proportion mortalities are shown in Table 12.

11.13.2.8.4 Because the smoothed observed proportion mortality

for the control is now greater than zero, the data in each

effluent concentration must be adjusted using Abbott's formula

114
(Finney, 1971). The adjustment takes the form::

pr = (Pf - pg) / (1 - pg)

Where: pg = the smoothed observed proportion mortality for the

control

Pf = the smoothed observed proportion mortality for

effluent concentration i

11.13.2.8.4.1 For the data in this example, the data for each

effluent concentration must be adjusted for control mortality

using Abbott's formula, as follows:

0.'020-0.020

1"-0.020

0.000

0.980

:: 0.0

0.520-:0.020

1-0.02,0

1. 000-0.020

1-0.020

0.500 a 0 ..510

0.980

0.980 a 1. ooq 0.980

The smoothed, adjusted response proportions for the,eff.luent

concentrations are, shown in Table 12.

11.13.2.8.5 Calculate the loglo of the estimated LC50, m, as

follows:

i-l 2

Where: pf the smoothed adjusted proportion mortality at

concentration i

Xi the lOglO of concentration i

115
k = the number of effluent concentrations tested, not

including the control

TABLE 12. DATA FOR EXAMPLE OF SPEARMAN~KARBER ANALYSIS

Copper Number of Smoothed Adjusted

Concentration Number Organisms Mortality Mortality Mortality

% of Deaths Exposed Proportion proportion proportion

Control 1 25 0.040 0.020 0.000

32.0 0 25 0.000 0.020 0.000

56.0 13 25 0.520 0.520 0.510

100.0 25 25 .1. 000 1.000 1.000

180.0 25 25 1.000 :i..ooo 1. 000

11.13.2.8.5.1 For this example, the 10910 of the estimated LC50,

m, is calculated as follows:

m =: [(0.510 - 0.000)

[(1.000 - 0.510)

[ (1.000 - 1. 000)

,

= 1.7479

(1.5051 + 1.7482)]/2 +

(1.7482 + 2.0qOO)]/2 +

(2.0000 + 2.2553)]/2 +

11.13.2.8.6 Calculate the estimated variance of m as follows:

k-1 P .. (l-p ") (X +X ) 2

V(m) =:E i i i.1 ~-1

i-2 4 (ni-1)

Where: Xi = the 10glo of concentration i

ni "" the number of organisms tested at effluent

concentration i

p~ = the smoothed adjusted observed proportion mortality

at effluent concentration i

k = the number of effluent concentrations tested, not

including the control

11.13.2.8.6.1 For this example, the est~mated variance of m,

V(m), is calculated as follows:

116
I

, '

I

V(m) = (0.5,10) (0.490) (2.0000 - 1.5051)2/4(24) +

(1. 000) (0. 000) (2.2553 - 1. 74.82) 2/4 (24)

0.0006376

11.13.2.8.7 Calculate the 95% confidence interval for m:m ±

2.0 J V(m)

11.13.2.8.7.1 For this example, the 95% confidence interval for

m is calculated as follows:

1.7479 ± 2 JO.0006376 = (1.6974, 1.7984)

11.13.2.8.8 The estimated LC50 and a 95% confidence interval' for

the estimated LC50 can be found by taking base10 antilogs of the

above values.

11.13.2.8.8.1 For this example, the estimated 1:"C50 is calcul,ated

as follows:

LC50 = antilog(m) antilog'1.7479) = 56.0 ~g/L ..

11.13.2.8.8.2 The limits of the 95% confidence interval for the

estimated LC50 are calculated by taking the antilogs of the upper

and lower limits of the 95% confidence interval.for m as follows.:

lower limit:

upper limit:

antilog(1~6974)49~8 ~g/L

antilog(1.7984) = 62~9 ~g/L

11.13.3 EXAMPLE OF ANALYSIS OF TOPSMELT, ATHERTNOPS AFFINIS,

GROWTH DATA

11.13.3.1 Formal statistical analysis of the gJ:~owth data is

outlined in Figure 4.

The response used in the' statis.tical analysis lEI mean weight per

surviving organism for each replicate. The IC2:; can be

calculated for the growth data via a point estinlation technique

(see Section 9, Chronic Toxicity Test Endpoints and Data

Analysis). Hypothesis testing can be used to obtain an'NOEC and

LOEC for growth. Concentrations above,the NOEC for su~vival are

excluded from the hypothesis test for growth effects.

11:7
11.13.3.2 The statistical analysis using hypothesis testing

consists of a parametric test, Dunnett's Procedure, and a

nonparametric test, Steel's Many-one Rank Test. The underlying

assumptions of the Dunnett's Procedure, normali~y and homogeneity

of variance, are formally tested. The test for normality is the

Shapiro-Wilk's Test and Bartlett's Test is used to test for

homogeneity,of variance. If either of these tests fails, ~he

nonparametric test, Steels' Many-one Rank Test, is used to

determine the NOEC and LOEC endpoints. If the assumptions of

Dunnett's Procedure are met, the endpoints are determined by the

parametric test.

11.13.3.3 Additionally, if unequal numbers of replicates occur

among the concentration levels tested there are parametric, and

nonparametric alternative analyses. The .parametric analysis is a

t test with the Bonferroni adjustment. The Wilcoxon Rank Sum

Test with the Bonferroni adjustment is the nonparametric

alternative. For detailed information on the Bonferroni

adjustment, see ~ppendix D.

11.13.3.4 The data, mean and variance of the observations at

each concentration. including the control are listed in Table 13.

A plot of the mean weights for each treatment is provided in

Figure 5. Since there is no survival in the 100 ~g/L and 180

~g/L copper concentrations, they are not considered in ,the'

growth analysis. Additionally, since there is significant

mortality in the 56.0 ~g/L concentration, its effect on growth is

not considered.

11.13.3.5 Test for Normality

11.13.3.5.1 The first step of the test for normality is,to

center the observations by subtracting the mean of all 'the

observations within a concentration from each observation in that

concentration. The centered observations are summarized in Table

14.

118
Figure 4. Flowchart for statistical analysis of the topsmelt,

Atherinops affinis, larval growth data.

~~9
TABLE 13. TOPSMELT, ATHERINOPS AFFINIS, GROWTH DATA

Copper Concentration (j.Lg/L)

Replicate Control 32.0 56.0 100.0 180.0

A 0.00134 0.00146

B 0.00153 0.00142

C 0.00134 0.00150

D 0.00146 0.00128

E 0.00144 0.00141

Mean (y!) 0.00142 0.00141

Sf 0.000000006 0.000000007

i 1 2 3 4 5

TABLE 14. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S

EXAMPLE

Replicate

A

B

C

D

E

Control

-0.00008

0.00011

-0.00008

0.00004

0.00002

32.0 ~g/L Copper

0.00005

0.00001

0.000.09

-0.00003

-0.00013

11.13.3.5.2 Calculate the denominator, D, of the test statistic:

n

r -2

D=L..t(X -X) i

i-l

Where: Xi = the ith centered observation

X = the overall mean of the centered observations

n = the total number of centered observations.

120
------------------*------~- -----" - -------------------------------------- 0'

E

f-

·r

<..'J

'-1

0.0015 " >k.

*

>k

* ;,.:. * IIWIXlM.R9'!JCATEMfAHWElOHf

COHtECT1lHEliQH VAW£ FOR liACH CONCEHTMTlON

~ll£~ VN.UER*:DUNtlETTIlarr

WltWElOtlTlBDWitIS VALUEYlOUlD IE

8lClHFICNm,YDl'FEREHfFJllOMntEODH'lROC.,I

I-'

t-.>

I-'

u.J

3: 0.0010

>-'

lY

o

Z

<C

w .,

:::;; 0.0005

00000 -1 ' • I I ?f :?Ie l

o 32 56

COPPER CONCENTRATION (ugll)

100 180

Figure 5. Plot of weight data from topsmelt, Atherinis affinis, larval survival and growth test.
For this set of data, n = 10

x = __1_(0.00) = 0.00

10

D = 0.000000055

11.13.3.5.3 Order the centered observations from smallest to

largest:

X11) X12) ••• X ln)

Where Xli) is the ith ordered observation. These ordered

observations are listed in Table 15.

11.13.3.5.4 From Table 4, Appendix B, for the number of

observations, n, obtain the coefficients au a 2 , ••• , a k where k

is n/2 if n is even and (n-l)/2 if n is odd. For the data in

this example, n = 10 and k = 5. The ai values are listed in

Table 16.

TABLE 15. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S

EXAMPLE

i Xli) i X(il

1 -0.00013 6 0.00002

2 -0.00008 7 0.00004

3 -0.00008 8 0.00005

4 -0.00003 9 0.00009

5 0.00001 10 0.00011

122
TABLE 16. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILlC'S EXAMPLE

i ai X ln- i +1) - Xli)

1 0.5739 '0.00024 X I1O) - X(11

2 0.3291 0.00017 X(9) - X(2)

3 0.2141 0.00013 XU) - X(3!

4 0.1224 0.00007 Xl7l - X(4)

5 0.0399 0.00001 XJ6) - X(S~

11.13.3.5.5 Compute the test statistic,W, as follows:

The differences X Cn -i +1 ) - Xli) are listed in Table 16. For this set

of data:

w = 1 (0.0002305)2 = 0.966

0.000006055

11.13.3.5.6 The decision rule for this test ,is to compare W with

the critical value found in Table 6, Appendix B. If the computed

W is less than the critical value, conclude that t~e.data are not

normally distributed. For this example, the critical value at a

significance level of 0.01 and 10 observations (n) is 0.781. '

Since W = 0.966 is greater than the crit~cal value, the conclude

that the data are normally distributed.

11.13.3.6 Test for Homogeneity of Variance

11.13.3.6.1 The test used to examine whether the variation in

mean dry weight is the same across all effluent c~ncentrations

including the control, is Bartlett's Test (Snedecor and Cochran,

1980). The test statistic is as follows:

B ='

p p

ULVi) In.? - LVi In s:] i-I ,t-l

c

123
Where: Vi = degrees of freedom for each effluent

concentration and control, Vi = (ni - 1)

ni = the number of replicates for concentration i

p = number of levels of' effluent concentration

including the control

In = loge

i = I, 2, ... , p where p is the number of

concentrations including the control

p

(LV1 S:)

? = _1-_1__

p p

C = 1+ [3 (p-l) r 1 [Ll/V _ (LV) -1] 1 . 1

1·1 1-1

11.13.3.6.2 For the data in this example (see Table 14), all

effluent concentrations including the control have the same

number of replicates (ni = 5 for alIi). Thus, Vi = 4 for all i.

11.13.3.6.3 Bartlett's statistic is therefore:

p

B= [(8)ln(6.5x10- 9 ) - 4Lln(s:)] /1.125

1-1

= [8(-18.851). - 4(-37.709)]/1.125

= 0.028/1.125

= .0.0249

124
11.13.3.6.4 B is approximately distributed as Chi-square with p

- 1, degrees of freedom, when 'the variances are in fact the same.

Therefore, the appropriate critical value for this test, a:t a

significance level 'of 0.01 with one degree of freedom, is 6.635.

Since B = 0.0249 is less than the critical value of 6.635,

conclude that the variances are not different.

11.13.3.7 Dunnett's Procedure

11.13.3.7.1 To obtain an estimate of the pooled'variance for the'

Dunnett's Procedure, construct an ANOVA table as described in

Table 17.

TABLE 17. ANOVA TABLE

Source

Between

Within

df

p - 1

N - P

Sum of Squares

(SS)

SSB

SSW

Mean Square(JI1S)

. (SS/df')

2

SB SSB/ (p-:1)

2

Sw = SSW/ (N-p)

---------------------~----:----'-----

Total N - 1 SST

Where: p = number of concentrat,ion levels' i~~cluding, the'

control

N = total number of observations, n 1 +. n 2 ••• + TIp

, ,

n i = number of observations in concentration i

p

SSB = 'ET:lni-G2 IN Between Sum of Squa:~es 1-1' -~

p' n i

SST = 'E'EY1~-G2 IN Total Sum of Squares

1-1:/-1

125
ssw = SST-SSB

G =

Within Sum of Squ~res

the grand total of all sample observations,

Ti = the total of the replicate measurements for

concentration i

Yij = the jth observation for concentration i

(represents the mean dry weight of the mysids for

concentration i in test chamber j)

11.13.3.7.2 For the data in this example:

N "" 10

G = T1 + T2 = 0.01415

p

SSB. ET:lnJ.-G2/N

J..1

= --L (1 . 001137 x 10-4 ) - ( 0 . 01415) 2

5 10

p nj

SST. EEYJ.2j-G2 IN

,1.1j.1

4.90 X 10-10

=: 0.0000201 - (0.01415) 2 = 7.775 X 10-8

10

126
ssw " SST-SSE :::: 7.775 X 1,0-8 - (4.9 x 10-1 °) ,- 7.726 X 10-8

2

SB :::: SSB/ (p-1) :::: (4.9 x 10-1 °) / (2-1) :::: 4.9 X 10-1 °2

Sw - SSW/ (N-p) :::: 7.726 x 3,.0-8 / (10-2) :::: 9.658 X 10-9

11.13~3.7.3 Summarize these calculations in theANOVA table

(Table 18).

TABLE 18. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXP.MPLE

Source df Sum of Squares Mean Square n~S)

(SS) (SS/df)

Between 1 4.90, x, 10-10 4.9 X 10-10 ,

Within 8 7.726 X 10-8 9.658 X 10-9

Total 9 7.775 X 10-8

11.13.3.7.4 To perform the individual comparisons, calculate the

t statistic for each concentration, and control combination as

follows:

t " 1

Where: Y i :::: mean dry weight for effluent concentration i

Y1 :::: mean dry weight for the control

Sw :::: squar~ root of the within m~an squal;~

n1 :::: number of replicates for the control

ni :::: number of replicates for concentrati,on i.

I

11.13.3.7.5 Table 19 includes the calculated t values for each

concentration and control combination. In this e:jcample there is

only one comparison, of the 32.0 ~g/L copper conc~ntratioh with

the control. 'The calculation is as follows:

127
t = 2

(0.00142 - 0.00141)

[9.828x10-S V(l/s) + (liS) ]

= 0.161

TABLE 19. CALCULATED t VALUES

Copper Concentration (~g/L)

32.0

i

2 0.161

11.13.3.7.6 Since the purpose of this test is to detect a

significant reduction in mean weight, a one-sided test is

appropriate. The critical value for this one-sided test is found

in Table 5, Appendix C. For an overall alpha level of 0.05, 8

degrees of freedom for error and one concentration (excluding the

control) the critical value is 1.86. The ·mean weight for

concentration i is considered significantly less than the mean

weight for the control if t i is greater than the critical value.

Since t 2 is less than 1..86, the 32. a f-Lg/L' concentration does not

have significantly lower growth than 'the control. Hence the NOEC

and the LOEC for growth cannot be calculated.

11.13.3.7.7 To quantify the sensitivity.of the test, the minimum

significant difference (MSD) that can be statistically detected

may be calculated:

MSD = d S ./(lin) + (lin) "V 1

Where: d = the critical value for Dunnett's Procedure

Sw = the square root of the within mean square

n = the common number of replicates at each

concentration

(this assumes equal replication at each

concentration)

n 1 = the number of replicates in the control.

1.28
11.13.3.7.8 In this example:

MSD = 1.86 (9.828x10-5 ) V(l/4) + (1/4)

:= 1. 86 (9 . 828 x 10-s) (0. 632)

:= 0.000116

11.13.3.7.9 Therefore, for this set of data, the minimum

difference that can be detected as statistically significant is

0.000116 mg.

11.13.3.7.10 This represents a 8.2% reduction in mean ~eight

from the control.

11.13.3.8 Calculation of the ICp

11.13.3.8.1 The growth data from Table 4 are utilized in this

example. As seen from Table 4 and Figure 6, the observed means

are monotonically non-increasing with respect to concentration

(mean response for each higher concentration is less than or

equal to the mean response ·for the previous concentration and 'the

responses between concentrations follow a linear trend) .

Therefore, the means ,do not require smoothing prior to

calculating the IC. In the following di~cussionj' the observed

means are represented by Yi and the smoothed mearis by Mi.

11~13.3.8.2 Since Ys = 0 < Y4 = 0 < Y3 := 0~00114 < Y2 = 0.00141

< Y 1 = 0.00142, set M1 := 0.00142, M2 = 0.00141, M3 = 0.00114, M4 =

o and Ms = o.

11.13.3.8.3 Table 20 contains the response means and smoothed

means and Figure 8 gives a plot of the smoothed response curve.

11.13.3.8.4 An IC25 can be estimated uSlng the Linear,

Interpolation Method. A 25% reduction in weight, compared to the

controls, would result in a mean dry weight of 0.001065 mg, where

M1 (1-p/100) = 0.00142(1-25/100). Examining the s~oothed means

and their associated concentrations (Table 20), the response,

0.001065 mg, is bracketed by C3 = 56.0 p.g/L copper and C4 = 100.0

p.g/L copper.

129
11.13.3.8.5 Using the equation from Section ,4.2 of Appehdix M,

the estimate of the IC25 is calculated as follows:

I C2 5 "" 5 6 . 0 + [0. 0 014 2 (1 - 25/10 0 ) - O. 0 0114 ] (10 0 . 0 - 5 6 • 0 )

(0.0 - 0.00114)

= 58.9 j1.g/L.

11.13.3.8.6 When 'the ICPIN program was .used to analyze this set

of data, requesting 80 resamples, the' estimate of the IC25, was, .

58.9089 p.g/L. The empirical 95% confidence interval for t,he true

mean was 44.2778 p.g/L to 67.0000 p.g/L. The computer program,

o~tput for the IC25 for this data set is shown in Figure 7.,

TABLE 20. TOPSMELT, ATHERINOPS AFFINIS, MEAN GROWTH

RESPONSE AFTER SMOOTHING

Copper Response Smoothed'

Cone. (j1.g/L) i Means Means

(mg) Yi (mg) Mi

Control 1 0.00142 0.00142

32.0 2 0.00141 0.00141

56.0 3 0.00114 0.00114

100.0 4 0.0 0.0

180.0 5 0.0 0.0

11.14.1 PRECISION

11.14.1.1 Single-Laboratory Precision

11.14.1.1.1 Data on the single-laboratory precision of t,he

topsmelt larval survival and growth test using copper chloride as

the reference toxicant are provided in Tables 21 an~ 22. In the

five copper tests presented here, the NOECs for survival were 100

p.g/L for all tests but one; this test had a NOEC of 180 f.l.g/L.

The coefficient of variation for copper pased on the LC25 is

17.3% for survival; the coefficient. of variation for copper based

130
on the LCSO is '9.7% for survival. The weight endpoint was' less

sensitive than survival in all but one test. An IC2S could be

calculated for three of five tests and the coeff:~'cient of

variation for these three tests was 60.69%, the (~oefficient of

variation based on the IC50 for these three ,testB was 4.75%.

11.14.1.2 Multilaboratory Precision

14.11.1.2.1 Qata on the interlaboratory, precision of the

topsmelt larval survival and growth test are provided in Table

23. Three separate interlaboratory tests were conducted. In the

first comparison both laboratories derived identical NOECs for'

copper (100~g/L). The coefficient of variation, based on LC50s

for survival was 36%. In the second comparison the NOEC for

effluent was 20% at both laboratories. The coeff'icient of

variation, based on the LC50sfor survival was 19%. In the third

comparison the NOEC for copper was 32 ~g/L at both laboratories.

The coefficient of variation, based on the LCSOs for survival was

3%.

11.11.2 ACCURACY

11.11.2.1 The accuracy of tox~city tests cannot be determined.

131
0000201 * * * ~1IQU:A1'I' fIUJf MIG'fT

--- COMICCTI l'HeCl-IMYDIlUlfVALUQ

------ ~JHI!wocmeIlUllV~.

i *

1><

0' 0.0015"1,

E * l- ;t ::c * (;l

-

w

5: 0.0010 J

>-

1

I-' e>::

lJJ 0

tv z

0000,1 <:

w

"::;;;

11

o.oOO0L,- I t

0 32 56 100 180

COPPER CONCENTRATION (IigIL)

Figure 6.. Plot of raw data, observed means, and smoothed means for topsmelt, Atherinis affinis, growth

data from Tables 4 a~d 21.
Conc. ID

Conc. Tested

Response 'J.

Response 2

Response 3

~esponse 4

Response 5

J.

o

.00134

.00153

.00134

.00J.46

.00J.44

2

32

.00J.46

.00J.42

.00J.50

.00J.38

.00J.28

3

56

o

.00J.47

.00J.70

.00J.24

.00130

4

100

o

o

o

o

o

5

180

o

o

o

o

o

*** Inhibition Concentration Percentage Estimate *** Toxicant/Effluent: Copper

Test Start Date: Test Ending Date:

Test Species: Atherinops affinis

Test Duration: 7 days

DATA FILE: wc_aa.icp

OUTPUT FILE: wc_aa.i25

Conc.

ID

Number

Replicates

Concentration

ug/L

Response

Means

Std.

Dev. '

Pooled

Response Means

-------------------~---------------------------~------ --_._-------------

J. ,5 0.000 O.OOJ. 0.000 O,OOJ.

2 5 32.000 0.001 O.OOl) 0.001

3 5 56.000 0.001 O.OO:L 0.001

4 5 J.OO.OOO 0.000 0.000 0.000

5 5 180.000 0.000 0.000 0.'000

The Linear 'Interpolation Estimate: . 58.9089 Entered P Value: 25

58.J.571

Lower:

Lower:

0.11

.,-

Number of Resamplings: 80

The Bootstrap Estimates Mean:

Original-Confidence Limits:

Expanded Confidence Limits:

Resampling time in Seconds:

Standard Deviation: 7,.9299

44.2778 Upper: 67.0000

36.9622 Upper: 71.0455

Random_Seed: -498847050

Figure 7. ICPIN program output for the IC25

133
TABLE 21. SINGLE LABORATORY PRECISION OF THE TOPSMELT, ATHERINOPS AFFINIS

SURVIVAL ENDPOINT WITH COPPER (CU~G/L) CHLORIDE AS A REFERENCE

TOXICANT

Test Number NOEC LC25 LC50

1 100 142.1 187.4

I

I 2 100 NC3 162.4

3 100 151.7 165.6 I

4 180 181.0 190.6

-- 5 100 119.2 204.0

,

# of Tests Statistic LC25 LC50

5 Mean 148.5 182.0

SD 25.6 17.6

I CV (%) 17.3 9.7%

TABLE 22. SINGLE LABORATORY PRECISION OF THE TOPSMELT, ATHERINOPS AFFINIS

GROWTH ENDPOINT WITH COPPER (CU ~G/L) CHLORIDE AS A REFERENCE

TOXICANT

Test Number NOEC LC25 LC50

1 180 222.1 264.2

i

2 180 NC4 NC4

I 3 >180 NC4 NC4 ,

!

4 56 47.6 NC4

5 >180 NC4 NC4

, # of Tests Statistic LC25 LC50

, 5 Mean 156.8

SD 95.2

CV (%) 60.7%

1Data from Anderson et al. 1994; po~nt est~mates calculated us~ng prob~t

analysis, except where noted.

2Five replicate exposure chambers with five larvae per chamber were used for

each treatment.

3LCSO calculated using Spearman-Karber method, this method does not calculate

an LC25.

4point estimate not calculated because the response was less than either 25 or

50%.

134
TABLE 23. MULTI-LABORATORY PRECISION OF THE TOJ?SMELT, .

ATHERINOPS AFFINIS, GROWTH AND SURVIVAL TEST

CONDUCTED WITH COPPER (CU /l-G/L) CHLORIDE AS A

REFERENCE TOXICANT

- Test Toxicant Laboratory Survival Growth

Number :

- NOEC Le50

1 Copper lb 100 162.0 NSc

Coppera 2d 100 274.0 NS

CV 36%

2 Effluent lb 20 31.4 NS

Effluent 2e 20 23.9 10

CV 19%

3 Coppera lb 32 55.7 NS

Coppera Ie 32 5.8.4 NS

CV 3:~

Two separate interlaborat()ry comparisons were cOl;1ducted, in

August 1990 and August 1991.

aThe August 1990 copper test was conducted at 34~ salinitYi the

August 1991 copper test was conducted at:: 20%0 salinity.

bMarine Pollution Studies Laboratory, Monterey County,

California.

CNot Significant.

dVantuna Research Group, Occidental College t Cali.fornia.

eChevron Research and Technology Co., Environmental Research

Group.

135
APPENDIX I. TOPSMELT TEST: STEP-BY-STEP SUMMARY

PREPARATION OF TEST SOLUTIONS

A. Determine test concentrations and appropriate dilution water

based on NPDES permit conditions and guidance from the

appropriate regulatory agency.

B. Prepare effluent test solutions by diluting well mixed

unfiltered effluent using volumetric flasks and pipettes.

Use hypersaline brine where necessary to maintain all' test

solutions at 34 ± 2~. Include brine controls in tests that

use brine.

c. Prepare a copper reference toxicant stock solution (10,000

~g/L) by adding 0.0268 g of copper chloride (CuCI 2 02H20) to 1

liter of reagent water.

D. Prepare zinc reference toxicant solution of 0 (control) 56,

100, 180, and 180 ~g/L by adding 0, 5.6, 10.0, 18.0, and

32.0 mL of stock solution, respectively, to a 1-L volumetric

flask and filling to 1-L with dilution water.

E. Sample effluent and reference toxicant solutions fqr

physical/chemical analysis. Measure salinity, pH and

dissolved oxygen from each test concentration.

F. Randomize numbers for test chambers and record the chamber

numbers with their respective test concentrations on a

randomization data sheet. Store the data sheet safely until

after the test samples have been analyzed.

G. Place test chambers in a water bath or environmental chamber

set to 20°C and allow temperature to equilibrate.

H. Measure the temperature daily in one. random replicate (or

separate chamber) of each test concentration. Monitor the

temperature of the water bath or environmental chamber

continuously.

I. At the end of the test, measure salinity, pH, and dissolved

oxygen concentration from each test concentration.

136
. PREPARATION AND ANALYSIS OF TEST ORGANISMS

Remove all dead

Artemia nauplii

A.

B.

C.

D.

E.

F.

G.

H.

I.

Obtain 9-15 day old larvae from a commeric!al supplier or inhouse cultures.

Larvae must' be randomized before placing them into the test

chambers. Be sure that all water used in culture, transfer,

and test solutions is' within 1°C of the test temperature.

larvae daily, and add 40 newly hatched

per larva twice daily; once in the morning , , "

and once in the afternoon. Adjust feeding to account for

larva mortality.

Renew test solutions daily using freshly ptepared solutions,

immediately after cleaning the test chambers.

After 7 days, count and record the number of live and dead

larvae in each chamber. After counting, ,use the

randomization sheet to assign the correct ~est concentration

to each chamber. Remove'all dead larvae.

The surviving larvae in each test chamber a.re immediately

prepared as a group for dry weight determination, or

preserved in 4% formalin then 70% ethanol .. Preserved

organisms are dried and weighed with 7 days.

Carefully transfer the larvae to a prenumbered, preweighed

micro-weigh boat using fine-tipped forceps. Dry for 24

hours at 60~C or at 105°C for a minimum of hours. Weigh

each weigh boat on a microbalance (accurate I to 1 I1-g).

Record the chamber number, larvae weight, wlaigh bo~t weight

(recorded previously), and number of larvae per weigh boat

(replicate) on the data sheet.

Analyze the data.

Include standard reference toxicant point estimate values in

the'standard quality control charts.

137
Test start Date:

Fish Species:

Data Sheet for Larval Fish Toxicity Test

Start Time:

Test End Date:

Collection/Arrival Date:

Reference Toxicant:

Broodstock Source:

Fish Age at Start:

End Time:

Test Concentration Numer Total Total Notes

Cont. Alive Number Number

II Alive at

Day Day Day Day 4 Day Day Day start

1 2 3 5 6 7.

l,;omputcr Data storage

Disk

File

Note: See larval we~ght data on separate sheet.

l38
Data Sheet for Weighing Larval Fish·

I Test Start Date:

Test End Date:

Toxicant:

Start Time:

End

Time:

Fish Species :

Collection!Arrival

Date:.

Fish Age at Start:

Sample Source:

Sample Type: Sediment Elutriate Porewater

Water

Test :site l:oae WeIght of Number welgnt per

Container or Foil Foil Weight Total Weight Larval of Fish Larval Fish

Number Concentration Number (mg) (mg) Fish I Larvae (mg)

(mg)

)

I

:l

6

1

1

I-

.3

t9

U

1

;2

3 . ,

~4

::15

l:omputer Data Storage Notes

Disk:

File:

Note: See larval mortality data on seDarate sheet.

139
SECTION 12

MYSID, Holmesimysis costata

SURVIVAL AND GROWTH TES~ METHOD

Adapted from a method developed by

John W. Hunt, Brian S. Anderson and Sheila L. Turpen

Institute of Marine Sciences, University of California

Santa Cruz, California

(in association with)

California Department of Fish and Game

Marine Pollution Studies Laboratory

34500 Coast Route 1, Monterey, CA 93940

TABLE OF CONTENTS

12.1

12.2

12.3

12.4

12.5

12.6

12.7

12.8

12.9

12.10

12.11

12.12

12.13

12.14

Scope and Application

Summary of Method

Interferences

Safety

'Apparatus and Equipment

Reagents and Supplies

Effluents and Receiving Water Collection,

Preservation, and Storage

Calibration and Standardization

Quality Control

Test Procedures

Summary of Test Conditions and Test

Acceptability Criteria

Acceptability of Test Results

Data Analysis

Precision and Accuracy

Appendix I Step-by Step Summary

140
SECT:ION 12

MYS:ID, HOLMBSIMYSIS COSTATA

SURV:IVAL AND GROWTH TEST'

12.1 SCOPE AND APPL:ICAT:ION

12.1.1 This method estimates the chronic toxicity of effluents

and receiving waters to the mysid, Holmesimysis costata~ ,using

three-to-fourday old juveniles in a seven-day, ,static-renewal

exposure. The effects include the synergistic, antagonistic,

and additive effects of a~l chemical, physical, and additive

components which adversely affect the physiological and

biochemical functions of the test,organisms.

12.1.2 Daily observations of mortality make it possible to also

calculate acute toxicity for desired exposure pE!riods (i.e., 24-

h, 48-h, 96-h LC50s) .

12.1.3 Detection limits of the toxicity of an e:ffluent or a pure

substance are organism dependent.

12.1.4" Brief excursions in toxicity may not be detected using

24-h composite samples. Also, because of the long sample

collection period involved in composite sampling and because test

chambers are not sealed, highly volatile 'and highly degradable

toxicants present in the source may not be detected 'in the test.

12.1.5 This method is commonly used in one of two forms:

(1) a definitive test, consisting"of a minimum of, five effluent

concentrations and a control, and (2) a receiving water test(s), / '

consisting of one or more receiving water concentrations and a

control., .

12.1.6 This method should be restricted to use by, or under the

supervision of, professionals experienced in aqtiatic toxicity

testing. Specific experience with any toxicity t:est, is usually

needed before acceptable results become routine.

12.2 SUMMARY OF METHOD

12.2.1 This method provides step-by-step instructions for

141
performing a 7-day static-renewal toxicity test using growth and

survival juvenile mysids to determine the toxicity of substances

in marine waters. The test endpoints are survival and growth.

12.3 INTERFERENCES

12.3.1 Toxic substances may be introduced by contaminants in

dilution water, glassware, sample hardware, and testing equipment

(see Section 5, Facilities, Equipment, and Supplies)., ,

12.3.2 Improper effluent sampling and handling may adversely

affect test results (see Section 8, Effluent and Receiving Water

Sampling, Sample Handling, and Sample Preparation for Toxicity

Tests) .

12.3.3 The test results can be confounded by (1) the presence of

pathogenic and/or predatory organisms in the dilution water,

effluent, and receiving water, (2) the condition of the brood

stock from which the test animals were taken, (3) the amount and

type of natural food in th~ ef'fluent, receiving water, or

dilution water, (4) nutritional value of ,the brine shrimp,

Artemia nauplii, fed during the test, and (5) the quality of the

brine shrimp, Artemia nauplii, or other food added during the

test, which may sequester metals and other toxic substances, and

lower the DO.

12.4 SAFETY

12.4.1 See Section 3, Health and Safety.

12.5 APPARATUS AND EQUIPMENT

12.5.1 Tanks, trays, or aquaria -- for holding and acclimating

adult mysids, e.g., standard salt water aquarium or Instant Ocean

Aquarium (capable of maintaining seawater at 10-20°C), with

appropriate filtration and aeration system.

12.5.2 Air pump, air lines, and air stones -- for aer~ting water

containing mysids for supplying air to test solutions with low

dissolved oxygen.

12.5.3 Constant temperature chambers or water baths -- for

maintaing test solution temperature and keeping dilution water

142
supply, juvenile mysids, and stock suspens{onsat test

temperature {1:3 or 15°C} prior to the test'.

12.5.4 Water purification system -- Millipore Super-Q, Deionized

water (DI) or equivalent.

12.5.5 Refractometer

12.5.6 Hydrometer{s}

for determining salinity.

for calibrating refraqtometer.

12.5.7 Thermometers, glass or electronic, laboratory grade

for measuring water temperatures. .

12.5.8 Thermometer, National Bureau of Standards Certified (see.

USEPA METHOD 170.1,. USEPA, 1979) -- to calibratl9 laboratory

thermometers.

12.5.9 pH and DO met,ers - - for routine physical and chemical

measurements.

1

12.5.10 Standard or micro.-Winkler apparatus --, for determining

DO (optional.) and calibrating the DO meter.

12.5.11 Winkler bottles -- for dissolved oxygen determinations.

12.5.12 Balance -- Analytical, capable of accu~ately weighing to

0.0001 g (for weighing reference toxicants) .

12.5.13 Microbalance -- Analytical, cap~ble of accurately

weighing to 0.000001 g (for weighing mysids) .

12.5.14 Fume hood -- to protect the analyst frdm effluent or

formaldehyde fumes.

12.5.15 Glass stirring rods for mixing test solutions.

12.5.16 Graduated cylinders - - Class A, 'borosilicate glass :or

non-toxic plastic labware, 50-1000 mL for making test solutions.

12.5.17 Volumetric flasks -- Class A, borosilicate glass or nontoxic plastic labware, 10-1000 mL for making test solutions. I

12.5.18 Pipets, automatic '-- adjustable, to cover a range of

143
delivery volumes from 0.010 to 100 mL.

12.5.19 Pipet bulbs and fillers -- PROPIPET@ or equivalen~.

12.5.20 Wash bottles -- for reagent water, for topping off

graduated cylinders, for rinsing small glassware and instrument

electrodes and probes.

12.5.21 Wash bottles -- for dilution water.

12.5.22 20-liter cubitainers or polycarbonate water cooler jugs

-- for making hypersaline brine.

12.5.23 Cubitainers, beakers, or similar chambers of non~toxic

composition for holding, mixing, and dispensing dilution water

and other general non-effluent, non~toxicant contact uses. These

should be clearly labeled and not used for other purposes.

12.5.24 Pipets, volumetric: 1, 10, 25, 50, and 100 mL -- for

dilutions.

12.5.25 Plastic randomization cups (approximately 100 mL, one

for each test chamber) .

12.5.26 Brine shrimp, Artemia, culture unit -- see Subsection

12.6.24 and Section 4, Quality Assurance.

12.5.27 Separatory funnels, 2-L -- two to four for culturing

Artemia.

12.5.28 Mysid culture apparatus (see Section 12.6.25.5). This

test requires 400 three-to four-day-old juvenile mysids.

12.5.29 Gear for collecting adult mysids, including a small

boat, 0.5 mm-mesh hand nets, plastic buckets, and portable air

supply (mysids may also be obtained from commercial suppliers;).

12.5.30 Pipet bulbs and glass tubes (4 mm diameter, with firepolished edges) for handling ad~lt mysids.

12.5.31

tubing)

Siphon tubes (fire polished glass with attached silicone

for test solution renewals.

144
12.5.32 Fire-polished wide-bore 10 mL pipet

juveniles.

for handling

12.5.33 Forceps with fine points -- for transferri~g juveniles

to weighing pans.

12.5.34 Light box for examining organisms. '

12.5.35 Drying'oven, 50-105°C range -- for drying organisms.

12.5.36 Desiccator -- for' holding dried organisms.

12.5.37 Clean NITEX® mesh sieves (~ 150 ~m, 500-1000~m) -- for

concentrating organisms. (NITEX® is available from Sterling

Marine Products, 18 Labe~ Street, Montclair, NJ 07042; 201~783­

9800) .

12.5.38 60 ~m NITEX® filter - for filtering re9~iving water..

12.6 REAGENTS AND SUPPLIES

12.6.1 Sample containers -- for sample shipment and storage (see

Section 8, Effluent and Receiving Water Sampling,and,Samp1e

H~ndling, and Sample Preparation for Toxicity Tests) .

12.6.2 Data sheets (one set per test) -- for data recording

(Figures 1 and 2) .

12.6.3 Tape, colored -- for labelling test chanmers and

containers.

12.6.4 Markers, water-proof -- for marking containers, etc.

12.6.5 Parafilm - - to cover gradu~ted cylinders: and vessels.

12 . 6 . 6 Gloves, disposable - - for personal proteiction from

contamination.

12.6.7 Pipets, serological 1-10 mL, graduated .

12.6.8 Pipet tips

12.6.9 Coverslips

for automatic pipets.'

for microscope slides.

145

. "
12.6.10 Lens paper -- for cleaning microscope optics.

12.6.11 Laboratory tissue wipes for cleaning and drying

electrodes, microscope slides, etc.

12.6.12 Disposable countertop covering -- for protection of work

surfaces and minimizing spi~ls and contamination.

12.6.13 pH buffers 4, 7, and 10 (or as per instructions of

instrument manufacturer) -- for standards and calibration check

(see USEPA Method 150.1, USEPA, 1979).

12.6.14 Membranes and filling solutions -- for dissolved oxygen

probe (see USEPA Method 360.1, USEPA, 1979), or reagents for

modified Winkler analysis.

12.6.15 Laboratory quality assurance samples and standards --

for the above methods.

12.6.16 Test chambers -- 1000 mL, five chambers per

concentration. The chambers should be borosilicate glass (for

effluents) or nontoxic disposable plastic labware (for reference

toxicants). To avoid contamination from the air and excessive

evaporation of test solutions during the .test, the chambers

should be covered during the test with safety glass plates or a

plastic sheet (6 rom thick) .

12.6.17 Micro-weighing pans, aluminum -- to determine the dry

weight of organisms. Weighting pan should be about 5 mg or less

to minimize noise in ,measurement of the small mysids.

12.6.18 Fronds 'of kelp (Macrocystis) for habitat ,in culture.

12.6.19 Reference toxicant solutions (see Subsection 1,2.10.2.4

and see Section 4, Quality Assurance) .

12.6.20 Reagent water -- defined as distilled or deionized water

that does not contain substances which are toxic to the test

organisms (see Section 5, Facilities, Equipment, and Supplies and

Section 7, Dilution Water).

146
12.6.21 Effluent and receiving water -- see Section 8, Effluent

and Surface Water Sampling, and Sample Handling, and Sample

Preparation for Toxicity Tests.

12.6.22 Dilution water and hypersaline brine .~- see Section 7,

Dilution Water and Section 12.6".24, Hyp.,.ersaline Brines." The

dilution water should be uncontaminated l-~m-filtered natural

seawater. Hypersaline brine should be prepared from dilution

water.

12.6.23 HYPERSALINE BRINES

12.6.23.1 Most industrial and sewage treatment effluents

entering marine and estuarine systems have little measurable

salinity. Exposure of larvae to these effluents will usually

require increasing the salinity of the test solutions. It is

important -t::0 maintain an essentially constant salinity across all

treatments. In some applications it may "be desirable to match

the test salinity with that of the receiving wc~ter (See Section

7.1). Two salt sources are available ~o adjust salini~ies --

artificial sea salts and .hypersaline brine (HSB) derived from

natural seawater. Use of artificial sea salts.is necessary only

when high effluent concentrations preclude sali.nity adjustment by

HSB alone.

12.6.23.2 Hypersaline brine (HSB) can be made 'by concentrating

natural seawater by freezing.or evaporation. HSB should be made

from high quality, filtered seawater, and can tie added "to the

effluent or to reagent water to increase salin~ty. HSB has

several desirable characteristics for use in effluent toxicity

testing. Brine derived from natural seawater C'on'l;:ains the

necessary trace metals, biogenic colloid~, and some of the

microbial components necessary for adequate gro;wth, survival,

and/or reproduction of marine and estuarine org-anisms, and it can

be stored for prolonged periods without any apparent degradation.

However, even if the maximum salinity HSB (100~) is used as a

diluent, the maximum concentration of effluent .(0%0) that can be

tested is 66% effluent at 34%0 salinity (see Table 1) .

12.6.23.3 High quality (and preferably high salinity) seawater

should be filtered to at least 10 ~m before placing into the

freezer or the brine generator. Water should be colleqted on an

incoming tide to minimize the possibility of contamination.

147-, . :~
12.6.23.4 Freeze Preparation of Brine

12.6.23.4.1 A convenient container. for making HSB by freez~ng is

one that has a bottom drain. One liter of brine can be made from

four liters of seawater. Brine may be collected by partially

freezing seawater at -10 to -20°C until the remaining liquid has

reached the target salinity. Freeze for approximately six hours,

then separate the ice (composed mainly of fresh water) from the

remaining liquid (which has now become hypersaline) .

12.6.23.4.2 It is preferable to monitor the water until the

target salinity is achieved rather than allowing total freezing

followed by partial thawing. Brine salinity should never exceed

100~. It is advisable not to exceed about 70~ brine salinity ,

unless it is necessary to test effluent concentrations greater

than 50%.

12.6.23.4.3 After the required salinity is attained, the HSB

should be filtered through a 1 ~m filter and poured directly into

portable containers (20-L cubitainersor polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and the date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after several months in storage.

12.6.23.5 Heat Preparation of Brine

12.6.23.5.1' The ideal container for making brine using heatassisted evaporation of 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 is to use a thermostatically controlled heat

exchanger made from fiberglass. If aeration is needed, use only

oil-free air compressors to prevent contamination.

12.6.23.5.2 Before adding seawater to the brine generator,

thoroughly clean the generator, aeration supply tube, heater, and

148
any. other materials that will be in direct contact with the

brine. A good quality biodegradable detergent should be used,

followed by several (at least three) thorough reagent water

rinses.

TABLE 1. MAXIMUM EFFLUENT CONCENTRATION' (%) TIt~T CAN BE TESTED

AT 34%'0 WITHOUT THE ADDITION OF DRY SA:C,TS GIVEN THE

INDICATED EFFLUENT AND BRINE SALINITIES.

Effluent Brine Brine Brine Brine Brine

Salinity 60 70 80 90 100

%"0 %"0 %'0 %'0 %'0 ~o

0 43.33 51.43 57.50 62.22 66.00

1 44.07 52.17 58.23 62:92 66.67

2 44.83, 52.94 58.97 63.64 67.35

3 45.61 53.73 59.. 74 64.37 68.04

4 46.43 54.55 . 60.53 65.12 68.75

5 47.27 55.38 61. 33 65.88 69.47

10 52.00 60.00 65.71 70.00 73.33

. 15 57.78 65.45 70.77 74.67 77.65

20 65.00 72.00 76.67 80.00 82.50

25 74.29 80.00 83.64 86.15 88.00

12.6.23.5.3 .Seawater should be filtered to at least 10 J,Lm before

being put into the brine generator. The temperature of the

seawater is increased slowly to 40°C. The water should be.

aerate~ 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 salin:ity·. <:ioes .

not exceed 100%0 and that the temperature cioes +lO't exceed 40°C.'

Additional seawater may be added to the brine to obtain the .

volume of brine required.

·12.6.23.5.4 After the required salinity is attained, the HSB

149
should be filtered through a 1 ~m filter and poured di~ectly ihto

portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled ~ith the salinity and the date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after several months in storage.

12.6.23.6 Artificial Sea Salts

12.6.23.6.1 No data from mysids using sea salts or artificial

seawater (e.g., GP2) are available for evaluation at this time,

and their use must be considered provisional.

12.6.23.7 Dilution Water Preparation from Brine

12.6.23.7.1 Although salinity adjustment with brine is the

preferred method, the use of high salinity brines and/or reagent

water has sometimes been associated with discernible adverse

effects on test organisms. For this reason, it is recommended

that only the minimum necessary volume of brine and reagent water

be used to offset the low salinity of the effluent, and that

brine controls be included in the test. The remaining ,dilution

water should be natural seawater. Salinity may be adjusted in

one of two ways. First, the salinity of the highest effluent

test concentration may be adjusted to an acceptable salinity, and

then serially diluted. Alternatively, each effluent

concentration can be prepared individually with appropriate

volumes of effluent and brine.

12.6.23.7.2 When HSB and reagent water are used, thoroughly

mix together the reagent water and HSB before mixing in the

effluent. Divide the salinity of the HSB by the expected test

salinity to determine the proportion of reagent water to brine.

For example, if the salinity of the brin~ is 100~ and the test

is to be conducted at 34~, 100~ divided by 34~ = 2.94. ~he

proportion of brine is 1 part, plus 1.94 parts reagent water. To

make 1 L of dilution water at 34~ salinity from a HSB of 100~,

340 mL of brine and 660 mL of reagent water are required. Verify

the salinity of the resulting mixture using a refractometer.

12.6.23.8 Test Solution Salinity Adjustment

150
12.6.23.8.1 Table 2 illustrates the preparation of test

solutions (up to 50% effluent) at 34%-0 by combining effl.uent,

HSB, and dilution water. Note: if the highest effluent

concentration does not exceed 50% effluent, it .is convenient to

prepare brine so that the sum of the effluent salinity and brine

salinity equals 68~i the required brine volume is then always

equal to the effluent volume needed foi each effluent

concentration as in the example in Table 2.

12.6.23.8.2 Check the pH of all brine mixtures and adj~st to

within 0.2 units of dilu:tio.n water pH by adding', dropwise, dilute

hydrochloric acid or sodium hydroxide(see Section 8.8.9, Effluent

and Receiving Water Sampling, Sample Handling, and Sample

Preparation for Toxicity Tests) .

12.6.23.8.3 To calculate the amount of brine tio add to' each

effluent dilution, determine the following quantities: -salinity

of the brine (SB, in ~), the salinity of the effluent (SE, in

~), and volume of the effluent to be added (VE, in mL). Then use

the following formula to calculate the volume o;f brine (VB, in

mL) to be added:

VB = VE x (34 - SE)/(SB - 34)

12.6.23.8.4 This calculation assumes that dilution water

salinity is 34 ± 2~.

12.6.23.9 Preparing Test Solutions

12.6.23.9.1 Two hundred mL of test solution ar'8 needed for each

test chamber. To prepare test solutions at low efflue~t

concentrations «6%), effluents may be added directly to dilution

water. For example, to prepare 1% effluent, add 10 mL of

effluent to a 1-liter volumetric flask using a volumetric pipet

or calibrated automatic pipet. Fill the volumetric flask to the

1-liter mark with dilution water, stopper iti and shake to mix.

Distribute equal volumes into the replicate test chambers.

12.6~23.9.2 To prepare a test solution at higher effltient

concentrations, hypersaline brine must usually be used. For

example, to prepare 40% effluent, add 400 mL of effluent to a 1-

liter volumetric flask. Then, assuming an effluent salinity of

2~ and a brine salinity of 66~, add 400 mL of b~ine (see

151
equation above and Table 2) and top off the flask with dilution

water. Stopper the flask and shake well. Distribute equal

volumes into the replicate test chambers.

12.6.23.10 Brine Controls

12.6.23.10.1 Use brine controls in all tests where brine is

used. Brine controls contain the same volume of brine as does

the highest effluent concentration using brine, plus the volume

of reagent water needed to reproduce the hyposalinity of the

effluent in the highest concentration, plus dilution water.

Calculate the amount of reagent water to add to brine controls by

rearranging the above equation, (See, 12.6.23.8.3) setting SE =

0, and solving for VE.

VE = VB x (SB - 34)/(34 - SE)

If effluent salinity is essentially O~, the reagent water volume

needed in the brine control will equal the effluent volume at the

highest test concentration. However, as effluent salinity and

effluent concentration increase, less reagent water volume is

needed.

12.6.24 BRINE SHRIMP, ARTEMIA SF., NAUPLII -- for feeding

cultures and test organisms.

12.6.24.1 Newly hatched Artemia sp. nauplii are used for food

for the stock cultures and test organisms. Although there are

many commercial sources of brine shrimp cysts, the Brazilian or

Colombian strains are preferred because the supplies examined

have had low concentrations of chemical residues and produce

nauplii of suitably small size. (One source that has peen found

to be acceptable is Aquarium Products, 180L Penrod Ct., Glen

Burnie, Maryland 21061). For commercial sources of brine shrimp,

Artemia, cysts, see Table 2 of Section 5, Facilities, Equipment,

and Supplies); and Section 4, Quality Assurance.

12.6.24.2 Each. new batch of Artemia cysts should be evaluated

for size <Vanhaecke and Sorgeloos, 1980, and Vanhaecke et al.,

1980) and nutritional suitability (Leger, et al., 1985,' Leger, et

al., 1986) against known suitable reference cysts by performing a

side-by-side larval growth test using the "new" and "reference"

cysts. The "reference" cysts used in the suitability test may be

152
a previously tested and acceptable batch'of cysts, or may be

obtained from the Quality Assurance Research Division, EMSL,

Cincinnati, OH 45268, 513-569-7325. A sample of ' newly-hatched

Artemia nauplii from each new batch of cysts should be chemically

analyzed. The Artemia cysts should not be used if the

concentration of total org~nochlorine pesticides 0.15 ug/g wet

weight or that the total concentration of organochlorine

pesticides plus PCBs exceeds 0.30 J-Lg/g wet weight: (For analytical

methods see USEPA, 1982).

12.6.24.3 Artemia nauplii are obtained as follO\lTs:

1. Add 1 L of seawater, or an aqueous unionized salt

(NaCI) solution prepared with 35 g salt or artificial

sea salts per liter, to a 2-L separatory funnel, or

equivalent.

2. Add 10 mL Artemia cysts to the separatory funnel and

aerate for 24 h at 27°C. Hatching time varies with

incubation temperature and the geographic strain of

Artemia used (see USEPA, 1985ai USEPA, 1993ai ASTM,

1993) .

3. After 24 h, cut off the air supply in t.he separatory

funnel. Artemia nauplii are phototactic, and will

concentrate at the bottom of the funnel if it is

covered for 5-10 minutes with a dark cloth or paper

towel. To prevent mortality, do not leave the

concentrated nauplii at the bottom of the funnel more

than 10 min without aeration.

4. Drain the nauplii into a funnel fitted with a ~150 J-Lm

NITEX® or stainless steel screen, and rinse with

seawater or equivalent before use.

12.6.24.4 Testing Artemia nauplii as food for toxicity test

organisms.

12.6.24.4.1 The primary criteria for acceptability of each new

supply of brine shrimp cysts is adequate .survival , and growth of'

the mysids. The mysids used to evaluate the acceptability of the

brine shrimp nauplii must be the same geographical origin and

stage of' development (3 to 4 days old) as those uised routinely in

the toxicity tests. Two 7-day chronic tests are performed sideby-side, each consisting of five replicate test vessels

containing 'five juveniles (25 organisms per test, total of 50

153
TABLE 2. EXAMPLES OF EFFLUENT DILUTION SHOWING VOLUMES OF

EFFLUENT (x~), BRINE, AND DILUTION WATER NEEDED FOR

ONE LITER OF EACH TEST SOLUTION.

FIRST STEP: Combine brine with deionized water or natural seawater to achieve

a brine of 68-x~ and, unless natural seawater is used for dilution water,

also a brine-based dilution water of 34~.

SERIAL DILUTION:

Step 1. Prepare the highest effluent concentration to be tested by adding

equal volumes of effluent and brine to the appropriate volume of dilution

water. An example using 40% is shown.

Effluent Cone. Effluent Brine Dilution Water*

, (t) 'x~ (68-x)~ 34~

I

40 800 mL 800 mL 400 mL

Step 2. Use e~ther ser~ally prepared d~lut~ons of the h~ghest test

concentration or individual dilutions of 100% effluent.

Effluent Cone. (%) Effluent Source Dilution Water* (34~)

20 1000 mL of 40% 1000 mL i

10 1000 mL of 20% 1000 mL

I

5 1000 mL of 10% 1000 mL

I

I

2.5 1000 mL of 5% 1000 mL ,.

I

Control none 1000 mL

INDIVIDUAL PREPARATION

Effluent Cone. Effluent x~ Brine (68-x) Dilution Water*

(%) 34~

40 400 mL 400 mL 200 mL

, 20 200 mL 200 mL 600 mL

10 100 mL 100 mL 800 mL

I

5 50 mL 50 mL 900 mL I

2.5 25 mL 25 mL 950 mL

: Control none none 1000 mL

*May be natural seawater or brine-reagent water equivalenb.

154
organisms). The juveniles in one set of test chambers is fed

reference (acceptable) nauplii and the other se't is fed nauplii

from the "new" source of Artemia cysts.

12.6.24.4.2 The feeding rate and frequency, test vessels, volume

of control water, duration of the tests, and age of the Artemia

nauplii at the start of the test, should be the same as used for

the routine toxicity tests.

12.6.,24.4.3 Results of the brine shrimp, Artem.ia, nauplii

nutrition assay, where there are only two treatrnents, can be

evaluated statistically by use of. a t test. ThE~ "new" food is

acceptable if there are no statistically significant differences

in the survival or growth of the mysids fed the'two sources of .

naupli,i.

12.6.25 TEST ORGANISMS

12.6.25.1 The test organisms for this method al:1e juveniles of

the mysid crustacean, Holmesimysis costata (Holmes 1900,;

previously referred to' as Acanthomysis sculpta) .' H. costata

occurs in the surface canopy of the giant kelp ~~crocystis

pyrifera where it feeds on zooplankters, kelp, epiphytes, and

detritus. There are few references to the ecology of this mysid

species (Holmquist, 1979; Clutter, 1967, '1969; Green, 1970;

Turpen et al., 1994). H. costata is numerically abundant in'kelp

fqrest habitats and is considered to be an important fqod source

for kelp forest fish (Clark 1971, Mauchline 1980). Mysids are

called opossum shrimp because females brood their young in an

abdominal pouch, the marsupium. H. costata eggs develop for

about ,20 days in the marsupium before the young are released as

juveniles; broods are released at night during molting. Females

~elease their first brood at 55 to 70 days post-release (at

12°C), and may have multiple broods throughout their

approximately 120-day life.

12.6.25.2 H. costata has been used in previous toxicity studies

with a variety of toxicants (Tatem and Portzer, :l985j Davidson et

al., 1986; Machuzac and, Mikel, 1987; ReiE!h and L~~may, 1988;'

Asato, 1988; Martin et al., 1989; Singer et al., ,1990j 1991; Hunt

et al., In Press). Mysids are useful as toxicity test organisms

'because of their widespread availability, ecologj.cal importance,

sensitivity to toxicants, and amenability to labc>ratory cultu,re

155
(Nimmo et al., 1977; Mauchline, 1980; Gentile et al., 1982;

Lussier et al., 1985).

12.6.25.3 Species Identification

12.6.25.3.1 Laboratories unfamiliar with the test organism

should collect preliminary samples to verify species

identification. Refer to Holmquist (1979) or send samples of

mysids and any similar co-occurring organisms to a qualified

taxonomist. Request certification of species identification from

any organism suppliers~ Records of yerification should, be

maintained along with a few preserved specimens.

12.6.25.3.2 There have been recent revisions to the taxonomy of

H. costata. Previous authors have referred to this species as

Acanthomysis sculpta. However, Holmquist's (1979) review

considers previous references to Acanthomysis sculpta in

California to be synonymous with Holmesimysis costata; we

consider Holmquist's designation to be definitive.

12.6.25.4 Obtaining Broodstock

12.6.25.4.1 H. costata can be collected by sweeping a small-mesh

(0.5 - 1 mm) hand net through the water just under the surface

canopy blades of giant kelp Macrocystis pyrifera. Although this

method collects mysids of all sizes, attention should be paid to

the number of gravid females collected because these are used to

produce the juvenile mysids used in toxicity testing. Mysids

should be collected from waters remote from sources of pollution

to minimize the possibility of physiological or genetic

adaptation to toxicants.

12.6.25.4.2 Mysids can be transported for a short time « 3

hours) in tightly covered 20 liter plastic buckets. The buckets

should be filled to the top with seawater from· the·col~ection

site, and should be gently aerated or oxygenated to maintain

dissolved oxygen above 60% sa~uration. Transport temperatures

should remain within 3°e of the temperature at the collection

site.

12.6.25.4.3 For longer transport times of up to 36 hours, mysids

can be shipped in sealed plastic bags filled with seawater. The

following transport procedure has been used successfully: 1)

156
· ,

fill the plastic bag with one liter of dilution 'water seawater,

2) saturate the seawater with oxygen by bubblinsr pure oxygen for. ,.

at least 10 minutes, 3) place 25-30 adult mysids, or up ,to 100

juvenile mysids in each bag, 4) for adults add about 20 Artemia

pauplii per mysid, for 100 juveniles add a pinch (10 to 20 mg) of

ground Tetramin® flake food and 200 newly-hatched Artemia '

nauplii, 5) seal the bag sec,urely, eliminating a:ny airspace, then

6) place it within a second sealed bag in an ice' chest. Do not

overfeed mysids in transport, as this may deplete dissolved

oxygen, causing stress or mortality in transported mysids. ,A

well insulated ice chest'should be cooled to approximately 15°C .'

by adding one l-liter blue ice block for every five l-liter bags

of mysids (a temperature range of 12 to 16°C is toleraple) . , Wrap

the ice in newspaper and a plastic bag to insulate it from the

mysid bags. Pack the bags tightly to avoid shif'!:ing within the

cooler.

12.6.25.5 Broodstock Culture and Handling

12.6.25.5.1 After collection, the mysids should be transported

directly to the laboratory and placed in seawater tanks or

aquaria equipped with flowing seawater or adequate aeration and'

filtration. Initi.al flow rates should be adjustE~d so that any

temperature change occurs gradually (O.5°C per hqur). The water

temperature should be 'held at 15 ± 1°C. Note: lII,tysids collected

north of Pt. Conception, California, should be held and tested at

13 ± 1°C.

12.6.25.5.2 Mysids,can be cultured in tanks'ransring from 4 to

1000 liters.. Tanks should be equipped with gentl€l aeration and

blades of Macrocystis to provide habitat. Static culture tanks

can 1?e used if there is constant aeration,temperature control,

and frequent water changes (one half the .water volume changed at·

least twice a week). Maintain culture density below 20 animals

per liter by culling out adult males or juveniles.

12.6.25.5.3 Adult mysids should be fed 100 Artem:ia nauplii per -'

my-sid per day. Juveniles should be fed 5 to 10 newly released

Artemia nauplii per juvenile per day and a pinch (10 to 20 mg) of

ground Tetramin® flake food per 100 juveniles per'day. Static

chambers should be carefully mpnitored arid rations adjusted to

'c,

157
prevent overfeeding and fouling of culture water. Refer to

section ~2.6.~9 for details ,of Artemia culture and quality,

control. '

~2.6.25.6 Culture Materials

~2.6.25.6.1 Refer to Section 5, Facilities and Equipment t for a

discussion of suitable materials to be used in laboratory culture

of mysids. Be sure all new materials are properly leached in

seawater before use. After use, all culture materials should be

washed in soap and water, then rinsed with seawater beforereuse.

12.6.25.7 Test Organisms

~2.6.25.7.1 Approximately 150 gravid femalemysidss~ouldbe

isolated to,provide approximately 400 juveniles for each set of

toxicity tests (5 juveniles/chamber x 30 reference toxicant

chambers and approximately 35 effluent chambers, plus additional

mysids so that only healthy active juveniles are used in the

test). Gravid females can be identified by their large, extended

marsupia filled with (visible) eyed juveniles. Marsupi,a appear.

distended and gray when females are ready to release young, due

to presence of the juveniles.

~2.6.25.7.2 Gravid females are easily isolated from other mysids

using the following technique: (1) use a small dip net to

capture about ~OO mysids from the culture tank, (2)transfer the

mysids to a screen-bottomed plastic tube. (150 ~m-mesh, 25-cm

diam.) partly immersed in a water bath or bucket, (3) lfft the

screen-tube out of the water to immobilize mysids on the damp

screen, (4)gently draw the gravid females off the screen with a

suction bulb and fire-polished glass tube (5-mm bore), (5)

collect the gravid females in a separate .screen tube. Re-immerse

the screen continuously during the isolation process; mysids

should not be exposed to air for more than a few seconds at a

time.

12.6.25.7.3 Four or five days before a toxicity test begins,

transfer gravid females into a removable, 2-mm-mesh screened

cradle suspended within an aerated 80-liter aquarium. Before

transfer, make sure there are no juveniles in with the adult

females. Extraneous juveniles are excluded to avoid

158
inadvertently mixing them with the soon-to-be released juvenilE:s

used in testing. Provide the gravid females with newly hatched

Artemia nauplii (approximately 200 per mysid) to help stimuiate

juvenile release. Artemia can be provided continuously

throughout the night from an aerated reservoir holding

approximately 75,000 Artemia. Direct the flow from the feeder

into the screened compartment with the females, and add a few

blades of Macrocystis for habitat. The females are plac.ed within

the screened compartment so that as the j uvenilel3 are released,

they 'can swim through the mesh into the bottom o:E the aquarium.

Outflows on flow-through aquaria should be screened (150.,-j1.m-mesh)

to retain juveniles and allow some Artemia to escape.

12.6.25.7.4 Juveniles are generally released at night, so it is

important to turn off all lights at night to promote release. In

the morning, the sGreened compartment containing the females

should be removed and placed in a ,separate aquarium. Juveniles

should be slowly siphoned through a wide-diameter hose into a

150.,...~m-mesh screen-bottom tube (25 cm diam.) immersed in a bucket

filled with clean seawater. Once the reJ,ease aquarium is

emptied, it should be wasped with hot fresh water to eliminate

stray juveniles that might mix with the next coh6rt.

l2.6.25.7.5 After collection, the number of juveniles should be

estimated visually or by counting subsamples with a small beaker.

If there are not enough juveniles to cond~ctthe necessary tests,

they can be mixed with juveniles from one previol.:ls or subsequent

release so that the test is initiated wit~ threeiand/prfour-day

old j.uveniles. .Initial experiments indicate that. my-sids 2-daysold and younger survive poorly in toxicity tests and th~t mysids

older than four days may vary in their toxicant sensitivity or

survival rate (Hunt et al., 1989; Martin et al. " 1989) .

12.6.25.7.6 Test juveniles should be transferred to additional

screen-tubes (or to 4-liter static beakers if flowing seawater is

unavailable). The screen-tubes are suspended in 'a 15-1ite~

bucket so that dilution water seawater' (0.5 liter/min) can flow

into the tube, through the screen, and overflow from the bucket.

Check water 'flow rates « one liter/min) to make sure that

juveniles or Artemia nauplii are not forced down onto. the screen.

The height of the bucket determines the level of water in the

screen tube. About 200 to 300 juveniles .can be h,eld in each

screen-tube (200 juveniles per static 4-liter beaker). Juven~les

159
should be fed 40 newly hatched Artemia nauplii per mysid per day

and a pinch (10 to 20 mg) of ground Tetramin® flake food per 100

juveniles per day. A blade of Macrocystis (well rinsed in

seawater) should be added to each chamber. Chambers should be

gently aerated and temperature controlled at 15 ± 1°C (or 13 ±

1°C if collected north of Pt. Conception). Half of the seawater

in static chambers should be changed at least once between

isolation and test initiation.

12.6.25.7.7 The day juveniles are isolated is designated d~y a

(the morning after their nighttime release). The toxicity test

should begin on day three or four. For example, if juveniles are

isolated on Friday, the toxicity test should begin on the

following Monday or Tuesday.

12.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND

STORAGE

12.7.1 See Section 8, Effluent and Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity Tests.

12.8 CALIBRATION AND STANDARIZATION

12.8.1 See Section 4, Quality Assurance.

12.9 QUALITY CONTROL

12.9.1 See Section 4, Quality Assurance~

12.10 TEST PROCEDURES

12.10.1 TEST DESIGN

12.10.1.1 The test consists of at least five efflue~t

concentrations plus a dilution water con~rol. Tests that use

brine to adjust salinity must also contain five replicates of a

brine control.

12.10.1.2 Effluent concentrations are expressed as percent

effluent.

12.10.2 TEST SOLUTIONS

160
12.10.2.1 Receiving waters

12.10.2.1.1 The sampling point is determined by the objectives

of. the test. At estuarine and ma~ine sites, saulples are usually

collected at mid-depth. Receiving water toxicity is det~rmined

with samples used directly as collected or with samples passed

through a 60 J.l.m NITEX® filter and compared without dilution,

against a control. Using five replicates chambers per test, each

containing 200 mL would require approximately 1 L or more of

sample per test per renewal.

12.10.2.2 Effluents

12.10.2.2.1 The selection of the effluent test concentrations

should be based on the objectives of the ·study. A dilution

factor of at least 0.5 is commonly used. A dilution factor of

o. 5 provides hypothesis test discrimination of :I: 100%, and

testing of a 16 fold range of concentrations. Hypothes'is test

discrimination shows little improvement as dilut.ion factors are

increased beyond 0.5 and declines rapidly if smaller dilution

factors are used. USEPA recommends that one of the five effluent

treatments must be a concentration of effluent rnixed with

dilution water which corresponds to the perm;l.tte,e's instream

waste concentration (IWC). At least two of the effluent

treatments must be of lesser effluent concentration than the IWC,

with one being at least one~half the concentration of the IWC.

If 100~ HSB is used as a diluent, the maximum concentration of

effluent that can be tested will be 66% at 34~ salinity. , \

• I .

12.10.2.2.2 If the effluent is known or suspect,ed to be highly

toxic, 'a lower range of effluent concentrations should be used

(such as 25%, 12.5%, 6.25%, 3.12% and 1.56%).

12.10.2.2.3 The volume of effluent required for, a 75% renewal of

five replicates per concentration for five concentrations of

effluent and two controls, each containing 200 m'L of test

solution, is approximately 370 mL.

12.10.2.2.4 Effluent dilutions'should be prepared for ~ll

replicates in each treatment in one container to minimize

variability among the replicates. Dispense into the appropriate

effluent test chambers. '

161
12.10.2.3 Dilution Water

12.10.2.3.1 Dilution water should be uncontaminated l-~mfiltered natural seawater or hypersaline brine prepared from

uncontaminated natural seawater plus reagent water (see Section

7, Dilution Water). Natural seawater may be uncontaminated

receiving water. This water is used in all dilution steps and as

the control water.

12.10.2.4 Reference Toxicant Test

12.10.2.4.1 Reference toxicant tests should be conducted as

described in Quality Assurance (see Section 4.7) .

12.10.2.4.2 The preferred reference toxicant for mysids is zinc

sulfate (ZnS04 0 7H20). Reference toxicant tests provide an

indication of the sensitivity of the test organisms and the

suitability of the testing laboratory (see Section 4 Quality

Assurance). Another toxicant may be specified by the appropriate

regulatory agency. Prepare a 10,000 ~g/L zinc stock solution by

adding 0.0440 g of zinc sulfate (ZnS04 0 7HzO) to one liter of

reagent water in a polyethylene volumetric ·flask. Alternatively,

certified standard solutions can be ordered from commercial

companies.

12.10.2.4.3 Reference toxicant solutions should be five

replicates each of 0 (control), 10, 18, 32, and 56, and 100 ~g/L

total zinc. Prepare one liter of each concentration by adding 0,

1.0, 1.8, 3.2, 5.6, and 10.0 mL of stock solution, respectively,

to one-liter volumetric flasks and fill with ,dilution water.

Start with control solutions and progress to the highest

concentration to minimize contamination.

12.10.2.4.4 If the effluent and reference toxicant tests are to

be run concurrently, then the tests must use juvenile originating

from or released from the same pool of gravid females. The tests

must be handled in the same way and test .solutions delivered to

the test chambers at the same time. Reference toxicant tests

must be conducted at 34 ± 2~.

12.10.3 START OF THE TEST

12.10.3.1 Prior to Beginning the Test

162
12.10.3.1.1 The test should begin as soon as possible,

preferably within 24 h of sample collection. Th,e maximum holding

time following retrieva~ of the sample from the sampling device

should not exceed 36 hfor off-site-toxicity tests unless

permission is granted by the permitting authority. In no case;

should the sample be used in a test more than 72 h a~ter sample

collection (see Section ,a. Effluent and Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity Test).,'

12.10.3.1.2 Just prior to test -initiation (apprQximCl;tely 1 h),

the temperature of a sufficient quantity of the Bample to make

the test solutions should be adjusted to the te~t temperature (13

or 15 ± 1°C) and maintained at that temperature during the.

addition of dilution water. .

12.10.3.1.3 Increase the temperature of .the watE~.r bath, .room, or

incubator to the required test temperature. (13 or 15 ± 1°C) .

12.10.3.1.4 Randomize the placement of test chaniliers in the

temperature-controlled water bath, room, or incut)ato~ at the

beginning of the test, using a position chart. }.ssign numbers

for the position of each test chamber u~inga. rar.ldom numbers Qr

similar process (see Append.ix A, for, an example of

randomization). Maintain the chambers in this cQnfiguration

throughout the test, using a position chart. Record these

numbers on a separat~ data sheet together with, th,e concentration,

and replicate numbers to which they correspond. Identify this

sheet with the date, test organism, test number,laboratory, and

investigator's name, -and safely store it away until after the

mysids have been examined at the end of the test.

12.10.3.1.5' Note: Loss of the randomization sheet would

invalidate the test by making it impossible to an~lyze ,the data

afterwards. Make a copy of the randomization sheet and store

separately. Take care to follow the numbering system exactly

while filling chambers with the test solutions.

12.10.3.1.6 Arrange the test chambers randomly in the water bath

or controlled temperature room. Once chambers have been labeled

randomly and filled with test'solutions, they canlbe arranged in

numerical order for "convenience, since this 'will also ensure

random placement of treatments.

163
12.10.3.2 Randomized Assignment of Mysids to Test Chambers

12.10.3.2.1 The juvenile mysids must ·be .randomized before

placing them into the test chambers. Pool all of the test

juveniles into a 1-liter beaker. Using a 10-mL wide-bore pipet

or fire-polished glass tube (approximately 2-3 mm inside

diameter), place one or two juveniles into as many plastic cups

as there are test chambers (including reference toxicant

chambers) . These cups should contain enough clean dilution

seawater to maintain water quality and temperature during the

transfer process (approximately 50 mL per cup). When.each of the

cups contains one or two juveniles, repeat the process, adding

mysids until each cup contains 5 animals.

12.10.3.2.2 Carefully pour or pipet off excess water in the

cups, leaving less than 5 mL with the test mysids. This 5·mL

volume can be estimated visually after initial measurements.

Carefully pour or pipet the juveniles into the test chambers

immediately after reducing the water volume. Gently rocking the

water back and forth before pouring may help prevent j~veniles

from clinging to the walls of the randomization cups. Juveniles

can become trapped in drops; have a squirt bottle ready to

gently rinse down any trapped mysids. If more than 5 mLs of

water are added to the test solution with the juveniles, report

the amount on the data sheet. Be sure that all water used in

culture, transfer, and test solutions is within 1°C· of the test

temperature. Because of the small volumes involved in the

transfer process, temperature control is best accomplis'hed in a

constant-temperature room.

12.10.3.2.3 Verify that all five animals are in the test

chambers by counting the number in each chamber after transfer.

This initial count is important because mysids unaccounted for at

the end of the test are assumed to be dead.

12.10.4 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE

12.10.4.1 The light quality and intensity should be at ambient

laboratory conditions are generally adequate. Light intensity

should be 10-20 ~E/m2/s, or 50 to 100 foot candles (ft-c), with a

16 h light and 8 h dark cycle. A 30 minute phase-in/out period

is recommended.

164
12.10.4.2 Tpe water temperature in the test chcimbers should be'

maintained at 13 or 15 ± 1°C. It is critical tnat the test water

.'temperature be maintained at 13 ± 1°C {for mysids collected north

6£ Pt. conception; California} or 15 ± 1°C (for, mysids collected'~

south of Pt • Conception, California). If a watE~r bath is used' to

maintain the test temperature, the water depth slUrrounding the

test cups should be as deep as possible without ,floating the

chambers.

,.

12.10.4.3· The test salinity should be in the range of 34 ± 2%0.,

The salinity should vary by no more than ±2%o among the chambers

on a given day. If effluent and receiving water tests are

conducted concurrently, the salinities of these tests should be

similar.

12.10.4.4 Rooms or incubators with high volume ventilation I

should be used with caution because the volatilization of the~

test solutions and evaporation of diluti~n water may cause wide

fluctuations in salinity. Covering the test chambers with clean

polyethylene plastic may help prevent \rblatiliza'tion and

evaporation of the test solutions.

12.10.5 DISSOLVED OXYGEN (DO) CONCENTRATION

12.10.5.1 Aeration may affect the toxicity of effluent and

should be used only as a last resort to maintain a 'satisfactory

DO. The DO concentration should, be measured on new solutions at

the start of the test (Day 0). The DO should not fall below 4.0

mg/L (see Section 8, Effluent and.Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity Tests). ',If

it is necessary to aerate, all treatments and thE~ control should

be aerated. The aeration rate should not exceed:that necessary'

to maintain a minimum acceptable DO and under no circumstances

should it exceed 100 bubbles/minute, using a pipe:t with a 1-2 mm

orifice, such as a 1 mL KIMAX@ serological pipet No. 37033, or

equivalent.

12.10.6 FEEDING

12.10.6.1 Artemia nauplii are prepared ~s described above.

12.10.6.2 The feeding rates in the test beakers should be

closely controlled to avoid overfeeding and fouling of 'test

165
solutions. Add 40 newly hatched Artemia nauplii per mysidper

day. Artemia nauplii should be well rinsed with clean seawater

and concentrated so that no more than one mL of seawater is added

during feeding. (Use a 100-~m-mesh screen tube for rinsing and

concentrating the nauplii; see Section 12.6.24.3). Test

performance may be enhanced by feeding half the ration twice

daily. If mysids die during the course of the experim~nt, the

ration should be reduced proportionally. The my~ids should not

be fed on day 7.

12.10.7 DAILY CLEANING OF TEST CHAMBERS

12.10.7.1 Before the renewal of test solutions, uneaten and dead

Artemia, dead mysids and other debris are removed .from the bottom

of the test chambers with a pipette. As much of the uneaten

Artemia as possible should be removed from each chamber to ensure

that the mysids eat primarily newly hatchednauplii. By placing

the test chambers on a light box, inadvertent removal of live

mysids can be greatly reduced because th~y can be more easily

seen. If a mysid is lost during siphoning, note the test chamber

from it came, and reduce the initial count from five to four for

that chamber when calculating survival at the end of the test.

12.10.8 OBSERVATIONS DURING THE TEST

12.10.8.1 Routine Chemical and Physical Observations

12.10.8.1.1 DO is measured at the beginning of the exposure

period in one test chamber at each test concentration and in the

control.

12.10.8.1.2 Temperature, pH, and salinity are measured at the

beginning of the exposure period in one test chamber at each

concentration and in the control. Temperature should also be

monitored continuously or observed and recorded daily for at

least two locations in the environmental.control system or the

samples. Temperature should be measured in a sufficient number

of test chambers at the end of the test to determine temperature

variation in the environmental chamber.

12.10.8.1.3 Record all the measurements .on the data sheet.

12.10.8.2 Routine Biological Observations

166
"

12.10.8.2.1 The number of live mysids are countt=d and recorded

each day. Dead animals and excess food should bt~ removed with a

pipette before test solutions are renewed. This is necessary to

avoid cannibalism and to prevent fouling of test solutions. :

12.10.8.2.2 Protect the mysids from unnecessary disturbance

during the test by carrying out the daily test observations,

solution renewals, and r~moval of the dead mysidH, carefully.

Make sure the mysids remain immersed during the performance of'

the above operations.

12.10.9 TEST SOLUTION RENEWAL

12.10.9.1 The test duration is 7 days~ Bec~use effluent

toxicity, may ch'ange, over short time periods in te;st chambers,' the

test solutions must be renewed after 48 hand 96 h. Prepare

renewal test, solutions in the same·way as initial test solutions.

Remove three quarters of the original test solution from each

chamber, taking care to avoid losing or da~aging ~ysids. This

can be done by siphoning with a small-bore (2 t03 mm) firepolished glass tube or pipet. Attach the glass tube to clear

plastic tubing fitted with a pinch clamp so that the s~phon flow

can be stopped quickly if necessary to release entrained mysids.

It is convenient to siphon old solutions into a small (500 mL)

chamber in order to check to make sure that no mysids have been

inadvertently removed during solution renewals. ~f a mysid is

siphoned, return it to the test chamber and note it on the data

sheet. Follow the chamber randomization sheet to' siphon first

from the controls, then work sequentially to the ~ighest test

concentration to avoid cross-contamination.

12.10.9.2 To minimize disturbance to the juvenile mysids,

refill the chambers to the 200-mL mark by carefully,siphoningnew

test solution into the test chambers using small diameter plastic

tubing attached to a bent clean glass rod that directs incoming

solution upward or to the side to slow the current and minimi~e

turbulence.

12.10.9.3 The effluent or receiving water used iri the test is

stored in an incubator or refrigerator at 4°C. Plastic chambers

such as 8-20 L cubitainers have proven suitable .fcir effluent

collection and storage. For on-site toxicity studies no more I

than 24h should elapse between collection of the effluent and.

167

i

.I
use in a toxicity test (see Section 8, Effluent and Receiving

Water Sampling, Sample Handl~ng, and Sample Preparation for

Toxicity Tests) .

12.10.9.4 Approximately 1 h before test initiation, a sufficient

quantity of effluent or receiving water sample is warmed to 13 ±

1°C or 15 ± 1°C to prepare the test solutions. A sufficient

quantity of effluent should be warmed to make the test solutions.

12.10.10 TERMINATION OF THE TEST

12.10.10.1 Ending the Test

12.10.10.1.1 Record the time the test is terminated.

12.10.10.1.2 Temperature, pH, dissolved oxygen, and salinity are

measured at the end of the exposure period in one test chamber at

each concentration and in the control.'

12.10.10.1.3 On the last day of the test, examine each test

chamber, and remove and record any dead mysids. Sum the

cumulative total of all mortalities observed in each test chamber

over the 7 days of the test, subtract this from the initial

number of mysids (5), and verify the number of survivors.

Immobile mysids that do not respond to a stimulus are considered

dead. The stimulus should be two or three gentle prods with a

disposable pipet. Mysids that exhibit any response clearly

visible to the naked eye are considered living. The most

commonly observed movement in moribund mysids is a quick

contraction of the abdomen. This or any other obvious movement

qualifies a mysid as alive.

12.10.10.2 Weighing

12.10.10.2.1 To prepare mysids for weighing at the en~of the

exposure period, remove any remaining dead mysids, then carefully

pour the contents of the test chamber through a small mesh screen

«300~m). Count the mysids before screening, and tak~ care to

keep track of them on the screen. Make sure mortality counts

have already been recorded. Briefly dip the screen containing

the mysids in deionized water to rinse away the salt. Using fine

point forceps, carefully transfer the mysids from the screen to a

preweighed and labelled micro-weigh boat. Carefully fold the

168
foil weigh boats over the mysids to avoid loss while drying test

organisms.

12.10.10.2.,2 To prepare weigh boats prior to t:esting, write the

test chamber number on each with a fine felt-t~pped marker, dry

the ink and 'weigh boat in a drying oven, allow;the dry weigh -, . boats to cool in a desiccator, weigh the weigh 'boats to the

nearest 1 microgram (ltg) on a'microbalance, and record the weight

and chamber number on the data sheet. ,Place the weighed weigh

boats in a clean ziplock bag until ready to USEl for weighing

mysids. The juvenile mysids-are very small, and light (60 ~g)

relative to the weigh boats (4 mg). Take all precautions to' make

sure weigh boats remain clean and dry during weighing and

subsequent storage, so that mysid weights may be accurately

determined by subtraction.

12.10.10.2.3 When all mysids are loaded onto weigh boats,

arrange them all in a dish; small tray o~ other small open

chamber, and place the~ in a clean drying oven. Dry for at least

24 hours at 60 DC or for at least 6 hours at 105"C. Remove the

weigh boats with mysids from the drying oven and place them in a

desiccator to cool for one hour. When cool, carefully weigh each

weigh boat on a microbalance (accurate to 1 ~g). Record the

chamber number, mysid weight, weigh boat weight (recorded

previously), and number of mysids per weigh boa1: (replicate) on

the data sheet.

12.10.10.3 Endpoint

12.10.10.3.1 Growth is measured as dry weight of survlvlng

mysids. All surviving mysids from a single replicate test

chamber are pooled together and weighed, then this total weight

is divided by the number of original mysids to obtain the mean

dry weight per'individual for each replicate, which is used for

statistical analysis.

12.10.10.3.2 The percentage of surviving mysids in each chamber,

~t the end of the test will 'be used for subsequent statistical

analysis.

169
12.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

12.11.1 A summary of te'st conditions and test acceptability

criteria is listed in Table 3.

12.12 ACCEPTABILITY OF TEST RESULTS

12.12.1 Test results are acceptable only if all the following

requirements are met:

(1) Control survival must be at least 75%.

(2) The average weight of control mysids must be at least

40 ~g per mysid.

(3) Between replicate variability in the mortality data

must be low enough that the minimum significant

difference (%MSD) is less than 40% in the reference

toxicant test.

(4) Between replicate variability in the weight data must

be low enough that the'%MSD is less than 50 ~g in the

reference toxicant test.

(5) Both the mortality NOEC and LC50 must be less than 100

~g/L zinc in the reference toxicant test.

12.13 DATA ANALYSIS

12.13.1 GENERAL

12.13.1.1 Tabulate and summarize the data. Table 4 presents a

sample set of survival and growth data.

12.13.1.2 The endpoints of the mysid 7-day chronic test are

based on the adverse effects on survival and growth. The LC50

and the IC25 are calculated using point estimation techniques

(see Section 9, Chronic Toxicity Test Endpoints and Data

Analysis). LOEC and NOEC values for survival and growth are

obtained using a hypothesis testing approach such as Dunnett's

Procedure (Dunnett, 1955) or Steel's Many-one Rank Test (Steel,

1959; Miller, 1981) (see Section 9). Separate analyses are

170
performed for the ~stimation of the LOEC and NOEC endpoints and • ': • - > "

for the estimation of the LC50 and IC25. Concentrations at which

there is no survival in .any of the test chambers' are excluded

from the statistical analysis of the NOEC.and.LOEC for survival.

and growth, but included in the estimation of th~'LC50 and IC25.

See the Appe~dices for examples of the manual computations, and

examples of data input and program output. . .

12.13.1.3 The statistical tests described here must be used with,·

a knowledge of the assumptions upon which the tests are

contingent. The assistance of a statistician is recommended for

analysts who are not proficient in statistics.

12.13.2 EXAMPLE OF ANALYSIS OF MYSID, HOLME$IMYSIS COST~TA,

SURVIVAL DATA

12.13.2 .1 Formal statistical analysis of the sm~vival .data is

outlined in Figures 1 and 2. ~he response used in the analysis is

the proportion of animals surviving in each test-or control

chamber. Separate analyses are performed for thE~. estimation of

the I\fOEC and LOEC endpoints and for the estimation of the LC50

. endpoint. Concentrations at which there.is no survival in any of

the test chambers are excluded from statistical Glnaly~is of the

NOEC and LOEC, but included in the estimation of the LC;jEC, and

IC endpoints~

'12.13.2.2 For .the case of equal numbers of replicates across all

concentrations and the control, the evaluation of the NOEC and

LOEC endpoints, is made via a parametric .t::est, Dunnett's

Procedure, or· a nonparametric test, Steel's Many-one Rank Test,

on the arc sine square root transformed data. Underlying

assumptions of Dunnett's.Procedure, normality and' homogeneity of

variance, are formally tested. The test for normality is the

Shapiro-Wilk's Test, and Bartlett's Test is used to test. for

homogeneity of variance. If either of these tests fails, the

nonparametric test, St~el's Many-one Rank: Test, is used to

determine the NOEC .and LOEC endpoints. If the as,sumptions of

Dunnett's Procedure are met, the endpoints are es-~imated by the·

parametric .procedure.

12.13.2.3 If equal numbers of replicates occur among the

concentration levels tested, there are parametric and

nonparametric alternative analyses. The parametr:~c analysis i's a

171
TABLE 3. SUMMAR~ OF TEST CONDITIONS TEST ACCEPTABILITY

CRITERIA FOR THE MYSID, HOLMESIMYSIS COSTATA, GROWTH

AND SURVIVAL TEST WITH EFFLUENTS AND RECEIVING,WATERS

J.. Test type: Static-renewal ,

2. Salinity: 34 ± 2%0

3. Temperature: 13 ± 1°C (mysids collected north

I of Pt. Conceptibn)

15 ±, 1°C (mysids collected south , of Pt. Conception)

, 4. Light quality: Ambient laboratory illumination

5. Light intensity: 10-20 flE/m2 / s (Ambient , laboratory illumination)

6. Photoperiod: 16 h light, 8 h darknes's

7. Test chamber: 1000 mL

, 8. Test solution volume: 200 mL

I

9. Renewal of test 75% renewal at 48 and 96 hours

solutibns:

J.O. Age of test organisms: 3 to 4 days post-hatch

,juvenile$

J.J.. No. organisms per test 5

chamber:

J.2. No. replicate chambers 5

per concentration:

J.3. No. mysids per 25

concentration:

: J.4. Source of food: Newly hatched Artemia nauplii I

(less than 24 h old)

I J.5. Feeding regime: Feed 40 nauplii per larvae

daily (dividing into morning

'and evening feedings)

172
16. Cleaning: Siphon during test solution

renewal

17. Aeration: None unless DO falls below 4.0

mg/L, then gently aerate in all

cups

18. Dilution water: Uncontami,nated l:"'P.hl-filtered

natural seawater or hypersaline

brine prepared f:rom natural

seawater ' .

19. Test concentrations: Effluents: Minimum of 5 and a

control

Receiving w'aters: 100%

receiving water and a control

20. Dilution factor: Effluents: ~0.5 series

Receiving waters: None,' or ~0.5

2l. Test duration: 7 days

22. Endpoints: Survival and grm'J'th

23. Test acceptability ~75% survival, a'ITerage dry

criteria: weight 0.40 JLg in the

controls; survival MSD .<40% ;

growth MSD <50 W;J; and both

survival and gro\~th NOECs must

be less than 100 p.g/L with zinc

24. Sampling requirements: For on-site tests, samples must

be used within 2,~ h of the time

they are removed from the

sampling device (see Section 8,

Effluent and Reqdving Water

Sampling, Sample Handling, and

Sample Preparation for Toxicity

Tests)

25. Sample volume required: , 2 L per renewal

173
TABLE 4. DATA FOR HOLMESIMYSIS COSTATA 7-DAY SURVIVAL AND GROWTH' TEST1

Treatment Replicate Total No. Prop. Mean

Chamber Mysids Alive Alive Weight

Control. Brine 1 5 5 1.00 0.051

2 5 5 1.00 0.050

3 5 5 1.00 0.040

4 5 5 1. 00 0.064

5 5 5 1. 00 0.039

Control. Dilution 1 5 5 1.00 0.048

2 5 5 1. 00 0.058

3 5 5 1. 00 0.047

4 5 5 1. 00 0.058

5 5 5 Lao 0.051

1.80% 1 5 5 1. 00

0.055

2 5 5 1. 00 0.048

3 5 5 1.00 0.042

4 5 4 0.80 0.041

5 5 5 1. 00 0.052

3.20% 1 5 5 1. 00

0.057

2 5 '4 0.80 0.050

3 5 5 1. 00 0.046

4 5 5 1. 00 0.043

5 5 4 0.80 0.045

t test with the Bonferroni.adjustment (see Appendix D). The

Wilcoxon Rank Sum Test with the Bonferroni adjustment is the

nonparametric alternative.

12.13.2.4 Probit Analysis (Finney, 1971 r see Appendix G) is used

to estimate the concentration that causes a specified percent

decrease in survival from the control. In this analysis, the

total mortality data from all test replicates at a give'n

concentration are combined. If the data do not fit the Probit

model, the Spearman-Karber method, the Trimmed Spearman-Karber

174
method, or trie Graphical method may be used to estimate the LC5"d'"

(see Appendiges H-K) .

12.13.2.5 The proportion of survival in each replicate must

first'betransformed by the arc s~ne square root transformation

procedure de~cribed in AppendixB. The raw and transfor~ed data,

means and variances of the transformed observations at each

concentration including the control are listed in Table 5. A

plot of the survival data is provided in Figure 3.

12.13.2.6 Test for Normality

12.13.2.6.1' The first step of the test for normality is .to

center the observations by subtracting the mean of all

observations within a concentration from each ol,servation in that

concentration. The centered observations are li.sted in Table 6.

12.13.2.6~2 Calculate the denominator, D, of the test statistic:

n . - 2

D = E (X.-X)

i.1

Where: Xi == the ith ce.ntered observation

x ;;:; the overall mean of the centered observations ,

n ;;:; the total number of centered observations. I

12.13.2.6.3 For this set of data, n 25

.,X == -L(0.OQ1) == 0.00

25

D ;;:; 0.227

12.13.2.6.4 Order the centered observations from smallest to

largest:

~'. x(n)

Where X(i)' is the ith ordered observation. These ordered

observations are listed,in Table 7.

175.
-

TABLE 5. MYSID. HOLMESIMYSIS COSTATA. SURVIVAL DATA

Concentration (%)

Replicate Control 1.80 3.20 5.60 10.00

1 1. 00 1. 00 1. 00 0.80 0.20

2 1. 00 1. 00 0.80 1. 00 0.00

RAW 3 1. 00 1. 00 1. 00 1.00 0.00

4 1. 00 0.80 1. 00 0.80 0.00

5 1. 00 1. 00 0.80 0.80 0.00

ARC SINE 1 1.345 1.345 1.345 1.107 0.464

SQUARE 2 1.345 1.345 1.107 1.345 0.225

ROOT 3 1.345 1.345 1.345 1.345 0.225

TRANS- 4 1.345 1.107 1.345 1.107 0.225

FORMED 5 1.345 1.345 1.107 1.107 0.225

Mean('T1) 1.345 1.297 1.250 1.202 ·0 .273

Sf 0.000 0.011 0.017 0.017 0.011

; 1 2 3 4 5

-

12.13.2.6.5 From Table 4, Appendix B, for the number of

observations, n, obtain the coefficients a 1 , a 2 , •••• , ak where k

is n/2 if n is even and (n-1)/2 if n is odd. For the data in

this example, n = 25 and k = 12. The a i values are listed in

Table 8.

12.13.2.6.6 Compute the test statistic, W, as follows:

1 k . 2

W : - [Ea (X (n-io1) -X (i)) ]

D i.1 i

The differences x(n-i+1) - Xli) are listed in Table 8.

176
, Figure 1. F~owchart for statistical analysis of mysid,

Holmesimysis costata, survival data by hypothesis testing.

177
,MORTALllYDATA -

iDEAD

TwOORMORE _

PARTIAL MORTALITIES?:

IS PROSIT MODEL . -

APPROPRIAi"E1

: (SIGNIFICANT x2 TEST)

Figure 2.Flowchart for statistical analysis of mysid,

Holrnesimysis costata, survival data by point estimation.

178
>kI

IfD'oUMLN!PtJCATEII.Wt'ML

CONfrlEtI'S1IEME:M\N.l.ER3fl6tlCH~T1DN

><

* ,;1<* *

* *

*

i

1

0.1 i

~.ot " - + +

1.0

0.9

0.8

z

0

- 0.1 >-

n:::

0 0.6 ,0...

0

n:::

0... 0.5

I-' -'

-...J .<l;

\D > 0.4 -

>

n:::

::J 0.3 (/)

0.2

0.0 1.8 3.2 5.6 10.0 18.0

, EFFLUENT CONCENTRATION (%)

Figure 3. Plot of survival of mysids, Holrnesimysis costata, at each treatment
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

Replicate Control

(Dilution)

Concentration

3.20 5.60 10.00

1 0.000 0.048 0.095 -0.095' 0.191

2 0.000 0.048 -0 ..143 . 0.143 -0.048

3 0.000 0.048 0.095 0.143 -0.048

4 0.000 -0.190 0.095 -0.095 -0.048

5 0.000 0.048 -0.143 -0.095 -0.048

TABLE 7. ORDERED CENTERED OBSERVATIONS FORSHAPIRO-WILK'S EXAMPLE

i X<il i

1 -0.190 14 0.000

2 -0.143 15 0.000

3 -0.143 16 0.048

4 -0.095 17 0.048-

5 -0.095 18 0.048

6 -0.095 19 0.048

7 -0.048 20 0.095

8 -0.048 21 0.095

9 -0.048 22 0.095

10 -0.048 23 0.143 ..

11 0.000 24 0.143

12 0.000 25 0.191

13 0.000

180
__________________________1 ."

TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S'EXAMPLE

a; xCn-;+l) - XCi)

1 0.4450 0.381 XC2S) ,_ X(l)

2 0.3069 0.286 X(24) - X(2)

3 0.2543 0.286 X(23) - X(3)

4 0.2148 0.190 X(22) - X(4)

5 0.1822 0.190 : X(21) - XCS)

6 0.1539 '0.190 X(20) - X(6)

7 0.1283 0.096 XCl9) - X(7)

8 0.1046 0.096 . XCl8) - X(8)

9 0.0823 0.096 ,'. X(l7) X(9)

10 0.0610 0.096 XCl6) - XClO)

11 0.0403 0.000 XClS) - XC1l)

12 0.0200 0.000 XCl4) X(12)

" "f,· -,,: ': ~~ i,·',' '.'

For this data in this example:

w 1 (0.4708)2

0.227

0.976

12.13.2.6.7 The decision rule for this test is to compare W as

calculated in Subsection 6.6 with the critical value found in

Table 6, Appendix B. If the computed W is less 1:han the critical

value, conclude that the data are not normally distributed. For

this set of data, the critical value ata significance level of

0.01 and n = 25 observations is 0.888. Since W = p.976 is

greater than the critical value, conclude that the qata are

~ormally distributed.

12. 13 .2 . 6 . 8 Since the variance of the control group is' zero ,

Bartlett's test statistic can not be calculated. Therefore, the

survival data variances are considered to be heterogeneous.

12.13.2.6.9 Since the data do not meet t:he assumption of

homogeneity of variance, Steel's Many-one Rank TE~St will be used

to analyze the survival data. ' " ,.i,'

181
12.13.2.7 Steel's Many-one Rank Test

12.13.2.7.1 For each contro'l and concentration combination,

combine the data and arrange the observations in order of size

from smallest to largest. Assign the ranks (1, 2, ... ,'10) to

the ordered observations with a rank ofi assigned to the

smallest observation, rank of 2 assigned to the next larger

observation, etc. If ties occur when ranking, assign the average

rank to each tied observation.

12.13.2.7.2 An example o£ assigning ranks to the combined data

for the control and 1.80% concentration is given in Table 9.

This ranking procedure is' repeated for each control/concentration

combination. The complete set of rankings is summarized in

Table 10. The ranks are then summed for each concentration

level, as shown in Table 11.

12.13.2.7.3 For this example, determine if the survival in any

of the concentrations is significantly lower than the survival in

the control. If this occurs, the rank ,sum at that concentration

would be significantly lower than the rank sum of thecontrpl.

Thus compare the rank sums for the survival at each of the

various concentration levels with some "minimum" or critical rank

sum, at or below which the survival would be ,considered

significantly lower than th~ control. At a significa~ce le~~i of

0.05, the minimum rank sum in a test with four concentrations

(excluding the control) is 17(See Table 5, Appendix E) .

12.13.2.8.1 The data used to calculate the LC50 is summarized in

Table 12. For this example, although there are two

concentrations with partial mortalities, the chi-square test for

heterogeneity was significant, indicating that Probit Analysis is

inappropriate for this set of data. Inspection of the data

reveals that the smoothed, adjusted proportion mortality for the

lowest concentration will not be zero, indicating that the

Trimmed Spearman-Karber Method is recommended to calculated the

LCSO for this dataset.

12.13.2.8.2 For the Trimmed Spearman-Karber

USEPA Trimmed Spearman-Karber program, TSK.

program output is provided in Figure 4.

182

analysis, r~~the

An example of the
TABLE 9. ASSIGNING RANKS TO THE CONTROL AND 1. 80% CONCENTRATION LEVEL

FOR STEEL'S MANY-ONE RANK TEST

Rank

1

6

6

6

6

6

6

6

6

6

Transformed Proporti'on

of Total Mortality

1.107

.1. 345

1.345

1.345

1.345

1.345

1.345

1.345

1.345

1.345

TABLE 10. TABLE OF RANKS1

'Concentration

1.80%

Control

Control

Control

Control

Control

1.80%

1.80%

1.80%

1.80%

Concentration (%)

Repli- Control 1.80 3.20 5,.60 10.0

cate

1 1.345(6.6.5.7.8) 1. 345(6) 1. 345(6.5) 1.107(2) 0.464(5)

2 1.345(6.6.5.7.8) 1. 345(6) 1.107(1.5) 1. 345(7) 0.225(2.5)

3 1.345(6.6.5.7.8) 1. 345(6) 1. 345(6. 5) 1. 345(7) 0.225(2.5)

4 1.345(6.6.5.7.8) 1.107(1) 1. 345(6.5) 1.1'07 (2) 0.225(2.5)

5 1.345(6.6.5.7.8) 1. 345(6) 1.107(1.5) 1.107(2) '0 .225 (2 .5)

lControl ranks are given in the order of theconcentrat'ion with which

they were ranked. I

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

1.83
TABLE 11.

concentration

1. 80

3.20

5.60

10.00

RANK SUMS

Rank Sum

25.0

22 ..5

20.0

15.0

TABLE 12. DATA FOR TRIMMED SPEARMAN-KARBER ANALYSIS

Concentration (%)

No Dead

No Exposed

Control

o

25

1.80

1

25

3.20

2

25

5.60

3

25

10.0

24

25

18.0

25

25

12.13.3 EXAMPLE OF ANALYSIS OF MYSID, HOLMESIMYSIS COSTATA

GROWTH DATA

12.13.3.1 Formal statistical analysis of the gro~th data is

outlined in Figure 5. The response used in the statistical

analysis is mean weight per surviving organism per replicate.

The IC25 can be calculated for the growth data via a point

184
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5

DATE: TEST NUMBER: 1 DURATION: 7 days

TOXICANT Eftl uent

SPECIES: Holmesimysis costata

RAW DATA: . Concentrati on Number Mortalities . --- ---- (%) Exposed

.00 25 0

1.80 25 1

3.20 25 2

5.60 25 3

10.00 25 24

18.00 25 25

SPEARMAN-KARBER TRIM: 4.00%

SPEARMAN-KARBER ESTIMATES: LC50: 6.95

95% LOWER CONFIDENCE: 6.22

95% UPPER CONFIDENCE: 7.76

Figure 4. Output for USEPA trimmed Spearman-Karber Program. version 1.5.

estimation technique (see Section 9, Chronic Toxicity Test

Endpoints and Data Analysis). Hypothesis testin9 can be used to

obtain an NOEC and LOEC for growth. Concentrations above the

NOEC for survival are excluded from the hypothesis test for

growth effects.

12.13.3.2 The statistical analysis using hypothesi.s tests

consists of a·parametric test, Dunnett's Procedure, and a

nonparametric test, Steel's Many-one Rank Test. : The underlying

185
ENDPOINT ESTiMATE

IC25

HOMOOENEOUSVARIANCE , .' :

'NO

tTESTWITH

BONFERRONI

ADJUSTMENT

Figure 5. Flowchart for statistical analysis of mysid,

Holmesimysis costata, growth data.

186
assumptions of the J;)unnett's Procedure, normali.ty and homogeneity

of variance, are formally tested. The test for, normality is the

Shapiro-Wilk's Test and Bartlett's Test is used to test for

homogeneity of variance. If either of these tests fails, the

nonparametric test, Steel's Many-one Rank Test, is used to

determine the NOEC and LOEC endpoints. If the assumptions of

Dunnett's Procedure are met, the endpoints are determined by the

parametric test.

12.13.3.3 Additionally, if unequal numbers of replicates occur

among the concentration levels tested, there are parametric and

nonparametric alternative analyses. The.parametric analysis is a

t test with the Bonferroni adjustment. The Wilcoxon Rank Sum

Test with the Bonferroni adjustment is the nonparametric

alternative. For detailed information on the Bonferroni

adjustment, see Appendix D.

12.13.3.4 The data, mean 'and variance of the ohservations at

each concentration including the control for this example are,

listed in'Table 13. A plot of the data is'provided in Figure 6.

Since there is significant mortality in the 10.0% concentration,

its effect on growth is not considered.

12.13.3.5 Test for Normality

12.13.3.5.1 The first step of the test for normality is to

center the observations by subtracting the mean of all

observations within.a concentration from each observation in that

concentration. The centered observations are listed in Table 14.

12.13.3.5.2 Calculate the denominator, D, of the statistic:

n

r - 2 D=L.t(X -X) 1.

1.-1

187
TABLE 13. MYSID. HOLMESIMYSIS COSTATA. GROWTH DATA

Concentration (%)

Replicate Control 1.80 3.20 5.60 10.0

1 0.048 0.055 0.057 0.041 0.033

2 0.058 0.048 0.050 0.040 0.000

3 0.047 0.042 0.046 0.041 0.000

4 0.058 0.041 0.043 0.043 0.000

5 0.051 0.052 0.045 0.040 0.000

0.052

0.0000283

1

0.048

0.0000373

2

0.048 0.041

0.0000307 0.0000015

3 4

0.007

0.000218

5

TABLE 14. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

Concentration (%)

Replicate Control 1.80 3.20 5.60

1 -0.004 0.007 0.009 0.000

2 0.006 0.000 0.002 -0.001

3 -0.005 -0.006 -0.002 0.00.0

4 0.006 -0.007 -0.005 0.002

5 -0.001 0.004 -0.003 . -0.001

188
'"E

0.06

0.05

0.04

*

*

*

* ------------ ---*"---- ------ ---$----- --

* * * IJCJlYDl,MLRfi!Il.Q\lE....waat1J'

cctfrEC'l'lnEWMWoUEfCIIlI!'IICHDONCENl'Mn:lH

JIiI'INiS8IfIllECNnCN.WI.llEFCaJMNm'l1QT

I/HtWSOtn'IS.OW'nt8\N.UEWOtlU)M:

~Y~fRQM1lEClQNJ1lIX,)

--- --:*: . - . - . -- - - - ---- - --- - - . --- - - . - .

· i-

:r:

0

-

W

:;::

f-1 >- en e::: \0 Cl

z

<

w

::2

0.03

*

0.02

0.01

0..001. I I l' ".1

0.0 1.8 3.2 ~8

.' EFFLUENT CONCENlRATION (%)

10.0· 18.0

Figure 6. Plot of growth data for mysid, Holmesimysis costata, test.
Where: Xi = the ith centered observation

X = the overall mean of the centered observations

n = the total number of centered observations'

12.13.3.5.3 For this set of data, n.= 20

x = (0.001) = 0.000

20

D = 0.000393

12.13.3.5.4 Order the centered observations from smallest to

largest

X(1) :!:: X (2 ) :!:: ••• :!:: X Cn )

where Xli) denotes the ith ordered observation. The ordered

observations for this example are listed in Table 15.

TABLE 15. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S 'EXAMPLE

i i

1 -0.007 11 0.000

2 -0.006 12 0.000

3 -0.005 13 0.000

4 -0.005 14 0.002

5 -0.004 15 0.002

6 -0.003 16 0.004

7 -0.002 17 0.006

8 -0.001 18 . 0.006

9 -0.001 19 0.007

10 -0.001 20 0.009

190
12.13.3.5.5 From Table 4, Appendix B, for the.number of

observations, n, obtain the coefficients a l , a 2,' '.' a k where k is

n/2 if n is even· and (n-1)/2 if n is odd. For the data in this

example, n = 20 and k = 10.' The ai values are l,isted in

Table 16.

12.13.3.5.6 Compute the test. statistic, W, as follows:

The differences xCn-i+1J - X(i) are listed in Table ;16. For this set

of data:

W = 1 (0.0194)2 = 0.958

0.000393

TABLE 16. COEFFICIENTS AND DIFfERENCES FOR SHAPIRO-WILK'S EXAMPLE

1

2

3

4'

5

6

.7

8

9

10

a-' · 1

0.4734

0.3211

0.2565

0.2085

0.1686

0.1334

0.1013

0.0711

0.0422

0.0140

X·<n-i+l)~.· X·m·,

0.016

0.013

0.011

0.011

0.008

0.005

0.004

0.001

0.001

0.001

'X(20) _. X(l)

.... X(19) X(2)

'; X<l8) _ X(3)

; XCl7l _ X(4)

: X(6) - XCS)

;XUS) X(6)

: X(4) - XC7J

X(13) _ X<S) . X(l2) X(9)

. XCll> XClO)

12.13.3.5.7 The decision rule for this test is to compare W as

calculated in Subsection '12.13.3.5.6 to a critical value found in

Table~, Appendix B. If 'the computed W is less than the critical

value, conclude that the data are not normally di:stributed. For

this set of data, the critical value at significance level of

191
0.01 and n = 20 observations is 0.868. Since W = 0.958 is

greater than the critical value, conclude that the data are

normally. distributed.

12.13.3.6 Test for Homogeneity of Variance

12.13.3.6.1 The test used to examine whether the variation in

mean weight of the mysids is the same across all concentration

levels including the control, is Bartlett's Test (Snedecor and

Cochran, 1980). The test statistic is as follows:

p p

[(LV) In "52 - LV1 In 51

2

]

B = __1._1 1._1 _

c

Where: Vi = degrees of freedom for each concentration and the

control, Vi = (ni - 1)

p = number of concentration levels including the

control

ln = loge

i = 1, 2, ... , p where p is the number of

concentrations including the control

ni = the number of replicates for concentration i.

p p

C 3 1+ [~(p-1) r 1 [L1/V1 - (LV1 ) -1]

1-1 1-1

12.13.3.6.2 For the data in this example (See Table 13,), all

concentrations including the control have the same number of

replicates (ni = 5 for all i). Thus, Vi = 4 for all i.

192
12.13.3.6.3 Bartlett1s statistic is therefore:

p

B = [(16)ln(0.0000245)-4,Eln(S:)]/1.104 1=1

=

=

=

[16(-10.617) - 4(-44.470)])1.104

[-169.872 - (-177.880)]/1.104

7.254

12.13.3.6.4 B is approximately distributed as chi-square with p

- 1 degrees of freedom, when the variances are in fact the same.

Therefore, the appropriate critical value for this test, at a

significance lev~l of O.pl with three de~rees of freedom, is

9.210. Since B = 7.254 is le~s than the critical value of 9.210,

conclude that the variances are not different.

12.13.3.7 Dunnett's Procedure

12.13.3.7.1 To obtain an estimate of the pooled variance for the

Dunnett's Procedure, construct an ANOVA table aB described in

Table 17.

TABLE 17. ANOVA TABLE

Source df Sum of Squares Mean Square(MS)

(SS) (SS/qf)

2

Between p - 1 SSB SB = SSB/(p-l)

2

Within N - P SSW Sw = SSW/ (N-p)

,

Where: p = number of concentration levels including the

control

N = total number of observations n l + n 2 ••• + n p

193
ni = number of observations in concentration i

p

SSE" ET1

2

/n1 -G2/N

1-1

p n1

SST s EEY12j-G2/N

1-lj-1

. SSW" SST-SSE

Between Sum of Squares

Total Sum of Squares

Within Sum of Squares'

G :: the grand total'of all sample observations,

p

G" ET1

1-1

,

Ti :: the total of the replicate measurements for

concentration i

Yij = the j th observation for concentration i

(represents the mean weight of the mysids for

concentration i in test chamber j)

12.13.3.7.2 For the data in this exampl~:'

N = 20

p

SSE" ET12/ni.-G2/N

i.·1

= _1_ ( 0 . 225 ) - ( 0 . 94 6 ) 2 = o. 0 0 02 54

5 20

p ni.

SST" EEY;j-G2/N

i..1j-1

= 0.0455 - (0.946)2

20

194

0.000754
ssw = SST-SSB

= 0.000754 - 0.000254 = O~.000500

S~ = SSB/(p-1) = 0.000254/(4-1} = 0.0000847

SI = SSW/(N-p) = 0.b00500/(20-4) = 0.0000313

12.13.3.7.3 Summarize these calculations in the ANOVA.table

(Table 18) .

TABLE 18. ANOVA TABLE FOR DUNNEIT'S PROCEDURE EXAMPLE

Source

Between

Within

Total

df

'3

16

19

Sum of Squares

(55)

'0.000254 .

0.000500

0.000754

Mean Square (MS)

(SS/df)

0.0000847

0.0000313

12.13.3.7.4. To perform the individual c~mparisons, calculate the

t statistic for each concentration, and control combination as

follows: .

Where: Yi = mean weight for concentrati'on i

Yl = mean weight for the control

. Sw = square root of the within mean square

n l = number of replicates for the control

195
ni = number of replicates for concentration i

~2.~3.3.7.5 Table 19 includes the calculated t values for each

concentration and control combination. In this example,

comparing the 1.80% concentration with the control the

calculation is as follows:

t = 2

(0.052-0.04B)

[0.00559V(1/S)+(1/5))

= 1.131

TABLE 19. CALCULATED t VALUES

Concentration (ppb)

1.80

3.20

5.60

2

3

4

1.131

1.131

3.111

~2.~3.3.7.6 Since the purpose of this test is to detect a

significant reduction in mean weight, a one-sided test is

appropriate. The critical value for this one-sided test is found

in Table 5, Appendix C. For an overall alpha level of 0.05, 16

degrees of freedom for error and three concentrations (excluding

the control) the approximate critical vaiue is 2.23. The mean

weight for concentration "i" is considered significantly less

than the mean weight for the control if t i is greater tha~ the

critical value. Therefore, the 5.60% concentration has

significantly lower mean weight than the control. Hence the NOEC

and the LOEe for growth are 3.20% and 5.60%, respectively.

~2.~3.3.7.7 To quantify the sensitivity'of the test, the minimum

significant difference (MSD) that can be detected statistically

may be calculated.

MSD = d S"V (1/n1 ) ... (lin)

196
critical value for Dunnett's Procedure'

square root· of the within mean:square

,common' number of replicates at J each

::entration

.ssumes equal repl~cation at .each concentration)

rlumber of replicates in the con~trol.

.his example:

.... .. .. ,' '.

,

- 2.23 (0.00559) (0.632)

- 0.00788

, .

'efore, for this set of data, the minimum

!an be detected as statistically significant is

.S represents a 15.2% reduction in mean weight

; '~., .' I • ~." 1

.ation of the rep ". t

growth data .from Table ~3. are utilized in, this

l in the table, the observed mea:!=ls are

l-increasingwith respect· to concentration~

loothed means will be simply the'r corresponding

j -

'he observed'means are represented by Yi and the

r Mi. Table 20 contains the smoc~thed means a,nd

plot of the smoothed response curve.'
12.13.3.8.5 Using the equation in Section 4.2 from Appendix L, '

the estimate of the IC25 is calculated as follows:

(C -C)

ICp : C + [M (l-p/lOO) -M ] (j.1) j

j 1 j (M -M)

(j.1) j

I C25 = 5. 60 + [0. 052 (1 - 25/100) - O. 041] (10 . 0 - 5. 60)

(0.0066 - 0.041)

= 5.86%.

12.13.3.8.7 When the ICPIN program was used to analyze this set

of data, requesting 80 resamples, the estimate of the IC25 was

5.86%. The empirical 95.0% confidence interval for the true mean

was 4.9440% to 6.2553%. The computer program output for the IC25

for this data set is shown in Figure 8.

-

TABLE 20. MYSID, HOLMESIMYSIS COSTATA, MEAN

GROWTH RESPONSE AFTER SMOOTHING

Toxicant Response Smoothed

Cone. Means Means

(%) i Yi (mg) Mi (mg)

Control 1 0.052 0.052

1.80 2 0.048 0.048

3.20 3 0.048 0.048

5.60 4 0.041 0.041

10.00 5 0.0066 0.0066

18.00 6 0.000 0.000

- 12.14 PRECISION AND ACCURACY

12.12.1 PRECISION

12.12.1.1 Single-Laboratory Precision

g

"

,

. .

12.12.1.1.1 Data on the single laboratory precision 'of the

Holmesimysis costata growth and survival test with zinc sulfate

198
are shown in Table 21. NOECs for mysid survival were either 32

or 56 Ilg!L Zn. There was also .·good agreement among LC50s, with a

coefficient of variation of 14%. Mysids did not exhibit a growth

response at zinc concentrations below those causing significant

mortality; NOEC.values for growth were always greater than or

equal to the highest zinc concentration. lC50 values for growth

could not be calculated.

12.12.1.2 Multi-laboratory Precision

12.12.1.2.1 The multi-laboratory data indicate a similar level

of test precision (Table 22). The four multi-laboratory tests

were conducted over a two year period, and each lJ.sed split

effluent samples tested at two laboratories. Survival NOEC

values were the same for both laboratories in three of the four

tests, with the NOECs varying by one concentra·tion in the fourth

test. The mean coefficient of variation between LC50 values from

different laboratories was '21%. The two' available comparison's' of

growth NOEC values indicate similar responses at both

laboratories. Growth was the more sensitive indLcator of

toxicity in three of the four effluent tests.

12.14.2 ACCURACY

12.14.2.1 The accuracy of toxicity tests cannot be determined.

199
0.06 J * * * 1IlMlUl~_YI8llHI'

* --- CtIMI:Ia,..~·teMWJ.8I

* - - - - - - CQIICTIHM)Q'ftC......

0.05k

* "

* 1 0'

o~j * E

:r:

>-- * <.:l \ * -

w 1 :;: 0.03

tv >- 0 0::

0 0

z 0.02 «

w

:::;;

<

0.01

i

j

j 0.00 l

l

0.0 1.8 3.2 5.6 10.0· 18.0

EFFLUENT CONCENTRATION (%)

Figure 7. Plot of raw data, observed means, and smoothe~ means for the mysid,

Holmesimysis costata, Growth data from tables 13 and 20
Cone. 10 1 2 3 4 5 6 --------------.----------------------------------------------------------

Cone. Tested 0 1.80 3.20 5.60 10.0 18.0

------------------------------------~----------------- ,------------------

Response 1 .048 .055 .057 .041 .. 033 0

Response 2 .058 .048 .050 .040 0 0

Response 3 .047 .042 .046 .041 0 0

Response 4 .058 .041 .043 .043 0 0

Response 5 .051 .052 .045 .040 0 0

*** Inhibition Concentration Percentage Estimate ***.

Toxicant/Effluent: Effluent

Test Start Date: Test Ending Date:

Test Species: mysid. Holmesimysis costata

Test Duration: 7 days

DATA FILE: mysid.icp

OUTPUT· FILE: mysid.i25

------------------------------------------------------ ------------~----

Cone. Number Concentration

10 Rep1i cates %

Response

Means

Std. Pooled

Dev. Response 'Means

1

2

3

4

5

6

5

5

5

5

5

5

0.000

1.800

3.200

5.600

10.000

18.000

0.052

0.048

0.048

0.041

0.00"7

0.000

0.005

0.006

0.006

0.001

0.015

0.000

0.052

0.048

0.048

0.041

0.007

0.000

The Linear Interpolation Estimate: 5.8174 Entered P Value: 25 ;'

Number of Resamplings: 80

The Bootstrap Estimates Mean:

Original Confidence Limits:

Expanded Confidence Limits:

Resampling time in Seconds:

5.8205 Standard Deviation: 0.2673

Lower: 4.9440 Upper: 6.2553

lower: 4.5073 Upper: 6.4743 '

0.22 Random Seed: 526805435

Figure 8. Output for USEPA Linear Interpolation Program'for the rc:25.

201
TABLE 21. SINGLE LABORATORY PRECISION DATA FOR THE MYSID,

HOLMESIMYSIS COSTATA GROWTH AND SURVIVAL TEST WITH

ZINC (ZN ~G/L) SULFATE AS THE REFERENCE TOXICANT.

I Test Survival Growth

NOEC LC50 NOEC

1 32 47 >32

I 2 32 59 >32

I 3 56 62 >56

I

4 56 65 >56

I

I N 4 4 4

Mean 44 58 >44

SD 7.9

CV (%) 14

No growth effect was observed in 'zinc concentrations below those

causing significant mortality (10, 18,32, 56 and 100 ~.g/L) .

All tests were conducted at MPSL.

202
· TABLE 22. MULTI-LABORATORY PRECISION DATA FOR ~mE MYSID,

HOLMESIMYSIS CO$TATA GROWTH AND SURVIVAL TEST WITH

SPLIT EFFLUENT (%) ON THE SAME DATE.

Test Effluent Lab Survival Growth

Type NOEC LCSO· NOEC

1 BKME OSU 1.0 1.8 0.5L

1 BKME MPSL 1.0 1.3 O. SL --

CV=26% -

2 POTW ATL 3.2 4.1 >3.2L

2 POTW MPSL 3.2 5.1 >3.2L

CV=14%

3 POTW SRH 10.0 12.8 na

3 POTW MPSL 10.0 11. 7, 3.2w

CV=6%

4 POTW SRH 10.0 15.8. 5.6w .

4 POTW MPSL 5.6 9.1 3.2w

CV=38%

Mean Interlaboratory CV= 21%

L Length was measured as the growth endpoint in tests 1 and 2,

w Weight was measured in test 3 and 4.

na Data was not available.

OSU is the Oregon State University Laboratory at the Hatfield

Marine Science Center in Newport Oregon.

ATL is Aquatic Testing Laboratory in Ventura, California.

SRH is S. R. Hansen and Associates in Concord, California.

MPSL is the Marine Pollution Studies Laboratory near Monterey,

California.

203
APPENDIX I. MYSID TEST: STEP-BY-STEP SUMMARY

PREPARATION OF TEST SOLUTIONS

A. Determine test concentrations and appropriate dilution water

based on NPDES permit conditions and guidance from the

appropriate regulatory agency.

B. Prepare effluent test solutions by diluting well mixed

unfiltered effluent using volumetric flasks and pipettes.

Use hypersaline brine where necessary to maintain all test

solutions at 34 ± 2~. Include brine controls in tests that

use brine.

C. Prepare a zinc reference toxicant stock solution (10,000

~g/L) by adding 0.0440 g of zinc sulfate (ZnS04 0 7H20) to 1

liter of reagent water.

D. Prepare zinc reference toxicant solution of a (control) la,

18, 32, 56 and 100 ~g/L by adding a, 1.0 1.8, 3:2, 5.6 and

10.0 mL of stock solution, respectively, to a 1-L volumetric

flask and filling to 1-L with dilution water.

E. Sample effluent and reference toxicant solutions f?r

physical/chemical analysis. Measure salinity, pH and

dissolved oxygen from each test concentration.

F. Randomize numbers for test chambers and record the chamber

numbers with their respective test concentrations on a

randomization data sheet. Store the data sheet safely until

after the test samples have been analyzed."

G. Place test chambers in a water bath or environmental chamber

set to 13 or 15°C and allow temperature to equilibrate.

H. Measure the temperature daily in one. random replicate (or

separate chamber) of each test concentration. Monitor the

temperature of the water bath or environmental chamber

continU01.l.sly.

I. At the end of the test, measure salinity, pH, and dissolved

oxygen concentration from each test concentration.

204
PREPARATION AND ANALYSIS OF TEST ORGANISMS

A. Four to five days prior to the beginning of the toxicity.

test, isolate approximately 150 gravid female mysids in a

screened (2-mm-mesh) compartment within an aerated BO-liter

aqua~ium (15°C). Add a surplus of Arternia :nauplii' (200 per

mysid, static; 500 per mysid, flow-through) to stimu:j.ate

overnight release of juveniles. Add' bladesl of kelp as

habitat.

B. Isolate the newly released juveniles by slowly siphoning

into a screen-tube (150-~m~mesh, 25cm diarrl.) immersed in a

bucket of clean seawater. Transfer juveniles into

additional screen-tubes or static 4-1iter beakers at a

density of approximately 50 juveniles per liter.

Juveniles should be fed five 'to ten newly released Arternia

nauplii per juvenile per day and a pinch (1:0 to 20 mg) of

ground Tetramin® flake food per 100 juveniles per day. I

Maintain the juveniles for three days at 13; to 15°C,

changing the water at least once in static chambers.

I .

C. After three days, begin randomized i~troduction of juveniles

into the test chambers. Place one or two mysids at a time

into as many plastic cups as there are test. chambers.

Repeat the process until each cup has exactly five' juvenile

mysids.

D. Eliminate excess water from the cups (no more than 5 mL

should remain) and pipet the mysids into the test chambers

using a wide bore glass tube or pipet (approximately 3 mm

ID). Make sure no mysids are left in the randomization • I

cups. Count the number of juveniles in each test chamber to

verify that each has five.

E. Remove all dead mysids daily, and add 40 newly hatched

Arternia nauplii/mysid/day, adjusting feedinsr to account for

mysid mortality.

F. At 48 and 96· hours, renew 75%- of the test solution in each

chamber.

G. After 7 days, count .and record the number of live and dead

mysids in each chamber. After counting, use the

randomization sheet to assign the correct test concentration

to each chamber. Remove all·dead mysids.

205
H. Carefully pour the contents of ·each ~est chamber through a

small mesh screen «300~m). Count the mysids and record

before screening. Briefly dip the screen containing the

mysids in fresh water to rinse away the salt. Carefully

transfer the mysids from the screen to a prenumbered,

preweighed micro-weigh boat using fine-tipped forceps. Dry

for 24 hours at 60°C. Weigh each weigh boat on a .

microbalance (accurate to 1 ~g). Record the chamber number,

mysid weight, weigh boat weight (recorded previously), and

number of mysids per weigh boat (replicate) on the data

sheet.

I. Analyze the data.

J. Include standard reference toxicant point estimate values in

the standard quality control charts.'

206
Data Sheet for Juvenile Holmesimysis'Toxicity test

Test Start Date:

Test End Date:

Reference Toxicant:

Sample Source:

Start Time:

End Tinie:

Mysid Source '

Collection!Arrival Date:

Mysid Age at Start:

'lest Number Alive 'I,Otal TOtal

Cont. Toxic Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 N\imber Number Notes and

# Cone. ).Jive at Start Initials

..

IS

9 ,

10

11

1:<

l~

1~

l'

21)

2 ' . 22 - 23

2<

2: ......... 21

2' - 2:

34

35

computer Vata storage

Disk: i

1

File: ,

: - Note: See juvenile growth data on separate sheet. ,

, . -

207
Data Sheet for Weighing Juvenile M;ysids

Test Start Date:

Start Time:

Mysid Source :

Test End Date:

End Time:

Collection!Arrival Date:

Reference Toxicant:

Mysid Age at Start:

Sample Source:

Sample Type:

Test :site \,;ooe Foil Foil Total Mysid Wt Number of WeIght per

Conllliner or Number Weight Weight (Total - Foil) Mysids Mysid

Number Concentration (I'g) (I'g) (mg) (I'g)

1

1

1

1

1

1,

11

I'

I,

14

15

Computer Data Storage

Disk:

File:

Note: See mysid mortality data on separate sheet.

208
Appendix I

13.1

13.2

13.3

13.4

13.5

13.6

13.7

13.8'

13.9

13.10

13.11

13.12

13.13

13.14

SECTION 13·

PACIFIC OYSTER, Crassostrea gigas

AND MUSSEL, Myti2us sp.

EMBRYO-LARVAL DEVELOPMENT TEST ME~HOD

Adapted from a method developed by

Gary A. Chapman, u.s. EPA, ORO' Newport, OR

and Debra L. Denton, U.S. EPA, Regi6n IX

TABLE OF CONTENTS

Scope and Applicatiori

Summary of Method

Interferences

Safety

Apparatus and Equipment

Reagents and Supplies

Effluents and Receiving ,Water CollectHm,

Preservation, and Storage

Callbration and Standardization

Quality Control

Test Procedure,s

Summary of Test Conditions and Test

Acceptability Criteria

Acceptability of Test Results

Data Analysis

Precision and Accuracy

Step-by Step Summary

209
SECTION 13

PACIFIC OYSTER, CRASSOSTREA GIG4S, AND ,MUSSEL, MYTILUS SPP.

EMBRYO-LARVAL DEVELOPMENT TEST

13.1 SCOPE AND APPLICATION

13.1.1 This method estimates the chronic toxicity of effluents

and receiving waters to the embryos and larvae of several bivalve

molluscs, the Pacific oyster (Crassostrea gigas) and the mussels

(Mytilus edulis, M. californianus, M. galloprovincialis, or M.

trossulus) in a 48-h static non-renewal 'exposure. The effects

include the synergistic, antagonistic, and additive effects of

all the chemical, physical, and biological components which

adversely affect the physiological and biochemical functions of

the test organisms.

13.1.2 Detection limits of the ,toxicity-of an effluent or

chemical substance are organism dependent.

13.1.3 Brief excursions in toxicity may not be detected using

24-h composite samples. Also, because of the long sample

collection period involved in composite sampling, and because the

test chambers are not sealed, highly volatile and highly

degradable toxicants in the source may nqt be detected in the

test.

13.1.4 This test is commonly used in one of two forms: (1) a

definitive test, consisting of a minimum of five effluent

concentrations and a control, and (2) a receiving water test(s),

consisting of one or more receiving water concentrations and a

control.

13.1.5 This method should be restricted to use by, or under the

supervision of, professionals experienced in aquatic toxicity

testing. Specific experience with any toxicity test is usually

needed before acceptable results become routine.

13.2 SUMMARY OF METHOD

13.2.1 The method provides step-py-step instructions for

performing a 48-h static non-renewal toxicity test using embryos

and larvae of the test species to determine the toxicity of

210
substances in marine and estuarine waters. The test endpoint is

normal shell development and should include mor1:ality as a

measure of adverse effect.

13.3 INTERFERENCES

13.3.1 Toxic substances may be introduced by contaminants in

dilution water, glassware, sample hardware, and testing equipment

(see Section 5, Facilities and Equipment, and S'llpplies) ..

13.3.2 Improper effluent sampling and handling may adversely

affect test results (see Section 8, Effluent and Receiving Water

Sampling, and Sample Handling, and Sample Preparation for

Toxicity Tests) .

13.4 SAFETY

13.4.1 See Section 3, Health and Safety

13.5 APPARATUS AND EQUIPMENT

13.5.1 Tanks, trays, or aquaria -- for holding and acolimating

adult pacific oysters and mussels, e.g., standard salt water

aquCl:rium or Instant Ocean Aquarium (capable of maintaining

seawater at 10-20°C), with appropriate filtration and aeration

system.

13.5.2 Air pump, air lines, and air stones -- for aerating water

containing broodstock or for supplying air to test'sol~tions with

low dissolved oxygen.

13.5.3 Constant temperature chambers or water baths -- for

maintaining test solution temperature and keeping dilution water

supply, gametes, and embryo stock suspensions at test temperature

prior to the test.

13.5.4 Water purification system -- Millipore Super-Q, Deionized

water (DI) or equivalent.

13.5.5 Refractometer

13.5.6 Hydrometer(s)

for determining salinitv.

for calibrating refractometer.

211
13.5.7 Thermometers, glass or electronic, laboratory grade

for measuring water temperatures.

13.5.8 Thermometer, National Bureau of Standards Certified (see

USEPA METHOD 170.1, USEPA, .1979) -- to calibrate laboratory

thermometers.

13.5.9 pH and DO meters -- for routine physical and chemical . measurements.

13.5.10 Standard or micro-Winkler apparatus -- for determining

DO (optional) and calibrating the DO meter.

13.5.11 Winkler bottles -- for dissolved oxygen determinations.

13.5.12 Balance -- Analytical, capable of accurately weighing to

0.0001 g.

13.5.13 Fume hood -- to protect the analyst from effluent or

formaldehyde fumes.

13.5.14 Glass stirring rods for mixing test solutions.

13.5.15 Graduated cylinders Class A, borosilicate glass or

non-toxic plastic labware, 50-1000 mL for making test solutions.

(Note: not to be used interchangeably for gametes or embryos and

test solutions).

13.5.16 Volumetric flasks -- Class A, borosilicate glass or nontoxic plastic labware, 100-1000 mL for making test solutions.

13.5.17 Pipets, automatic -- adjustable, to cover a range of

delivery volumes from 0.010 to 1.000 mL.

13.5.18 Pipet bulbs and fillers -- PROPIPET@ or equivalent.

13.5.19 Wash bottles -- for reagent water, for topping off

graduated cylinders, for rinsing small glassware and instrument

electrodes and probes.

13.5.20 Wash bottles -- for dilution water.

212
13.5.21 20-liter cubitainers or polycarbonate water cooler jugs

-- for making hypersaline brine.

13~5.22 Cubitainers, beakE:rs, or similar chambers of non-toxic

composition for holding, ~ixing, and dispensing dilution water

and other general non-effluent, non-toxicant contact uses .. These

should be clearly labeled and not used for other purposes.

13.5.23 Beakers, 50 mL -- for pooling sur~ogate water samples

for chemist~y measurements at the end of the test.

13.5.24 Beakers, 250 mL borosilicate glass -- for preparation of

test solutions.

13.5.25 Beakers, 1,000 mL borosilicate glass -- for mixing

gametes for fertilization of eggs.

13.5.26 Inverted or compound microscope -- for inspecting

gametes and making counts of embryos and larvae. The use of an

inverted scope is not required, but recommended., Its use reduces

the exposure of workers to hazardous fumes (forrr~lin or

glutaraldehyde) duringtl1e counting of larvae and reduces sample

examination time. Alternatiyely, a Sedgewick-Ra:fter cell may be

used on a regular compound scope.

13.5.27 Counter, two unit, 0-999-- for recording counts of

embryos and larvae.

13.5.28 A perforated plunger -- for maintaining a homogeneous

suspension of embryos.

13.5.29 Nytex screens, ca. 75 ~m and ca. 37 ~m, for rinsing

gametes to separate individual gametes from larger material; for

retaining eggs, embryos, or larvae.

13.5.30 60 ~m NITEX® filter -- for filtering receiving water.

13.6 REAGENT~ AND SUPPLIES

13.6.1 Sample containers -- for sa~ple shipment and storage (see

Section 8, Effluent and Receiving Water Sampling, and Sample

Handling, and Sample Preparation for Toxicity Tests) .

213
13.6.2 Data sheets (one set per test)

Figure 1) .

for data recording (see

13.6.3 Tape, colored -- for labelling test chambers and

containers.

13.6.4 Markers, water-proof -- for marking containers, etc.

13.6.5 Parafilm to cover graduated cylinders and vessels

containing gametes, embryos.

13.6.6 Gloves, disposable -- for personal protection from

contamination.

13.6.7 Pipets, serological -- 1-10 mL, graduated.

13.6.8 Pipet tips

13.6.9 Covers1 ips

for automatic pipets.

for microscope slides.

13.6.10 Lens paper -- for cleaning microscope optics.

13.6.11 Laboratory tissue wipes for cleaning and drying

electrodes, microscope slides, etc.

13.6.12 Disposable countertop covering -- for protection of work

surfaces and minimizing spills and contamination.

13.6.13 pH buffers 4, 7, and 10 (or as per instructions of

instrument manufacturer) -- for standards and calibration check

(see USEPA Method 150.1, USEPA, 1979).

13.6.14 Membranes and filling solutions -- for dissolved oxygen

probe (see USEPA Method 360.1, USEPA, .1979) f or reagents for

modified Winkler analysis.

13.6.15 Laboratory quality assurance samples and standards --

for the above methods.

13.6.16 Test chambers -- 30 mL, four chambers per concentration.

The chambers should be borosilicate glass or nontoxic disposable

214
plastic labware. The test may be performed in other sized

chambers as long as the density of embryos is the same.

13.6.17 Formaldehyde, 37% (Concentrated Formalin) -- for

preserving larvae. 'Note: formaldehyde has been identified as a

carcinogen and is irritating to skin and mucous membranes. It

should not be used at a concentration higher than necessary to

achieve morphological preservation of la:r::vae for counting and

only under conditions of maximal ventilation and minimal

opportunity for volatilization into room air.

13.6.19 Reference toxicant solutions (see Section 13.10.2.4 and

Section 4, Quality Assurance) .

13.6.20 Reagent water -- defined as distilled or deionized water

that does not contain' substances which are toxic to the test

organisms (see Section 5, Facilitiesi Equipment., and Supplies and

Section 7, Dilution Water) .

13.6.21 Effluent and receiving water -- see Section 8, Effluent

'and Surface Water Sampling, 'and Sample Handling, and Sample

Preparation for Toxicity Tests.

13.6.22 Dilution water. and. hypersaline brine. -- see' Section 7,

Dilution Water and Section 13.6.24, Hypersaline Brines., The

dilution water should be uncontaminated l-~m-filtered natural

seawater. Hypersaline brine should be prepared from dilution

water.

13.6.23 HYPERSALINE BRINES

13.6.23.1 Most industrial and. sewage treatmen~ effluents

entering marine and estuarine systems have little measurable

salinity. Exposure of larvae to these effluent$ will usually

require increasing the salinity of the test ,solutions. It is

important to maintain an essentially constant salinity across all

treatments. In some applications it may ,be desirable to match

the test salinity with that of the receiving water (See Section

7.1). Two salt sources are available to adjust, salinities --

artificial sea salts and hypersaline brine (HSB) derived from

natural seawater. Use of artificial sea salts is necessary only

when high effluent concentrations preclude salirity adjustment by

, HSB alone.

215
13.6.23.2 Hypersaline brine (HSB) can be made by concentrating

natural seawater by freezing or· evaporation. HSB should be made

from high quality, filtered seawater, and can be added to the

effluent or to reagent water to increase salinity. HSB has

several desirable characteristics for use in effluent toxicity

testing. Brine derived from natural seawater contains the

necessary trace metals, biogenic colloids, and some of the

microbial components necessary for adequate growth, survival,

and/or reproduction of marine and estuarine organisms, and it can

be stored for prolonged periods without any apparent degradation.

However, even if the maximum salinity HSB (100~) is used as a

diluent, the maximum concentration of effluent (0%0) ·that can be

tested is 66% effluent at 34%0 salinity (see Table 1) .

13.6.23.3 High quality (and preferably high salinity) seawater

should be filtered to at least 10 ~m befqre placing into the

freezer or the brine generator. Water should be collected on an

incoming tide to minimize the possibility of contamination.

13.6.23.4 Freeze Preparation of Brine

13.6.23.4.1 A convenient container for making HSB by freezing is

one that has a bottom drain. One liter of brine can be made from

four liters of seawater. Brine may be collected by partially

freezing seawater at -10 to -20°C until the remaining liquid has

reached the target salinity. Freeze for approximately .six hours,

then separate the ice (composed mainly of fresh water) from the

remaining liquid (which has now become hypersaline) .

13.6.23.4.2 It is preferable to monitor the water until the

target salinity is achieved rather than allowing total freezing

followed by partial thawing. Brine salinity should never exceed

100%0. It is advisable not to exceed about 70%0 brine s~linity

unless it is necessary to test effluent concentrations greater

than 50%.

13.6.23.4.3 After the required salinity is attained, the HSB

should be filtered through a 1 ~m filter-and poured directly into

portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and the date the brine

was generated. Containers of HSB should be stored in the dark at

216
I-'

-..,J

TABLE 1. MAXIMUM EFFLUENT CONCENTRATION (%) THAT CAN BE TESTED BY ADDING DILUTION WATER

ONLY OR BRINE ONLY (WITHOUT ADDITION OF DRY SEA SALTS), GIVEN VARIOUS EFFLUENT

SALINITIES, DILUTION WATER SALINITIES~ AND BRINE SALINITIES, AND MAINTAINING

30~ TEST SALINITY.

Effl. Dilution Water Salinity %0 Brine Salinity %0

31 32 33 34 35 60 70 80 90 100 00

0 3.23 6.25 9.09 11.76 14.29 50.00 57.14 62.50 66.67 70.00

1 3.33 6.45 9.38 12.12 14.71 50.85 57.97 63.29 67.42 70.71

2 3.45 6.67 9.68 12.50 15.15 51.72 58.82 64.10 68.18 71.43

3 3.57 6.90 10.00 12.90 15.63 52.63 59.70 64.94 68.97 72.16

4 3.70 7.14 10.34 13.33 16.13 53.57 60.61 65.79 69.77 72.92

5 3.85 7.41 10.71 13.79 16.67 54.55 61.54 66.67 70.59 73.68

10 4.76 9.09 13.04 16.67 20.00 60.00 66.67 71.43 75.00 77.78

15 6.25 11.76 16.67 21. 05- 25.00 66.67 72.73 76.92 80.00 82.35

20 - 9.09 16.67 23.08 28.57 33.33 75.00 80.00 83.33 85.71 - 87.50

25 16.67 28.57 37.50 44.44 50.00 85.71 88.89 90.91 92.31 93.33
4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after several months in storage.

13.6.23.5 Heat Preparation of Brine

13.6.23.5.1 The ideal container f~r making brine using heatassisted evaporation of 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 intb the

seawater, ensure that the heater materials do not corrode or

leach any substances that would contaminate the brine. One

successful method is to use a thermostatically controlled heat

exchanger made from fiberglass. If aeration is needed, use only

oil-free air compressors to prevent contamination.

13.6.23.5.2 Before adding seawater to the brine generator,

thoroughly clean the generator, aeration supply tube, heater, and

any other materials that will be in direct contact with the

brine. A good quality biodegradable detergent should be used,

followed by several (at least three) thorough reagent water

rinses.

13.6.23.5.3 Seawater should be filtered to at least 10 ~m before

being put into the brine generator. 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~ an that the temperature does not exceed 40°C.

Additional seawater may be added to -the brine to obtain the

volume of brine required.

13.6.23.5.4 After the required salinity is attained, the HSB

should be filtered through a 1 ~m filter and poured directly into

portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitqble). The brine storage containers should

be capped and labelled with the salinity'and the date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after several months in storage.

218
13.6.23.6 Artificial Sea Salts

13.6.23.6.1 No. data from mussel or oyster tests using sea salts

or artificial seawater (e.g., GP2) are'available for evaluation

at this time,.and their use must be considered. provisional.

13.6.'23.7 Dilution Water Preparation from Brine'

13.6.23.7.1 Although salinity adjustment with brine is the

preferred method, the use of high salinity brines and/or reagent

water has sometimes been associated' with discernible,adverse

effects on test organisms. For this reason, it:i.s recommended

that only the minimum necessary volume of brine and reagent water

be used to offset the low salinity of the effluent, and that

brine controls be included in the test. The remaining dilution

water should be natural seawater. Salinity may Jpe adjusted in

one of two ways. First, the salinity of the highest effluent

test concentration may be adjusted to an acceptable salinity, and

then serially diluted. Alternatively, each effluent

concentration can be prepared individually with appropriate

volumes of effluent and brine.

13.6.23.7.2 When HSB and reagent water are used, thoroughly

mix together the reagent water and HSB before mixing in the

effluent. Divide the salinity of the HSB by the expected test

salinity to determine the proportion of reagent ,~ater to brine.

For example, if the salinity of the brine is 100~io and the test

is to be conducted at 30~, 100~ divided by 30~ ='3.33. The

proportion of brine is 1 part in 3.33 (one part brine t'o 2.33

parts reagent water). To make 1 L of dilution water at 30~

salinity from a HSB of 100~, 300 mL of brine and 700 mL of

reagent water are required. Verify the salinity of the resulting

mixture using a refractometer.

13.6.23.8 Test Solution Salinity Adjustment

13.6.23.8.1 Table 2 illustrates the preparation;of test

solutions (up to 50% effluent) at 34~ by combining effluent,

HSB t and dilution water. Note: if .the highest ej:fluent

concentration does not exceed 50% effluent, it is convenient to

prepare brine so that the sum of the effluent salinity and brine

salinity equals 68~i the required brine volume is then always

219
equal to the effluent volume needed for each effluent

concentration as in the example in Table 2.

13.6.23.8.2 Check the pH of all test solutions and adjust to

within 0.2 units of dilution water pH by·adding, dropwise, dilute

hydrochloric acid or sodium hydroxide (see Section 8.8.9,

Effluent and Receiving Water Sampling, Sample Handling, and

Sample Preparation for Toxicity Tests) .

13.6.23.8.3 To calculate the amount of brine to add to each

effluent dilution, determine the following quantities: salinity

of the brine (SB, in %0) ,.. the salinity of .the effluent (SE, in

%0), and volume of the effluent to be added (VE, in mL). Then

use the following formula to calculate the volume of brine (VB,

in mL) to be .added:

VB = VE x (30 - SE)/(SB - 30)

13.6.23.8.4 This calculation assumes that dilution water

salinity is 30 ± 2%0 ..

13.6.23.9 Preparing Test Solutions

13.6.23.9.1 Ten mL of test solution are ,needed for each test

container. To prepare test solutions at low effluent

concentrations «6%), effluents may be added directly to dilution

water. For example, to prepare 1% effluent, add 1.0 mL of

effluent to a 100-mL volumetric flask using a volumetric pipet or

calibrated automatic pipet. Fill the volumetric flask .to the

100-mL mark with dilution water, stopper it, and shake to mix.

Pour into a (150-250 mL) beaker and stir. Distribute equal

volumes into the replicate test chambers. The remaining test

solution can be used for chemistry.

13.6.23.9.2 To prepare a test solution at higher effluent

concentrations, hypersaline brine must usually be used. For

example, to prepare 40% effluent, add 400 mL of effluent to a 1-

liter volumetric flask. Then, assuming an effluent salinity of

2%0 and a brine salinity of 66%0, add 400 mL of brine (see

equation above and Table 2) and top off the flask with dilution

water. Stopper the flask and shake well. Pour into a (1.00-250 . /

220
TABLE 2. EXAMPLES OF EFFLUENT DILUTION SHOWING 'VOLUMES OF

EFFLUENT (ATX~), BRINE, AND DILUTION 'WATER NEEDED FOR

ONE LITER OF EACH TEST SOLUTION.

FIRST STEP: Combine brine with reagent water or natural seawater

to achieve a brine of 68-x%o and, unless natural'seawater is used

for dilution water, also a brine-based dilution 'water of 34%0.

SERIAL DILUTION:

Step 1. Prepare the highest effluent concentration to be tested

by adding equal volumes of effluent and brine to the appropriate

volume of dilution water. An example using 40% is shown.

Effluent Cone. Effluent Brine Dilution

(%) x~o (68 -x) %'0 Water* 34%'0

40 800 mL 800 mL " 400 mL

Step 2. Make serial dilutions from the highest test

concentration.

Effluent Cone. (%) Effluent Source Dilution Water*

(34%'0)

20 1000 mL of 40% 1000 mL·

10 1000 mL of 20% 1000 mL

5 1000 mL of 10% 1000 mL

2.5 1000 mL of 5% 1000 mJ.J

Control none 1000 mL

.

Effluent Cone. Effluent x~o Brine (68-x)%'0 Dilution

(%) Water* 34%0

40 400 mL 400 mL 20b mL

20 200·mL 200 mL 600 mL

10 100 mL 100 mL 800 mL

5 50 mL 50 mL 900 mL

2.5 25 mL 25 mL 950 mL

Control none none 1000 mL

INDIVIDUAL PREPARATION·

*May be natural seawater or brine-reagent water equivalent.

221
roL) beaker and stir. Distribute equal volumes into the replicate

test chambers. The remaining test solution can be used for

chemistry.

~3.6.23.~0 Brine Controls

~3.6.23.~0.1 Use brine controls in all tests where brine is

used. Brine controls contain the same volume of brine as does

the highest effluent concentration using brine, plus the volume

of reagent water needed to reproduce the hyposalinity of the

effluent in the highest concentration, plus dilution water.

Calculate the amount of reagent water to add to brine controls by

rearranging the above equation, (See, 13.6.23.8.3) setting SE = 0, and solving for VE.

VE = VB x (SB - 30)/(30 - SE)

If effluent salinity is essentially O~, the reagent water volume

needed in the brine control will equal the effluent volume at the

highest test concentration. However, as effluent salinity and

effluent concentration increase, less reagent water volume is

needed.

13.6.24 TEST ORGANISMS, OYSTERS AND MUSSELS

~3.6.24.1 The test organisms for this test'are the Pacific,

oyster, Crassostrea gigas, or mussels, Mytilus spp. (at least

twelve per test). Pacific oysters are native to Japan, but have

been cultured commercially on the west coast of the United States

for over a century.

~3.6.24.2 Species Identification

~3.6.24.2.1 The three species of mussels included in this method

are presumably native to the west coast. The California mussel

(Mytilus californianus) is distributed a~ong the exposed rqcky

coast from Alaska to Baja California and is found from intertidal

areas to 150 feet depth. The other two mussels included in this

method (M. trossulus and M. galloprovinciallis) are common in

sheltered waters such as bays and estuaries and were previously

considered to be west coast populations of Mytilus edulis. The

two species are both present in central California, with M.

222
galloprovincialis reported from San.Francisco Bay to Baja

California, and M.. trossulus reported from Monterey to Alaska.

13.6.24.2.2 Test organisms should be identified to species using

morphological ·features in recognized keys. Separation of the "M.

edulis" complex, (M. trossulus, and M. galldprovinciallis) may

not be possible without electrophoretic characterization. The

geographic source of the Mytilus spp. broodstock must be

reported.

13.6.24.3 Obtaining Broodstock

13.6.24.3.1 Adult oysters (Crassostrea gigas) and mussels

(Mytilus spp.) can be. obtained from commercial I:mppliers and the

mussels can also be collected from the field. Organisms are best

shipped in c;iamp towels or seaweed .and kept cool (4-12°C). Note:

if practical, check the sex ratio of brood stock or request such

information from a commercial supplier. A highly skewed sex

ratio could result in poor embryo yield.

13.6.24.4. Broodstock Culture and Handling

13.6.24.4.1 The adult bivalves are maintained in glass aquaria

or fiberglass troughs or tanks. These are supplied continuously

(approximately 5 L/min) with ~atural seawater, or salt water

prepared from·commercial sea salts is recirculated. The animals

are checked daily and any obviously unhealthy an~mals are

discarded. Prior to. spawning, the animals should be brushed or

gently scraped to remove barnacles and other encrusting

organisms; this alleviates problems of egg and sperm

contamination, especially through potential barnacle spawning.

13.6.24.4.2 Although ambient temperature seawater is usually

acceptable for holding, recommended temperatures are 14-15°C for

oyster and 8°C for mussels; conditioning bivalves to spawning

condition usually requires holding for from 1-8 weeks at a higher

temperature (20°C for oysters, 15-18°C for mussels) .

,

13.6.24.4.3 Natural seawater (~30~) is used to maintain the

adult animals and as·a control water. in the tests.

13.6.24.4.4 Adult animals used in field studies are transported

in insulated boxes or coolers packed with wet kelp or paper

223
toweling. Upon arrival at the field sit~, aquaria are filled

with control water~ loosely covered with a styrofoam sheet and

allowed to equilibrate to the holding temperature before animals

are added.

13.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND

STORAGE

13.7.1 See Section 8, Effluent and Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity Tests.

13.8 CALIBRATION AND STANDARDIZATION

13.8.1 See Section 4, Quality Assurance.

13.9 QUALITY CONTROL

13.9.1 See Section 4, Quality Assurance.

13.10 TEST PROCEDURES

13.10.1 TEST DESIGN

13.10.1.1 The test co~sists of at l~ast four replicates of five

effluent concentrations plus a dilution water control. Tests

that use brine to adjust salinity must also contain four

replicates of a brine control. In addition, at least six extra

count controls are prepared in dilution water and the number of

embryos in each are counted at the time of test initiation ..

These counts provi~e an average initial embryo ~ensity that is.

used in the calculation of test results (see 13.13.1.3). Extra

replicates are recommended for water chemistry during the tests

(see Section 13.8 and Table 3).

23.10.1.2 Effluent concentrations are expressed as percent

effluent.

13.10.2 TEST SOLUTIONS

13.10.2.1 Receiving waters

13.10.2.1.1

of the test.

The sampling point is determined by the objectives

At estuarine and marine sites, samples are usually

224
collected at mid-depth .. Receiving, water toxicity is determined

with samples used directly as collected or with samples passed

through a 60 lim NITEX® filter and compared ,without dilution,

against a control. Using four replicate chambeJ~s per test, each

containing 10 mL, and 400 mL for chemical ancHysis, would require

approximately 440 mL of sample per test.

13.10.2.2 Effluents

14.10.2.2.1 The selection of the effluent test lconcentrations

~hould be based on the objectives of the study. I A dilution

factor of at least 0.5 is commonly used. A dilution factor of

0.5 provides hypothesis test discrimination of ± 100%, and,

testing of a 16 fold range of concentrations. Hypothesis t~st

discrimination shows little improvement as dilut,ion factors are

incre~sed beyond 0.5 and declines rapidly if sma1ler diluti.on

factors are used~ USEPA recommends that 'one of ,the five effluent

treatments must be a concentration of effluent mixed with

dilution water which corresponds to the permitte~ls in~tream

waste concentration (IWC). At least two of the effluent

treatments must be of lesser effluent concentration than the lwe,

with one being at least one-half the concentration of the IWC.

If 100~ HSB is used as a diluent, the maximum concentration of

effluent that can be tested will be 70% at 30~ salinity.

13.10.2.2.2 If the effluent is known or suspect~d t9 be highly

,toxic, a lower range of effluent concentrations should be used

(such as 25%, 12.5%, 6.25%, 3.12% and 1.56%).

13.10.2.2.3 The volume in each test chamber ,is 10 mL.

13.10.2.2.4 Effluent dilutions should beprepar~d for all

replicates in, each treatment in one container to minimize

variability among the replicates. Dispense into the appropri.ate

effluent test chambers.

13.10.2.3 Dilution Water

13.10.2.3.1 Dilution water should be uncontaminated 1-J1.mfiltered natural seawater'or hypersaline brine (prepared from

uncontaminated natural seawater) plus reagent wat,er (see Section

225
7, Dilution Water). Natural' seawater may be uncontaminated

receiving water. This water is used in all dilution steps and as

the control water.

~3.10.2.4 Reference Toxicant Test

13.10.2.4.1 Reference toxicant tests should be conducted as

described in Quality Assurance (see Section 4.7).

13.10.2.4.2 The preferred reference toxicant for oysters and

mussels is copper chloride (CuCI 2 0H20). Reference toxicant tests

provide an indication of the sensitivity of the test organisms

and the suitability of the testing laboratory (see Section.4

Quality Assurance). Another toxicant may be specified by the

appropriate regulatory agency. Prepare a copper reference

toxicant stock solution (2,000 mg/L) by adding 5.366 g of copper

chloride (CuCI 202H20) to 1 liter of reagent water. For each

reference toxicant test prepare a copper sub-stock of 3mg/L by

diluting 1.5 mL of stock to one liter with reagent water.

Alternatively, certified standard solutions can be ordered from

commercial companies.

13.10.2.4.3 Prepare a control (0 ~g/L) plus four replicates each

of at least five consecutive copper reference toxicant solutions

(e.g., from the series 3.0, 4.4, 6~5, 9.~, 13.9, 20.4, and 30.0

~g/L, by adding 0.10, 0.15, 0.22, 0.32, 0.46, 0.68, and 1.00 mL

of sub-stock solution, respectively, to 100-mL'volumetric flasks

and filling to 100-mL with dilution water). Start with control

solutions and progress to the highest concentration to minimize

contamination.

13.10.2.4.4 If the effluent and reference toxicant tests are to

be run concurrently, then the tests must use embryos from the

same spawn. The tests must be handled in the same way .and test

solutions delivered to the test chambers at the same time.

Reference toxicant tests must be conducted at 30 ± 2~.

13.10.3 COLLECTION OF GAMETES FOR THE TEST

13.10.3.1 Spawning Induction

13.10.3.1.1 Select at least a dozen bivalves and place them into

a container filled with seawater (ca. 20°C for oysters, 15°C for

226
mussels} and allow time for them to resume pumping (ca. 30.

minutE?s). Mussels will often start pumping following immersion

if they have been kept out of water and refrigerated overnight

prior to spawning.

,

TABLE 3. EXAMPLE OF TYPICAL TEST ARRAY SHOWING NUMBER AND TYPES

OF TREATMENT CHAMBERS REQUIRED.

TREATMENT Test Vials Chemistry

Vials -

Count Control 6 0

Brine Control 4 1~3

Dilution Water Control 4 1-3

Effluent cone. 1 4" , . 1-3 - Effluent cone. 2 4- 1-3

Effluent cone. 3 4 1-3

Effluent cone. 4 4 1-3

Effluent cone. S 4· 1-3

TOTAL Chambers = 41-55 34 7-21

13.10.3.1.2 , Over a 15-20 minute period, increase the temperature

(do not exceed 32°C for oysters, or 20°C for muss'els), yheeking,

for spawning.

13.10.3.1.3 If no spawning occurs after 30 minut;es, replace the

water with some at the original temperature and after 15 minutes

again increase the temperature as in 13.10.3.2. Although ASTM

(1993) cautions against it, the addition of algae into the water

can often stimulate spawning of bivalves; if thiS! method is used,

the organisms should be moved to clean water one€~ spawning

begins. Mussels can also be induced to spawn by injection,of 0.5

M KCl into the posterior adductor muscle. Oyste:r:s. can be induced

to spawn by the addition of heat-killed sperm about one hour

after initial temperature increase.

13.10.3.2 Pooling Gametes

227
13.10.3.2.1 When individuals are observed to be shedding

gametes, remove each spawner from the tank and place each in a

separate container (20°C water for oysters, 15°C for mussels) .

Alternatively, bivalves can be placed into individual~"Chambers

initially (at temperatures per 13.10.5.2) and these placed into a

water bath that provides the desired maximum temperature.

13.10.3.2.2 Early in the spawning process, examine a small

sample of the gametes from each spawner to confirm sex and to see

if the gametes are of adequate quality.

13.10.3.2.3 Place a small amount of sperm from each male onto a

microscope slide (well slides work nicely). Examine the sperm

for motility; use sperm from those males wi·th the better sperm

motility.

13.10.3.2.4 A small sample of the eggs from each female should

be examined for the presence of significant quantities of poor

eggs (vacuolated, small, or abnormally shaped). If good quality

eggs are available from one or more females, questionable batches

of eggs should not be used for the test. It is more important to

use high quality eggs than it is to use a pooled population of

eggs.

13.10.3.2.5 Sperm and egg suspensions that are to be used for

preparing the embryo stock should be pas~ed through Nytex screen

(ca. 75 ~m) to separate out clumps of gametes or extraneous

material.

13.10.3.2.6 The pooled eggs are placed into alL beaker and

sufficient dilution water added to achieve an egg density of

about 5,000-8,000 eggs/mL. (objects are just discernible when

viewed through the egg suspension) in about 800-900 mL water

volume.

13.10.3.3 Fertilization

13.10.3.3.1 Sperm density may vary from one spawning to the

next. It is important to use enough sperm to achieve a high

percent egg fertilization, but too many sperm can cause

polyspermy with resultant abnormal development. To achieve an

acceptable level of sperm, several egg suspensions of equal

density should be fertilized using a range of sperm volumes,

228
e.g., 100 mLof egg suspension plus 1, 3, and 10 mL of sperm

suspe~sion; This test 'fertilization should be aC90mplished

within 1 hour of spawning. Use the eggs with the lowest amount

of sperm giving normal embryo development after 1.5-2.5 hours

after fertilization, as determined by miqroscopic examination.

Usually >90% of the eggs should be fertilized; oysters should

have changed from the tear-drop shaped egg to a round single cell

zygote; mussels should show a single polar body;, or embryos of

either species should have advanced to the two-cell stage.

13.10.4 START OF THE TEST

13.10.4.1 Prior to Beginning the Test

13.10.4.1.1 The test should begin as soon as possible,.

preferably within 24 h of sample collection. The maximum holding

time following retrieval of the sample from the 13ampling device'

should not exceed 36 h for off-site toxicity tests unless

permi,ssion is granted by the permitting authority. In no case

should the sample be used in a test. more ,than 72,h after sample

collection (see Section 8 Effluent and Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity Test) .

13.10.4.1.2 Just prior to test initiation (approximately 1 h),

the temperature of a sufficient quantity of the sample to make

the test s~lutions should be adj'usted to the test: temperature (18

or 20 ± 1°C) and maintained at that temperature during the

addition of dilution water.

13.10.4.1.3 Increase the temperature of the water bath, room, or

incubator to the required test temperature (18 ,or 20 ± 1. °C) .

13.10.4.1.4 Randomize the placement of test champers in the

temperature-controlled water bath, room, or incubator at the

beginning.of the test, using a position chart. A~sign numbers

for the position of each test chamber using a random numbers or

similar process (see Appendix A, for an example of

randomization). Maintain the chambers in this cohfiguration

throughout the test, using a position chart. Recbrd these

numbers on Cl. separate data sheet together with the concentration

and replicate numbers to which they correspond. Identify this

sheet with the date, test organism, test 'number, ,;Laboratory, and

229
investigator's name, and safely store it away until aft.er the

oysters or mussels have been examined at the end of the test.

13.10.4.1.5 Note: Loss of the randomization sheet would

invalidate the test by making it impossible to analyze the data

afterwards. Make a copy of the randomization sheet and store

separately. Take care to follow. the numbering system exactly

while filling chambers with 'the test solutions.

13.10.4.1.6 Arrange the test chambers randomly in the water bath

or controlled temperature room. Once chambers have been labeled

randomly, they can be arranged in numerical order for

convenience, since this will also ensure random placement of

treatments.

13.10.4.2 Estimation of Embryo Density

13.10.4.2.1 Adjust the embryo suspension to a density of 1,500-

3,000/mL. Confirm by counting chamb~r counts on 1 mL subsamples

from a stirred suspension of embryos. F~nal larval density ~f

15/mL will provide reasonable precision (150 larvae) and be

easier to count than 300 larvae. Add 0.1 mL of the embryo

suspension to 10 mL of test solution into each of the rangomized

test vials. It is extremely important (for a consistent embryo

density among test chambers) to maintain a homogeneous

distribution of embryos in the stock suspension by regular, slow

oscillation of a perforated plunger during embryo distribution.

13.10.4.3 Initial Density Counts

13.10.4.3.1 If tests are conducted on small volumes, using an

inverted microscope, the total number of embryos injected into

the count controls should be determined as soon as the test has

been started. If larger test volumes are used, with counts based

upon subsamples, the embros should be resuspended in the water.

using a perforated plunger. Then subsamples are taken (e.g., 5-

10 mL) and the total number of embryos counted in the subsample.

Two methods for these counts are to use a counting chamber of the

same volume as the subsample, or to screen ~he embryos using a 37

~m screen and backwash with a smaller volume for small counting

chambers. In either procedure, appropriate multiple rinsing'is

needed to achieve quantitative transfer of embryos.

230
13.10.4.3.2 Initial counts are required to determine survival, in

the controls and other treatments. High coefficients of

variability in initial counts make survival estimates inexact and

may actually decrease the sensitivity of the test.

13.10.4.4 Incubation

13.10.4.4.1 Cover and incubate the chambers in an environmental

chamber or by partial immersion in a temperature-(rontrolled water

bath for 48 hours.

13.10.4.4.2 At the end of the 48-hour 'incubation period, examine

a count control test chamber (or control test vial if the count

controls were transferred to a counting chamber t(i> make the

initial counts) under a microscope to check for complete

development of control organisms. If development is complete,

the test should be ended. If development 'does not: appear to be

complete, the test should be continued until complete development

occurs (but not beyond 54 hours total test duratic>n) . '

13.10.5 LIGHT, PHOTOPERIOD, SALINITY ANDTEMPERA1?URE

13.10.5.1 The light quality and intensity should:be at ambient

laboratory conditions. Light intensity should be '10-20. p,E/m2/s,

or 50 to 100 foot candles (ft-c), with a 16 h light and 8 h dark

cycle. ;,

13.10.5.2 The water temperature in the test champers should be

maintained at 18 or 20 ± l°C. If a water bath is used to

maintain the test temperature, the water depth surrounding the

test cups s,hould be as deep as possible without floating the

chambers.

13.10.5.3 The test salinity should be in the range of 30 ± 2~.

The salinity should vary by no more than ±2~ amon~ the chambers

on a given day. If effluent and receiving water tests are

conducted concurrently, the salinities of these tests should be

similar.

I

13.10.5.4 Rooms or incubators with high volume ventilation

should be used with caution because the volatilization of the

test solutions and evaporation of dilution water may cause wide

fluctuations in salinity. Covering the test chambers with clean

231
polyethylene plastic may help prevent volat'ilization and

evaporation of the test solutions.

13.10.6 DISSOLVED OXYGEN (DO) CONCENTRATION

13.10.6.1 Aeration may affect the toxicity of effluent and

should be used only as a last resort to maintain a satisfactory

DO. The DO concentration should be measured on new solutions at

the start of the test (Day 0). The DO should not fall below 4.0

mg/L (see Section 8, Effluent and Receiv.ingWater Sampling,

Sample Handling, and Sample Preparation',for' Toxicity Tests). If

it is necessary to aerate, all treatments and the control should

be aerated. The aeration rate should not exceed that necessary

to maintain a minimum acceptable DO and under no circumstances

should it exceed 100 bubbles/minute, using a pipet with a 1-2mm

orifice, such as a 1 mLKIMAX@ serological pipet No. 37033, or

equivaient.

13.10.7 OBSERVATIONS DURING THE TEST

13.10.7.1 Routine Chemical and Physical Observations

13.10.7.1.1 DO is measured at the beginning of the exposure

period in each test concentration and in the control.

13.10.7.1.2 Temperature, pH, and salinity are measured at the

beginning of the exposure period in each test concentration and

in the control. Temperature should also be monitored continuously

or observed and recorded daily for at least two locations in the

environmental control system or the samp~es. Temperature should

be measured in a sufficient number of test chambers at the end of

the test to determine temperature variation in the environmental

chamber.

13.10.7.1.3 Record all the measurements on the data sheet.

13.10.8 TERMINATION OF THE TEST

13.10.8.1 Ending the Test

13.10.8.1.1 Record the time the test is terminated.

13.10.8.1.2 The pH, dissolved oxygen, and salinity are measured

232
at the. end of the exposure period in one.test chamber at each

concentration and in the control. If small electrodes are used,

these measurements can be performed in a single extra replicate

vial set up specifically for this measurement. Measurements

should not be made in vials that are to be counted, as larvae may

adhere to 'electrodes, possibly biasing larval c!ounts.

13.10.8.2 Sample Preservation

13.10.8.2.1 To terminate the test, add 0.25 mLI of concentrated

formalin (37% formaldehyde). It is advisable not to shake the

contents at any time following test termination because the

larvae may stick to the edge of the chambers. Simply allow the

preservative to mix passively and the larvae to settle out. The

use of glutaraldehyde instead of formalin is likely to be.

acceptable, but as no record of its use with this test is known,

care should be taken to confirm· that glutaraldehyde kills,

preserves, and produces no artifacts that wouldi confound the test

results.

13.10.8.2.2 Note: Formaldehyde has been identified as a

carcinogen and both glutaraldehyde and formaldehyde are

irritating to skin and mucus membranes. .Neither should be used

at higher concentrations than needed to achieve morphological

preservation and only under conditions of maximal ventilation and

minimal opportunity for volatilization into room air.

13.10.8.3 Counting

13.10.8.3.1 After addition of preservative, obl3erve all the

larvae in each test vial. This can be done by E~xamining the

contents of each test vial with an inverted microscope at about

40X-50X magnification or by quantitative, transfer of al.l larvae

onto' a counting chamber and counting using a cornpound microscope

at about 100X. Using the mechanical stage, carefully count and

score all larvae as either normal or abnormal. If substantial

numbers of completely developed shells without nneat are observed

(i. e., > 5 percent of normal larvae), then thesE~ shells should be

enumerated separately (as dead larvae). "LarvaE~ possessing

misshapened or otherwise malformed shells are considere.d normal,

provided development has been completed" (ASTM, '1994). Record

the final counts on the data sheet.

233
13.10.8.3.2 If the number of larvae observed appears to be low

in relation to the number inoculated at the beginning of the

test, this signifies either mortality and dissolution, or

possible adherence to the walls of the vials or incomplete

transfer to the counting chamber. Inspect the vials for evidence

of the latter two occurrences.

13.10.8.4 Endpoint

13.10.8.4.1 The percentage of embryos that did not survive and

develop to live larvae with completely developed shells (i.e.,

abnormal or dead organisms) is calculated for each treatment

replicate (See 13.13.1.3). All larvae are considered live unless

they are merely empty shells "without meat" (ASTM, 1994) i embryos

and larvae that are not yet in the D-hinge stage are counted as

abnormal, even if they may have died during the. test. Embryos

and larvae that die and disintegrate during the test are

estimated from initial embryo counts (See N' in 13.13.1.3).

13.10.8.4.2 Unless used as the dilution water, natural seawater

controls are only used to check the relative performance of the

dilution water controls (e.g., brine controls) required·for

salinity adjustment. Statistical analysis should use the

appropriate dilution water control data.

13.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

13.11.1 A summary of test conditions and test acceptability

criteria is listed in Table 4.

TABLE 4. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY

CRITERIA FOR, CRASSOSTREA GIGAS and MYTILUS SPP.,

EMBRYO-LARVAL DEVELOPMENT TEST WITH EFFLUENTS AND

RECEIVING WATERS

1. Test type: Static non-renewal

2. Salinity: 30 ± 2 %-0

,

3. Temperature: 20 ± 1°C (oysters)

: 15 or 18 ± 1°C (mussels) *

I 4. Light quality: Ambient laboratory light

234
5. Light intensity: 10-20 uE/m2/s U~mbient

laborat~ry levels}

6. PhOtoperiod: 16 h light, 8 h darkness

7. Test chamber size: 30 mL

8. Test solution volume: 10 mL

9. No. larvae per chamber: 150-300 ,

10. No. replicate chambers 4 (plus 3 chemistry vials)

per concentration:

1l. Dilution water: Uncontaminated 1,-Jlm-filtered

natural seawate!: or hypersaline

brine prepared from natural

seawater

12. Test concentrations: Effluents: Minimum of 5 and a

control

Receiving waters: 100%

receiving water and a control

13. Dilution factor: Effluents: ~0.5

Receiving waters: None or ~0.5

14. Test duration: 48 hours (or until complete

developm(:nt up to 54 hours) ,

15. Endpoint: Survival and normal shell

development

16. Test acceptability Control survival must be ~70%

criteria: for oyster embryos or ~50% for

mussel embryos ih control

vials; ~90% normi3.1 shell

development in s'llrviving

controls; and must achieve a

%MSD of <25%

235
~7. Sampling requirements: One sample collected at test

initiation, and preferably used

within 24 h of the' time it is

removed from the sampling

- device (see Section, 8, Effluent

and Receiving Water Sampling,

I Sample Handling, and Sample

Preparation for Toxicity Tests)

~8. Sample volume required: 1 L per test

*Mussel embryo-larval tests were commonly conducted at 15°C

(ASTM, ~994). Experience has shown that many laboratories in

northern Californa, Oregon, and Washington often fail to achieve

adequate control development at 15°C in 48 hours. It is

acceptable to conduct the test at 15°C with the permission of the

regulatory authority. Developmental rates may be dependent upon

species, local population c~aracteristics, or other factors.

~3.~2 ACCEPTABILITY OF TEST RESULTS

~3 .12 .1 For tests to be considered acceptable, the fol.lowing

requirements must be met:

(1) The mean survival must be at least 70% for oysters or

at least 50% for mussels in ·the controls.

(2) The percent normal must be at least 90% in the

surviving controls.

(3) The minimum significant difference (%MSD) is <25%

relative to the control.

~3.~3 DATA ANALYSIS

13.13.1 GENERAL

~3.~3.1.1

proportion

sample set

Tabulate and summarize the data. Calculate the

of normally developed larvae for each replicate.

of test data is listed in Table 5.

236

A
13.13.1.2 Final calculations are based upon count~ of normal

larvae and total larvae at test termination, and mean initial

embryo count.

13.13~1.3 The percentage of embryos that did not survive or

develop to live larvae with completely developed shells (i.e.,

abnormal or dead organisms) is calculated for each treatment

replicate (including controls) using the formula:

where:

A = 100 (N' B I )

\ .

,

A = percent abnormal and dead organisms

B I = the adjusted number of normal larvae a1: the end of the

test

N' = the initial number of embryos in the tE~st chambers

. expressed as the mean of the initial counts;

and: ifN > N', where

N = the actual number of larvae at the end of th~ test

then: :8' = B (N' / N)

where: B = the actual number of normal larvae at the end of the

test but, when N N' I then: Bl = B

The means of "A" are obtained for each treatment concentration,

and the latter are corrected for control.response using Abbott's

formula, as follows:

E = 100 (A M)

100 - M

·where:

E·= the mean percent abnormal/dead corrected for controls

A = the mean percent abnormal/dead

M = the value of A for the controls.

13.13.1.4 The statistical tests described here must be used with

a knowledge of the assumptions upon which the tests are

contingent. The assistance of a statistician is recommended for

analysts who are not proficient in statistics.

237
TABLE 5. DATA FROM BIVALVE DEVELOPMENT TEST.

Copper

Concentration Initial Number Number Proportion

(/lg/L) Replicate Density Surviving Normal Normal

Control A 25 22 22 1. 00

B 25 25 24 0.96

C 25 25 25 1. 00

D 30 30 29 0.97

0.J.3 A 25 23 22 0.96

B 30 30 29 0.97

C 25 25 25 1. 00

D 25 24 '23 0.96

0.25 A 25 25 23 0.92

B 25 J.9 J.8 0.95

C 25 2J. J.9 0.90

D 25 23 22 0.96

0.50 A 25 J.J. J.O 0.9J.

B 25 J.4 J.3 0.93

C 25 J.7 J.5 0.88

D 25 J.5 J.4 0.93

1.00 A 25 8 7 0.88

B 25 6 5 0.83

C 25 8 7 0.88

D 25 J.J. 9 0.82

2.00 A 25 2 2 1. 00

~3.~3.~.5 The endpoints of toxicity tests using bivalves are

based on the reduction in proportion of normally developed

larvae. The IC25 is calculated using the Linear Interpolation

Method (see Section 9, Chronic, Toxicity Test Endpoints and Data

Analysis). LOEC and NOEC values 'for larval development are

obtained using a hypothesis testing approach such as Dunnett's

Procedure (Dunnett, 1955) or Steel's Many-one Rank Test (Steel,

~959; Miller, 1981) (see Section 9). Separate analyses are

performed for the estimation of the LOEC and NOEC endpoints and

for the estimation of the IC25. See the 'Appendices for examples

of the manual computations, and examples of data input and

program output.

~3.13.2 EXAMPLE OF ANALYSIS OF BIVALVE EMBRYO-LARVAL

DEVELOPMENT DATA

238
13.13.2.1 Formal statistical analysis of the embryo-larval

development is outlined in Figure 1. The response use~ in the

analysis is the proportion of normally developed surviving larvae

in each test or control chamber. Separate analyses are performed

for the estimation of the NOEC and LOEC endpoin~:s and for the

estimation of. the IC25 endpoint. Conc.entratiQns, at which there

is no normal development in any of the test chamlpers are excluded

from statistical analysis of the NOEC and LOEC, but included in.

the estimation of the IC25.

13.13.2.2 For the case of equal numbers of repl:~cates across all

concentrations and the control, the evaluation of the NOEC and

LOEC endpoints is made via a parametric test, Dunnett's

Procedure, or a nonparametric test, Stee~' s Many·-one Rank Test,

on the arc sine square root transformed data. Underlying

assumptions of Dunnett's Procedure, normality and homogeneity of

variance, are formally tested. The test for norrrrality ·is the

Shapiro-Wilk's Test, and Bartlett's Test is used'to test for

homogeneity of variance. If either of these tes~s fails, the

nonparametric test,..Steel's Many-one Rank Test, is, used to

determine the NOEC and LOEC endpoints.· If the assumptions of

Dunnett's Procedure are met, the endpoints are eSltimated by the

parametric procedure.

13.13.2.3 If unequal numbers of. replicates occur among the

concentration levels tested, there are parametric and

nonparametric alternative analyses. The parametric analysis is a

t test with the Bonferroni adjustment (see Appendix D). The

Wilcoxon Rank Sum Test with the Bonferroni adjustment is the

nonparametric alternative.

13.13.2.4 Example of Analysis of Embryo-Larval r:'evelop·ment Data

13.13.2.4.1 Since the response of interest is the proportion of

normally developed surviving larvae, each replicate must first be

transformed by the arc sine square root t,ransform:ation procedure

described in Appendix B. Because there are varying numbers of

survivors in .the replicates, the adjustment for response

proportions of zero or one will not be made. The. raw and

transformed data, means and variances of the transformed

observations at each effluent concentration and control are

listed in Table 5. The data are plotted in Figure 2.

239
_ NO

lTESTWITH

SONFERRONI. ' .ADJUSTMENT .

Figure ~. Flowchart for statistical analysis of the pacific

oyster, Crassostrea gigas, and mussel, Mytilus spp.,

development data.

240
13.13.2.5 Test for Normality

13.13.2.5.1 The first step of the test for normality 'is to .

center the observations by subtracting the mean fof all

observations within a concentration from each observation in that

concentration. The centered observations are summarized in

Table 6.

TABLE 6 . BIVALVE EMBRY0-LARVAL DEVELOPMENT DATA

Copper Concentration (fJ-g/L )

Replicate Control 0.13 0.25 0.50 1. 00 2.00

A 1. 00 0.96 0.92 0.91 0.88 1. 00

RAW B 0.96 0.97 ' 0.95 0.93 0.83 0.67

C 1. 00 1. 00 0.90 0.88 0.88 0.75

D 0.97 0.96 0.96 0.93 0.82 0.40

ARC SINE A 1.571 1.369 1.284 1.266 1.217 1.571

SQUARE ROOT B 1.369 1.397 1.345 1.303 1.146 0.959

TRANSFORMED C 1. 571 1.571 1.249 1.217 1.217 1.047

D 1.397 1.369 1.369 1.303 1.133 0.685

Mean ('Ii) 1.477 1.427 1. 312' 1.272 1.178 1. 066

si 0.01191 0.0094'5 0.00303 0.00l66 0.00203 0.l3733

i 1 2 3 4 5 6

!

13.13.2.5.2 Calculate the denominatorj D, of the statistic:

Where: Xi = the ith centered observation

X = the overall mean of the centered observations

n = the total number of centered observations

13.13.2.5.3 For this set of data,' n 24

241

X - ----L- (-0.002)

24

D = 0.4963

0.000
13.13.2.5.4 Order the centered observations from smallest to

largest

where Xli) denotes the ith ordered observation .. The ordered

observations for this example are listed in Table 7

TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S

EXAMPLE

i

1

2

3

4

5

6

7

8

9

10

11

12

Xli)

-0.381

-0.108

-0.107

-0.080

-0.063

-0.058

-0.058

-0.055

-0.045

-0.032

-0.030

-0.028

i

13

14

15

16

17

18

19

20

21

22

23

24

Xli)

-0.019

-0.006

0.031

0.031

0.033

0.039

0.039

0.057

0.094

0.094

0.144

0.505

13.13.2.5.5 From Table 4, Appendix B, for the number o~

observations, n, obtain the coefficients a l , a 2 , ••• ak where k is

n/2 if n is even and (n-l)/2 if n is odd. For the data in this

example, n = 24 and k = 12. The ai values are listed in Table 8.

13.13.2.5.6 Compute the test statistic, W, as follows:

k > 2

W = ~[L·a.(x(n-i+l)_x(i))]

D i-l l.

The differences, Xln- i +l ) - Xli), are listed in Table 8. For the

data in this example:

242
W = 1

0.4963

(0.6322)2 = 0.805

TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'$

EXAMPLE

i ai X (n-i+l) - Xli)

J. 0.4493 0.886 X(24) - X(l)

2 0.3098 0.252 , X(23) - X (2)

3 0.2554 0.20J. X(22) - X(3)

4 0.2J.54 0.J.74 X(21) - X(4)

5 0.J.807 0.J.20 X(20) - Xes)

6 0.J.5J.2 0.097 X(19) - X(6)

7 0.J.245 0.097 I X(lS) - X(7)

8 0.0997 0.088 X(17) - Xes)

9 0.0764 0.076 X(16) - X(9)

J.O 0.0539 0.063 X(B) - X(lO)

J.J. 0.032J. 0.024 X(14) - X(ll)

J.2 0.0J.07 0.009 X(13) - X(12)

13.13.2.5.7 The decision rule for this test is to compare W as

calculated in Subsection 5.6 to a critical valuE~ found in

Table 6, Appendix B. If the computed W is less than the critical

value, conclude that the data are not normally distributed. For

the data in this example, the critical vqlue ata significance

level of 0.01 and n = 24 observations is 0.884. Since W = 0.805

is less than the critical value, conclude that t:hedata are not

normally distributed.

13.13.2.5.8 Since the data do not meet the assumption of

normality, Steel's Many-one Rank Test will be used to analyze the

embryo-larval development data.

13.13.2.6 Steel's Many-one RankT~st

13.13.2.6.1 For each control and concentration combination,

combine the data and arrange the observations in order of size

from smallest to largest. Assign the ranks (1, 2, ... , 8) to

the ordered observations with a rank of 1 assigned to the

smallest observation, rank of 2 assigned "to the next larger

observation, etc. If ties occur when ranking, assign the average

rank to each tied observation.

243
*

*

*

0.50 1.00 2.00

COPPER CONCENTRATION (ugIL)

--sk * * * IlCMl'oIM.~TI-..DI'WCf _11&__'........._ 1.0

Cl 0.9

w

0..

0 0.8 -'

w

>

w 0.7 Cl

>- 0.8 -'

-'

<r:

::;; 0.5 0::

t\) 0

z: ,0.4 z:

0

-

f- 0.3 0::

0

0.. 0.2 0

0::

0..

0.1

0.0

0.00 0.13 0.25

•

Figure 2. Plot of mean proportion of normaly developed bivalve larvae .

•-- •
13.13.2.6.2 An example of assigning ranks to the combined data

for the control and-.'0.13 Jlg/L concentration is!:riven in Table 9.

This ranking procedure is repeated for each con'trol/concentration

combination. The complete set of rankings is summarized in

Table 10. The ranks are t-hen summed for each concentration

level, as shown in Table 11.

TABLE 9. ASSIGNING RANKS TO THE CONTROL AND 0.13 Jlg/L

CONCENTRATION LEVEL FOR STEEL.' S MANY-ONE RANK TEST

Rank

2

2

2

4.5

4.5

7

7

7

Transformed

Proportion

Normal

1.369

1.369

1.369

1.397

1.397

1.571

1. 571

1.571

concentration

0.13 p.g/L

0.13 p.g/L

Control

0.13 p.g/L

Control

0.13 p.g/L

Control

Control

13.13.2.6.3 For this example, determine if the survival in any

of the concentrations is significantly' lower than the survival in

the control. If this occurs, the rank sum at that concentration

would be significantly lower than the rank sum (jf the control.

, Thus compare the rank sums for the survival at t~ach of the

various concentration levels with some "minimum'" or critical rank

sum, 'at or below which the survival would be considered

significantly lower than the control. At a significance level of

0.05, the minimum rank sum in a test with five concentrations

(excluding the control) and four replicates is 10 (See Table 5,

Appendix E) .

13.13.2.6.4 Since the rank sums for the 0.50 Jlg/L and 1.00 Jlg/L

concentration levels are equal to the critical 'value, the

proportions of normal development in those concf~ntrations are,

considered significantly less than that in the control.. Since no

other rank sum is less than or equal to the crH:.ical value, no

245
TABLE 10. TABLE OF RANKS1

Copper Concentration (~g/L)

Replicate

J.

2

3

4

Control

J..57J.{7,7.5,7.5,7.5,7)

1.369{2,4.5,5,5,4)

J..57J.{7,7.5,7.5,7.5,7)

J..397{4.5,6,6,6,5)

0.J.3

, J.. 369 (2)

1.397{4.5)

1. 571 (7)

J..369(2)

0.25

1.284(2)

1.345(3)

1.249 (J.)

1.369(4.5)

Copper Concentration (~g/L) (Continued)

Replicate 0.50 1.00 2.00

J. 1. 266 (2) 1. 2J.7 (3.5) 1.571(7)

2 1.303(3.5) 1. J.46 (2) 0.959(2)

3 1.2J.7{J.) 1.'2J.7(3.5) 1.047(3)

4 1.303{3.5) 1.J.33{J.) 0.685{J.)

lControl ranks are given in the order of the concentration with which

they were ranked.

TABLE 11.

Concentration

Jlg/L Copper)

0.13

,0.25

0.50

RANK SUMS

Rank Sum

15.5

10.5

10.0

other concentration has a significantly ~ower proportion normal

than the control. Because the 0.50 Jlg/L concentration shows

significantly lower normal development than the control while the

246
higher 2.00 Jlg/L concentration does not·, these test results are

considered to have an anomalous dose-response relationship and it

is recommended that the test be repeated. If an NOEC and LOEC

must be determined for this test, the lowest concentration with

significant growth impairment versus the control is considered to

the LOEC for growth. Thus, for this test, the NOEC and LOEC

would be 0.25 Jlg/L and 0.50 ~g/L, respectively.

13.13.2.7 Calculation of the ICp

13.13.2.7.1 The embrYo-larval development data in Table 4 are

utilized in this example. As can be seen from Table 4 and Figure

2, the observed means are monotonically non-increasing with

respect to concentration (mean response for each higher

concentration is not less than or equal to the mean response for

the previous concentration and the responses between

concentrations do n~t follow a linear· trends) . : Therefore, it is

not necessary to smooth the means prior to calculating the IC.

The observed means, represented by Yi become the corresponding

smoothed means, Mi. Table 12 contains the response means and

smoothed means and Figure 3 gives a plot .of the smoothed response

curve.

TABLE 12. BIVALVE MEAN LARVAL DEVELOPMENT' RESPONSE AFTER

SMOOTHING

Response , Smoothed i

Copper Means, Yi Means, Mi

Cone. (Jlg/L) i (proportion) .(proportion)

Control 1 0.983 0.983

0.13 2 0.973 0.973

0.25 3 0.932 0.932

0.50 4 0.913 0.913

1. 00 5 0.852 0.852

2.00 6 0.705 0.705

13.13.2.7.2 An IC25 can be estimated using the Linear

Interpolation Method. A 25% reduction in mean proportion of

normally developed larvae, compared to the controls, would result

247
1.0 i'l' >t' * * 'Ie .....,.,.~T!1lIMI..CIIB1'

C!IlllIICI'ITHI.......\IlrlLU *

Sf' ClOMCTI".~ ....W1G

Cl 0.91 >i< * t.U

0..

0 0.8 ...J

t.U

>

t.U 0.7 j

* Cl

>- 0.6 ...J

...J

« t\) ::::;: 0.5 0:::

(Xl 0

z 0.4"-1 z * 0

-

t- 0.3

O:::

0

0... 0.2 0

0:::

0...

0.1

0.0

0.00. 0.13 0.25 0.50 1.00 2.00

COPPER CONCENTRATION (ugIl)

Figure 3. Plot of raw data, observed means, and smoothed means for the bivalve

development data from Tables 4 and 12.
in a mean proportion of 0.737, where M1 (1-p/100),= 0.983(1- . ,

25/100). Examining the means and their associatied c;:oncentrations

(Table 12), the response, 0.737, is bracketed bi C4 = 1.00 fJ.g/L

copper and Cs = 2.00 fJ.g/L copper.

13.13.2.7.3 Using the equation from Section 4.2 in Appendix L,

the estimate of the IC25 is calculated as follows:

IC25 = 1.00 + [0.983(1 - 25/100) - 0.852] (,2.00 - 1.00)

(0.705 - 0.852)

= 1. 78 fJ.g/L.

13.13.2.7.4 When the ICPIN program was used to analyze this set

of data, requesting 80 resamples,. the estimate of the I.C25 was

1.7839fJ.g/L., The empirical 95.0% confidence interval for the

true mean was not available because the number of resamples which

generated an IC25 estimate was not an even multiple of 40. The

computer program output for the IC25 for this data set is shown

in Figure 4.

13.14 PRECISION AND ACCURACY

13.14.1 PRECISION

13.14.1.1 Single-Laboratory Precision

13.14.1.1.1 Single-laboratory precision data for the Mytilus

spp. with the reference toxicant cadmium and lyophilized pulp

mill effluent with natural seawater are provided in Tabl~s 4-5.

The coefficient of variation, based on EC25, is 32.8% to 45.0%

for cadmium and 14.2% to 30.6% for lyophilized ~~lp mill

effluent. Single-laboratory precision data for 'the Crassostrea

gigas with the reference toxicant cadmium and l~jphilized pulp

mill effluent with natural seawater are provided in Tables 6-7.

The coefficient of variation, based on EC25, is 18.5% to 80.4%

for cadmium and 20.8% to 43.3% for lyophilized pulp mil.l

effluent.

13.14.1.2 Multi-laboratory Precision

249
Cone. ID

Cone. Tested

Response J.

Response 2

Response 3

Response 4

J.

o

1. 00

0.96

1.00

0.97

2

0.J.3

0.96

0.97

1. 00

0.96

3

0.25

0.92

0.95

0.90

0.96

4

0.50

0.91

0.93

0.88

0.93

5

1. 00

0.88

0.83

0.88

0.82

6

2.00

1. 00

0.67

0.75

0.40

*** Inhibition concentration Percentage Estimate *** Toxicant/Effluent: Copper

Test Start Date: Test Ending Date:

Test Species: bivalve

Test Duration: 48 hours

DATA FILE: bivalve.icp

OUTPUT FILE: bivalve.i25

Cone. Number Concentration Response Std. Pooled

ID .Replicates ug/L Means Dev. Response Means

------------------------------------------------------ --~--------.------

J. 4 0.000 0.983 0.021 0.983

2 4 0.130 0.973 0.019 0.973

3 4 0.250 0.932 0.028 0.932

4 4 0.500 0.913 0.024 0.913

5 4 1.000 0.852 0.032 0.852

6 4 2~000 0.705 0.247 0.705

The Linear Interpolation Estimate: 1. 7839 Entered P Value: 25

Number of Resamplings: 80Those resamples not used had estimates

above the highest concentration/ %Effluent.

The Bootstrap Estimates Mean: 1.6188 Standard Deviation: 0.J.758

No Confidence Limits can be produced since the number of resamples

generated is not a multiple of 40.

Resampling time in Seconds: 0.17 Random Seed:-232404862

Figure 4. ICPIN program output for the IC25.

250
13.14.1.2.1 Multi-laboratory precision data for Mytilus spp.

with the reference toxicant, cadmium and lyophilized pulp mill

effluent are pr.ovided in Tables 12-13. The coefficient of

variation for cadmium EC25 is 23.6%, and 'for effluent EC25 is

14.4% based on five laboratories. Multi-laborat.ory precision

data for Crassostrea gigas with the reference t6xicant, cadmium,

and lyophilized pulp mill effluent are provided in Tables 14-15.

The coefficient of variation is 21.3% for cadmiu,m EC25 and 14".2%

for lyophilized pulp mill effluent EC25, based on results .from

five laboratories.

13.14.2 ACCURACY

13.14.2.1 The accuracy of toxicity tests cannot be determined.

TABLE 12. SINGLE AND MULTI-LABORATORY PRECISION·OF.THEMUSSEL,

MYTILUS SPP., DEVELOPMENT TEST PERFORMED WITH CADMIUM

CHLORIDE (CD MG/L) AS A REFERENCE TOXICANT

Month Lab A Lab B Lab C Lab D .Lab E

Oct-92 2.35 1.06 2.42 4.20 4.77

Nov-92 0.86 3.49 3.89 2.21' 2.39

Dec-92 1. 79 2.51 no data 2.27 3.73

Jan-93 3.69 2.25 6.77 no data 1.57

Feb-93 2.81 2.91 5.85 3.75 3.05

Mar-93 3.71 2.64 2.62 4.89 no data

Mean 2.54 2.48 4.31 3.46 : 3.10

SD 1.11 0.81 1. 94 1.19 1.23

CV (%) 43.9 32.8 45.0 34.3 40.0

# of Labs Statistic EC25

5 Mean (N=5) 3.18

SD 0.75

CV(%} 23.6 .

These data are from: Pastorok, et al. (1994), West Coast Mar~ne Spec~es

Chronic Protocol Variability Study, PTI Environmental Serv;ices, Prepared for

Washington Department of Ecology, Feqruary, 1994.

251
TABLE 13. SINGLE AND MULTI-LABORATORY PRECISION OF THE MUSSEL,

MYTILUS SPP., DEVELOPMENT TEST PERFORMEP WITH

LYOPHILIZED PULP MILL EFFLUENT (%) AS' THE TOXICANT

Month Lab A Lab B Lab C Lab D Lab E

Oct-92 1. 78 1.40 2.02 1. 83 1. 85

Nov-92 1.57 1. 94 2.70 1. 98 no data

Dec-92 1.74 1. 88 3.08 no data 1. 87

!

Jan-93 3.17 2.03 2.46 1. 07 data I no

i Feb-93 1.66 no data no data no data no data

Mar-93 1.85 1.66 1. 72 1. 82 ,no data "

Mean 1.96 1. 78 2.40 1.68 1. 86

i SD 0.60 0.25 0.54 0.41 0.28

CV (%) 30.6 14.2 22.5 24.5 1.4

# of Labs Statistic EC25 '"

5 Mean (n=5) 1. 93

SD 0.28

CV(%) 14.4

These data are from: Pastorok~ et al. (1994) West Coast Marine

Species Chronic Protocol Variability Study, PTI Environmental

Services, Prepared for Washington Department of Ecology,

February, 1994.

252
TABLE 14. SINGLE AND MULTI-LABORATORY PRECISIONbF THE OYSTER,

CRASSOSTREA GIGAS, DEVELOPMENT TEST PE:RFORMED WITH

CADMIUM CHLORIDE (CD MG/L) AS A REFERENCE TOXICANT

Month Lab A Lab B Lab C Lab D Lab E

July-92 1. 04 1. 54 0.50 0.41 0.56

Aug-92 0.31 1. 38 0.30 0.35 no data

Sept-92 0.68 0.20 0.49 no data no data'

Apr-93 no data 0.45 0.51 no data 0~95

May-93 0.46 0.30 1. 05 0.52 0.83

June-93 0.26 1. 55 0.93 no data '0.83

July-93 0.28 0.82 0.66 1. 56 -0.90

Mean 0.51 0.89 0.63 0.71 0.81

SD 0.31 0\59 0.27 0.57 0.15

CV (% ) 60.6 66.7 42.1 80.4 18.5

# of Labs Statistic EC25

5 Mean (n=5) 0.71

SD 0.15

CV (%) 21.3'

These data are from: Pastorok,_ et al. (1994), West Coast Marine

Species Chronic Protocol Variability Study, PTI Environmental

Services, Prepared for Washington Department of Ecology,

February, 1994.

253
TABLE 15. SINGLE AND MULTI-LABORATORY PRECISION OF THE OYSTER,

CRASSOSTREA GIGAS, DEVELOPMENT TEST PERFORMED WITH

LYOPHILIZED PULP MILL EFFLUENT (%) AS THE TOXICANT

Month Lab A Lab B Lab C Lab D Lab E

July-92 no data 0.91 1. 28 no data 1.43

Aug-92 1.21 1.09 0.98 0.61 no data

Sept-92 0.76 1.66 0.83 no data no data

. Apr-93 0.80 1.10 1.61 1. 66 no data

May-93 1.21 0.65 1. 90 0.93 0.93

June-93 1.09 1.32 1. 72 0.83 0.98

July-93 0.82 0.80 1. 56 1. 67 1. 04

: Mean 0.98 1. 08 1.41 1.14 1 .. 10 ,

SD. 0.21 0.34 0.40 0.49 0.23

, CV (%) 21.6 31.4 28.0 43.3 20.8

# of Labs Statistic EC25

5 Mean (n=5) 1.14

SD 0.16

cy (%) 14.2

These data are from: Pastorok( et al., (1994), West Coast Marine

Species Chronic Protocol Variability Study, pTI Environmental

Services, Prepared for Washington Departmeht of Ecology,

February, 1994.

254
APPENDIX ~. ,BIVALVE TEST: STEP-BY-STEP SUMMARY,

PREPARATION OF TEST SOLUTIONS

A. Determine test concentrations and appropria~te dilution water

based on NPDES permit conditions and guidance from the

appropriate regulatory agency.

B. Prepare effluen~ test solutions by dilutingr well mixed

unfiltered effluent using volumetric flasks: and pipettes.

Use hypersaline brine where necessary to maintain all test

solutions at 30 ± 2~. Include brine controls in tests that

use brine.

C. Prepare a copper reference toxicant stock solution.

D. Prepare a series copper refe,rence toxicant concentrations.

E. Sample effluent and reference toxicant solutions f.or

physical/chemical analysis., Measure salinity, pH and

dissolved oxygen from each test concentration.

F. Randomize numbers for test chambers and rec,ord the chamber

numbers with their respective test concentrations on a

randomization data sheet. Store the data sheet safely until

after the test samples have been analyzed.

G. Place test chambers in a water bath or environmental chamber

set to 18 or 20°C and allow temperature to equilibrate.

H. Measure the temperature daily in one random replicate (or

separate chamber) of each test concentration. Monitor the

temperature of the water bath or environmental chamber

continuously.

I. At the end of the test; measure salinity, pH, and dissolved

oxygen concentration from each test concent~ation.

PREPARATION AND ANALYSIS OF TEST ORGANISMS

A. Determine test concentrations and appropriate dilution water

based on NPDES permit requirements and guid~nce from the

appropriate regulatory agency.

255
B. Prepare test solutions by diluting well mixed unfiltered

effluent using volumetric pipettes. Use hypersaline brine

where necessary to maintain all test solutions at 30 ± 2~.

Include, brine controls in tests that use brine.

C. Sample effluent and reference toxicant solutions for

physical/chemical analysis., Measure salinity, pH and

dissolved oxygen from each test concentration.

D. Randomize numbers for test chambers .and record the chamber

numbers with their respective test concentrations on a

randomization data sheet. Store the data sheet safely until

after the test samples have been analyzed.

E. Place test chambers in a water bath or environmental chamber

set to 18 or 20°C as appropriate for the test species and

allow temperature to equilibrate.

F. Measure the test solution temperature daily in a randomly

located blank test chamber. Monitor the temperature of the

water bath or environmental chamber continuously.

PREPARATION AND ANALYSIS OF TEST ORGANISMS

A. Obtain test organisms and hold or condition as necessary for

spawning.

B. On day of test, spawn organisms, examine gametes, pool good

eggs, pool good sperm.

C. Fertilize subsets of eggs with a range of sperm

concentrations to obtain >90% embryogenesis without

polyspermy.

D. Adjust embryo stock suspension density to 1500-300·0/mL.

E. Introduce organisms to test chambers (150-300 embryos in 0.1

mL of stock) .

F. Count all embryos in each of six extra controls set up for

determining mean embryo density and variation. Return these

to the test for later examination for developmental rate in

controls.

256
G. Near the end of the 48-hour incubation period examine

several of the extra controls to determine if development

has reached the prodisoconch stage. If yes, terminate the

test at 48 hours; if no, continue the test :for up to 54

hours as requi~ed for complete development.

H. Terminate the test by addition of formalin.

I. Count larvae and record the number of norma~ prodisoconch

larvae and other larvae in each test vial.

J. Analyze the data.

K. Include standard reference toxicant point estimate values in

the standard quality control charts.

257
Sample data sheet for embryo microscopic examination.

BIVALVE DEVELOPMENT TEST: RESULTS

Bioassay No. _

Counter _

Date---------

Number Sample Abnormal Normal %Normal I Notes

258
SECTION 14

RED ABALONE, Haliotis rufescens

LARVAL DEVELOPMENT TEST METHOD

Adapted from a method developed by

John W. Hunt 'and Brian S. Anderson

Institute of- Marine Sciences, University of California

Santa Cruz, California

(in association with)

California Department of Fish and Game

Marine Pollution· Studies Laboratory

34500 Coast Route 1, Monterey, CA 93940

TABLE OF CONTENTS

14.1

14.2

14.3

14.4

14.5

14.6

14.7

14.8

14.9

14.10

14.11

14.12

14.13

14.14

Scope and Application

Summary of Method

Interferences

Safety

Appa'ratus and Equipment

Reagents and Supplies

Effluents and Receiving Water Collection,

Preservation, and Storage

Calibration and Standardization

Quality Control

Test Procedures

"Summary of Test Conditions and Test

Acceptability Criteria

Acceptability of Test Results

Data Analysis

Precision and Accuracy

Appendix I Step-by Step Summary

259
SECTION 14

RED ABALONE, HALIOTUS RUFESCENS

LARVAL DEVELOPMENT TEST METaOD

14.1 SCOPE AND APPLICATION

14.1.1 This method estimates the chronic toxicity of effluents

and receiving waters 20 the larvae of red abalone, Haliotis

rufescens during a 48-h statiG non-renewal exposure. The effects

include the sYnergistic, antagonistic, and additive effects of

all chemical, physical, and biological components which adversely

affect the physiological. and biochemical functions of the test

organisms.

14.1.2 Detection limits of the toxicity of an effluent or

chemipal substance are organism dependent.

14.1.3 Brief excursions in toxicity may not be detected using

24-h composite samples. Also, because of the long sample

collection period involved in composite sampling and because the

test chambers are not sealed, highly volatile and highly

degradable toxicants in the source may not be detected in the

test.

14.1.4 This method is commonly used in one of two forms: (1) a

definitive test, consisting of a minimum of five effluent

concentrations and a control, and (2) a receiving water test(s),

consisting of one or more receiving water concentrations and a

control.

14.1.5 This method should be restricted to use by, or under the

supervision of, professionals experienced in aquatic toxicity

testing. Specific experience with any toxicity test is usually

needed before acceptable results become routine.

14.2 SUMMARY OF METHOD

14.2.1 This method provides the step-by-step instructions for

performing a 48-h static non-renewal test using early development

of abalone larvae to determine the toxicity of substances in

marine and estuarine waters. The test endpoint is normal shell

development.

260
14.3 INTERFERENCES

14.3.1 Toxic substances may be introduced by contaminants in

dilution water, glassware, sample hardware, and testing equipment

(see Section 5, Facilities and Equipment, and Supplies) .

14.3.2 Improper effluent sampling and handling lnay adversely

affect test results (see Section 8, ~ffluent and Receiving Water

Sampling, and Sample Handling, and Sample Prepariation for

Toxicity Tests) .

14.4 SAFETY

14.4.1 See Section 3, Health and Safety.

14.5 APPARATUS AND EQUIPMENT

14.5.1 Tanks, trays, or aquaria - - for holding (~nd acclimating

adult red abalone, e.g., standard salt water aquarium or Instant

Ocean Aquarium (capable of maintaining seawater at 10-20°C), with

appropriate filtration and aeration system.

14.5.2 Air pump, air lines, and air stones for aerating water

containing broodstock or for supplying air. to test solutions with

low dissolved oxygen.

14.5.3 Constant temperature chambers or water baths --for

maintaining test solution temperature and keepinSf dilution water

supply, gametes, and embryo stock suspensions attest temperature

(15°C) prior to the test.

14.5.4 Water purification system -- Millipore Super-Q, Deionized

water (DI) or equivalent.

'14.5.5 Refractometer

14.5.6 Hydrometer{s)

for determining salinity.

for calibrating refractometer.

14.5.7 Thermometers, glass br electronic, labor~tory grade

for measuring water temperatures.

261
14.5.8 Thermometer/ National Bureau. of Standards Certified (see

USEPA METHOD 170.1/ USEPA/ 1979) -- to calibrate laboratory

thermometers.

14.5.9 pH and DO meters -- for routine physical and chemical

measurements.

14.5.10 Standard or micro-Winkler apparatus

DO (optional) and calibrating the DO meter.

for determining

14.5.11 Winkler bottles -- for dissolved oxygen determinations.

14.5.12 Balance -- Analytical, capable of accurately weighing to

0.0001 g.

14.5.13 Fume hood -- to protect the analyst from effluent or

formaldehyde fumes.

14.5.14 Glass stirring rods for mixing test solutions.

14.5.15 Graduated cylinders Clas~ A, borosilicate glass or

non-toxic plastic labware, 50-1000 mL for making test solutions.

(Note: not to be used interchangeably for gametes or embryos and

test solutions).

14.5.16 Volumetric flasks -- Class A, borosilicate glass or nontoxic plastic labware, 10-1000 mL for making test solutions.

14.5.17 Pipets, automatic -- adjustable, to cover a range of

delivery volumes from 0.010 to 1.000 mL.

14.5.18 Pipet bulbs and fillers -- PROPIPET@ or equivalent.

14.5.19 Wash bottles -- for reagent water, for topping off

graduated cylinders, for rinsing small glassware and instrument

electrodes and probes.

14.5.20 Wash bottles -- for dilution water.

14.5.21 20-1iter cubitainers or polycarbonate water cooler jugs

-- for making hypersaline brine.

262
I

14.5.22 Cubitainers, beakers, or similar chambers of non-toxic

composition for holding, mixing, and dispensing dilution water·

and other general non-effluent, non-toxicant contact uses. These

should be clearly labeleq and not used for other purposes.

I

14.5.23 Beakers, 1,000 mL borosilicate glass -- for mixing

gametes for fertilization of eggs.

14.5.24 Beakers, 250 mL borosilicate glass ~- for preparation of

test solutions.

14.5.25 Counter, two unit, 0-999 -- for recording counts of

larvae.

14.5.26 Inverted or compound microscope -- for inspect·ing

gametes and making counts of larvae.

14.5.27 Perforated plunger -- for stirring egg solutions.

14.5.28 Supply of Macrocystis or other macroalgae (if holding

broodstock for longer than 5 days) -- for feeding I abalone.

14.5.29 Stainless steel butter knife, rounded smooth-edged blade

(for handling adult abalone). Abalone irons and plastic putty

knives have also been used successfully.

14.5.30 Sieve or screened tube, approximately 37 ~m-mesh -- for

retaining larvae at the end of the test.

14.5.31 60 ~m NITEX® filter -- for filtering receiving water.

14.6 REAGENTS AND SUPPLIES

14 . 6 . 1 Sample containers - - for sample shipment a,nd storage (see

Section 8, Effluent and Receiving Water Sampling, .and Sample

Handling, and Sample Preparation for Toxicity Tests) .

14.6.2 Data sheets (one set per test) -- for data recording (See

Appendix I) .

14.6.3 Tape, colored -- for labelling test chambers and

containers.

263
14.6.4 Markers, water-proof -- for marking containers, etc.

14.6.5 Parafilm to cover· graduated cylinders and vessels

containing gametes, embryos.

14.6.6 Gloves, disposable

contamination.

for personal protection from

14.6.7 Pipets, serological -- 1-10 mL, graduated.

14.6.8 Pipet tips

14.6.9 Coverslips

for automatic pipets.

for microscope slides.

14.6.10 Lens paper -- for cleaning microscope optics.

14.6.11 Laboratory tissue wipes for cleaning and drying

electrodes, microscope slides, etc.

14.6.12 Disposable countertop covering for protection of work

surfaces and minimizing spills and contamination.

14.6.13 pH buffers 4, 7, and 10 (or as per instructions of

instrument manufacturer) -- for standards and calibration check

(see USEPA Method 150.1, USEPA, 1979).

14.6.14 Membranes and filling solutions ._- for dissolved oxygen

probe (see USEPA Method 360.1, USEPA, 1979), or reagents for

modified Winkler analysis.

14.6.15 Laboratory quality assurance samples and standards --

for the above methods.

14.6.16 Test chambers -- 600 mL, five cnambers per

concentration. The chambers should be borosilicate glass (for

effluents) or nontoxic disposable plastic labware (for reference

toxicants). To avoid contamination from the air and excessive

evaporation of test solutions during the test, ~he chambers

should be covered during the test with safety glass plates or a

plastic sheet (6 mm thick) .

14.6.17 Formaldehyde, 37% (Concentrated "Formalin) -- for

preserving larvae. Note: formaldehyde has been identified as a

264
carcinogen and is irritating to skin and mucous membranes. It

should not be used at a concentration higher than necessary to

achieve morphological preservation of larvae for counting and

only under conditions of maximal ventilation and minimal

opportunity for volatilization into room.air.

14.6.18 Tris (hydr6xYmethyl) aminomethane ~nd hydrogen peroxide

(for H20 2 spawning method) -- for spawning abalone.

14.6.19 Reference toxicant ,solutions (see Subsection 14.10.2.4

and see Section 4~ Quality Assurance) .

14.6.20 Reagent water -- defined as distilled or deionized water

that does not contain substances which are toxic to the test

organisms (see Section 5, Facilities, Equipment; ,and Supplies and

Section 7, Dilution Water) .

14.6.21 Effluent and receiving water -- see Section 8, Effluent

and Surface Water Sampling, and Sample Handling, and Sample

Preparation for Toxicity Tests.

14.6.22 Dilution water and hypersaline brine see Section 7,

Dilution Water anq Section 14.6.24, Hypersaline Brines.' The

dilution water should be uncontaminated l-j.tm-fil:tered natural

seawater. Hypersaline brine should be prepared from dilution

water.

14.6.23 HYPERSALINE BRINES

14.6.23.1 Most industrial and sewage treatment effluents

entering marine and estuarine systems have littl(: measurable

salinity. Exposure of larvae to these effluents. will usually

require increasing the salinity of the test solu1:ions. It is

~mportant to maintain an essentially constant salinity across all

treatments. In some applications it may'be desirable to match

the test salinity with that of the receiving watE:r (See Section

7.1). Two salt sources are available to adjust salinit,ies --

artificial sea salts and hypersaline brine (HSB) derived from

natural seawater. Use of artificial sea salts if~necessary only

when high effluent concentrations preclude salinity adjustment by

HSB alone.

265
14.6.23.2 Hypersaline brine (HSB) can be made by concentrating

natural seawater by freezing or evaporation. HSB should be made

from high quality, filtered seawater, and can be added to the

effluent or to reagent water to increase salinity. HSB has

several desirable characteristics for use in effluent toxicity

testing. Brine derived from natural seawater contains, the

necessary trace metals, biogenic colloids, and some of the

microbial components necessary for adequate growth, survival,

and/or reproduction of marine and estuarine organisms,and it can

be stored for prolonged periods without any apparent degradation.

However, even if the maximum salinity HSB (100~) is used as a

diluent, the maximum concentration,of effluent (O~) that can be

tested is 66% effluent at 34~ salinity (see Table 1) .

14.6.23.3 High quality (and preferably high salinity) seawater

should be filtered to at least 10 ~m before placing into the

freezer or the brine generator. Water should be collected on an

incoming tide to minimize the possibility of contamination.

14.6.23.4 Freeze Preparation of Brine

14.6.23.4.1 A convenient container for making HSB by freezing is

one that has a bottom drain. One liter of brine can be made from

four liters of seawater. Brine may be collected by partially

freezing seawater at -10 to -20°C until the remaining liquid has

reached the target salinity. Freeze for approximately six hours,

then separate the ice (composed mainly of fresh water) from the

remaining liquid (which has now become hypersaline) •

14.6.23.4.2 It is preferqble to monitor the water unt~l the

target salinity is achieved rather than allowing total freezing

followed by partial thawing. Brine salinity should never exceed

100~. It is advisable not to exceed about 70~ brine salinity

unless it is necessary to test effluent concentrations greater

than 50%.

14.6.23.4.3 After the required salinity is attained, the HSB

should be filtered through a 1 ~m filter and poured directly into

portable containers (20-L cubitainersor polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and the date the brine

was generated. Containers of HSB should ,be stored in the dark at

266
4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after several months j~n storage.

14.. 6.23.5 Heat Preparation of Brine

TABLE 1. MAXIMUM EFFLUENT CONCENTRATION (%). THAT CAN BE TESTED

AT 34%0 WITHOUT THE ADDITION OF'DRY SALII'S GIVEN THE

INDICATED EFFLUENT AND BRINE SALINITIE£;.

Effluent Brine Brine Brine Brine Brine

Salinity 60 70 80 90 100

g., %0 g., 00 00 %'0 %'0 %'0

0 43.33 51.43 57.50 62.22 66.00

1 44.07 52.17 58.23 62.92 66.67

2 44.83 52.94 58.97 63.64 67.35

3 45.61 53.73 59.74 64.37 68.04

4 .46.43 54.55 60.53 65.12 I 68.75

5 47.27 55.38 61.33 65.88 69>17

10 52.00 60.00 65.7"1 70.00 73.33

15 57.78 65.45 70.77 74.67 77.65

20 65.00 72.00 76.67 80.00 82.5.0

25 74.29 80.00 83.64 86.15 88.00

14.6.23.5.1 The ideal container for making brine using heatassisted evaporation of 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 corning 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 is to use a thermostatically controlled heat

267
exchanger made from fiberglass. If aeration is needed, use only

oil-free air compressors to prevent contamination.

14.6.23.5.2 Before adding seawater to the brine generator,

thoroughly clean the generator, aeration supply tube, heater, and

any other materials that will be in direct contact with the

brine. A good quality biodegradable detergent should be used,

followed by several (at least three) thorough reagent water

rinses.

14.6.23.5.3 Seawater should be filtered to at least 10 ~m before

being put into the brine generator. 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.

14.6.23.5.4 After the required salinity ,is attained, the HSB

should be filtered through a 1 ~m filter and poured directly into

portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and the date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after several months in storage.

14.6.23.6.1

artificial

this time,

14.6.23.6

14.6.23.7

Artificial Sea Salts

No data from red abalone tests using sea salts or

seawater (e.g., GP2) are available for evaluation at

and their use must be considered provisional.

Dilution Water Preparation from Brine

14.6.23.7.1 Although salinity adjustment with brine is the _

preferred method, the use of high salinity brines and/or reagen~'

water has sometimes been associated with discernible adverse'

effects on test organisms. For this reason, it is recommended

that only the minimum necessary volume of brine and reagent water

be used to offset the low salinity of the effluent, and that

268
brine controls be included in the test. The rema~n~ng dilution

water should be'natural seawater. Salinity may be adj~sted in

one of two ways. First, the salinity of the highest effluent

test concentration may be adjusted to an acceptable salinity, and

then serially diluted. Alternatively, each effluent

. concentration can be p~epared individually with appropriate

volumes of effluent and brine..

14.6.23.7.2 When HSB and reagent water are used, thoroughly

mix together the reagent water and HSB before mixing iri the

effluent. Divide the salinity of the HSB by the expected test

salinity to determine the proportion of reagent· water to b~ine.

For example, ·if the salinity of the brine is 109%0 and the test

is to be conducted at 34%'0, 100%0 divided by 34%0 :=' 2.94. The

proportion of brine is 1 part plus 1.94 parts r~agent water. To

make 1 L of dilution water at 34%0 salinity from a HSB,of 100%0,

340 mL of brine and 660 mL of reagent water are required. Verify

the salinity of. the resulting mixture using a refractometer.

14.6.23.8 .Test Solution Salinity Adjustment

14.6.23.8.1 Table 2 illustrates the preparation of test

solutions '(up to SO~ effluent) at 34%0 by combining effluent,

HSB, and 'dilution water. Note: if the highest e:ffluent,

concentration does not exceed so~ effluent, it i,s convenient to

prepare brine so that the sum of the effluent salinity and brine

salinity equals 68%0; the required brine volume is then always

equal to the effluent· volume needed for each effluent

concentration as in the example in Table ,2..

14.6.23.8.2 Check the pH of all test solutions and adjust to

within 0.2 units of dilution water pH by adding, dropwlse, dilute

hydrochloric acid or sodium hydroxide (see Section 8.8.9,

Effluent and Receiving Water Sampling, Sample Handling, and

Sample Preparation for Toxicity Tests) .

14.6.23.8.3 To calculate the amount of brine to add to each

'effluent dilution, determine ,the following qu~ntities: salinity

of the brine (SB, in %0), the salinity of the effluent (BE, in

%0), and volume of the effluent to be added (VE,: in mL) . Then'

use the following formula to calculate the volumE~ of brine (VB,

.in mL) to be added:

269
VB = VB x (34 - SE)/(SB - 34)

14.6.23.8.4 This calculation assumes that dilution water

salinity is 34 ± 2~.

14.6.23.9 preparing Test Solutions

14.6.23.9.1 Two hundred mL of test solution are needed for each

test chamber. To prepare test solutions at low,effluent

concentrations «6%), effluents may be added directly t'o dilution

water. For example, to prepare 1% effluent, add 10 mL of

effluent to a .1-liter volumetric flask using a volumetric pipet

or calibrated automatic pipet. Fill the volumetric flask to the

1-L mark with dilution water, stopper it, and shake to mix.

Distribute equal volumes into the replicate test chambers.

14.6.23.9.2 To prepare a test solution at higher effluent

concentrations, hypersaline brine must usually be used. For

example, to prepare 40% effluent, add 400 mL.of effluent to a 1-

liter volumetric flask. Then, assuming an effluent salinity of

2~ and a brine salinity of 66~, add 400 mL of brine (see

equation above and Table 2) and top off the flask with dilution

water. Stopper the flask and shake well.' Distribute equal

volumes into the replicate test chambers.

14.6.23.10 Brine Controls

14.6.23.10.1 Use brine controls in all tests where brine is

used. Brine controls contain the same volume of brine as does

the highest effluent concentration using brine, plus the volume

of reagent water needed to ,reproduce the hyposalinity of the

effluent in the highest concentration, plus dilution water.

Calculate the amount of reagent water to add to brine controls by

rearranging the above equation, (See, 16.6.23.8.3) setting SE =

0, and solving for VE.

VE = VB x (BB - 34)/(34 - SE)

If effluent salinity is essentially' 0%0, the reagent wat,er volume

needed in the brine control will equal the effluent volume at the

highest test concentration. However, as effluent salinity and

effluent concentration increase, less reagent water volume is

needed.

270
14.6.24 TEST ORGANISMS

14.6.24.1 The test organisms used for this test ar~ red abalone,

Haliotis rufescens. This large gastropod mollusc is harvested

commercially in southern California and supports a popular

recreational fishery throughout the state. It, cqnsumesa variety

of seaweeds and small incidental organisms, and is an important

food source for sea otters, lobsters, and octopods (Hines and

Pearse 1892). Abalone are "broadcast" spawners that reproduce by

equiva1ent.ejecting large numbers of gametes into the water

column, whe~e fertilization takes place external~y. Freeswimming larvae hatch as trochophores, then undergo torsion while

passing through a veliger stage. Abalone larvae do not feed

during their one to three weeks in the plankton, ,but exist on

energy st'ored in the yolk sack, supplemented perhaps by the

uptake of dissolved amino acids. Once <larvae come into cont.act

with suitable substrate, they metamorphose and bE~gin to consume

benthic algae using a rasp:-like tongue (the radula). Red abalone

become reproductive after about two years at a length of about 7

cm, and can live for at least 25 years, growing to 30 cm in

length. Refer to Hahn (1989) for a review of abalone lif~ history

and culture to Martin et al. (1977), Morse et al (1979) and JIunt

and Anderson (1989 and 1993) for previous toxicit~y studies.

14.6.24.2 Species Identification

14.6.24.2.1 Broodstock should be positively identified to

species. Epipodal characteristics provide the best means of

identification. All California haliotids have a lacey epipodial

fringe,' except for the red and black abalone, which have smoot.h,

lobed epipodia. The red abalone can be distinguished from the

black by shell coloration and by the number of respiratory pores

in the shell (reds have 3 to 4, blacks have 5 to 8). For further.

information on abalone taxonomy consult Owen et al. (1971), and

Morris et al. (1980).

14.6.24.3 Obtaining ~roodstock

14.6.24.3.1 M~ture red abalone broodstock can be collected from

rocky substrates from the intertidal to depths exceeding 30

meters. They are found most commonly in crevices in areas where

there is an abundance of macroalgae. State collection permits

are usually required for collecting abalone. Collection of

271
TABLE 2. EXAMPLES OF EFFLUENT DILUTION SHOWI~G VOLUMES OF

EFFLUENT (x~), BRINE, AND DILU~ION WATER NEEDED FOR

ONE LITER OF EACH TEST SOLUTION.

fIRST STEP; Combine brine ~ith reagent water or natural seawater .to achieve a

brine of 6B-x~ and, unless natural seawater is used for dilution water, also

a brine-based dilution water of 34~.

SERIAL DILUTION;

Step 1. Prepare the highest effluent concentration to be tested by adding

equal volumes of effluent and brine to the appropriate volume of dilution

water. An example using 40% is shown.

, Effluent Cone. Effluent Brine Dilution Water*

(t) x~ (68-x)~ 34~

: 40 800 mL 800 mL 400 mL

step 2. Use either serially prepared dilutions of the highest test

concentration or individual dilutions of ~OO% effluent.

Effluent Cone. (%) Effluent Source Dilution Water* (34~)

I

20 1000 mL of 40% 1000 mL

J.O ~000 mL of 20% ~OOO mr;

5 1000 mL of 10% 1000 mL

2.5 ~ooo mL of 5% 1000 mL·

Control none 1000 mL

INDIVIDUAL PREPARATION

i Effluent Cone. Effluent x~ Brine (68-x) Dilution Water*

(t) 34~

: 40 400 mL 400 mL 200 mL

!

, 20 200 mL 200 mL 600 mL

, 10 ~00 mL 100 mL 800 mL

I 5 50 mL 50·mL 900 mL

2.5 25 mL 25 mL 950 mL

Control none none 1000 mL

*May be natural seawater or brine-reagent water.

272
abalone is regulated by California law. Collectors must obtain a

scientific collectors permit from the California Department of

Fish and Game and observe any regulations regarding collection,

transfer, and maintenance of abalone broodstock.

14.6.24.3.2 While abalone captured in t~e wild can be induced to

spawn, those grown or conditioned in the laborat6ry have been

more dependable. Commercial mariculture facilities in California

produce large.numbers of abalone, and distribution systems exist

to supply live spawners to a num~er of market areas~ In any

case, broodstock should be obtained from sources free of

contamination by' toxic substances to avoid gen$1:ic or

physiological preadaptation to pollutants. )

14.6.24.3.3 Abalone broodstock can be transported for short time

periods from the field or supply facility in clE:an covered

plastic buckets filled with seawater. Use compressed air, or

battery powered pumps to supply aeration. Compressed oxygen is

not recomm~nded becaul3e bubbled oxygen may indu(~e unintended

spawning (Morse et al., 1977). 'Maintain water t:emperatures

within 3°C of the temperature at the collecting site~ Four

abalone in a 15-literbucket should remain healtlhy for up to four

hours. under these conditions. I

14 . 6 .24 . 3 .4 Abalone can be transported for up t.O 3a hours in

sealed, oxygen-filled plastic bags containing moist (seawater)

polyfoam sponges (Hahn, 1989). Cut the polyfoam into s.ections

(about 20 X 40 cm) and allow them to soak in clean seawater for a

few minutes. New sponges should be leached in seawater for at

least 24 hours. Rinse the sponges in fresh seawater and wring

them out well. Place the polyfoam inside double plastic trash.

bags, then place the abalone on the moist foam. i It is important

that t~ere is no standing water in the bags. Put the abalone,

bags into an ice chest (10 to 15 liter), fill the bags with pure

oxygen, squeeze the bags to. purge out all the air, then refill

with oxygen (approximately three liters of oxygen gas will

support eight abalone). Seal the bags (air-tight) with a tie or

rubber band. Wrap two small (one-liter) blue iC.e blocks in

sections of 'newspaper (about 15 pages thick) for insulation, and

place the wrapped blue ice in a sealed plastic bag in the chest

on top of the abalone bags. Fill any remaining space with

packing and seal the box for shipping. Avoid transporting the

ice chest in temperatures below freezing ,or abQ~e 30°C.

273
14.6.24.4 Broodstock Cuiture and Handling

14.6.24.4.1 At the testing facility, pla~e the abalone in

aerated tanks with flowing seawater (1 to 2 liter/min). With

high water quality, water flow, and aeration, abalone 8 to 10 cm

long can be kept at a density of one per liter of tank space or

one per 100 cm2 of tank surface area, whichever provides the

lower density. Density should be cut to a maximum of 0.5 per

liter in recirculating systems and to a maximum of 0.25 per liter

in static tanks. Tanks should be covered for shade and to

prevent escape. Drain and rinse culture tanks twice weekly to

prevent build-up of detritus. Remove any dead abalone

immediately, and drain and scrub its tank.

14.6.24.4.2 Ideal maintenance temperature is 15 ± 1°C, the

toxicity test temperature (see also Leighton, 1974). If

broodstock are to be held for longer tha~ 5 days at the testing

facility, feed broodstock with blades of the giant kelp,

Macrocystis. Feed to slight excess; large amounts of uneaten

algae will foul culture water. If Macrocystis is unavailable"

other brown algae (Nereocystis, Egregia, Eisenia), or any 'fleshy

red algae can be substituted (Hahn, 1989).

14.6.24.4.3 Recirculating tanks should be equipped with

biological or activated carbon filtration systems and oyster

shell beds to maintain water quality. Measure the ammonia

content of static or recirculating seawater daily to monitor the

effectiveness of the filtration system. Un-ionized ammonia

concentrations should not exceed 20 ~g/liter and total ammonia

concentrations should not exceed 1.0 mg/liter. Supply constant

aeration and temperature control. Add only a few blades of algal

food at each cleaning to prevent its accumulation and decay.

14.6.24.4.4 When handling abalone, use a rounded, dUll-bladed

stainless-steel butter knife, abalone iron, or plastic putty

knife to release the animal's grip on the substrate. ,Gently

slide the flat dull blade under the foot at the -posterior end

near the beginning of the shell whorl, and slide it under about

two-thirds of the foot. Apply constant pressure to keep the

front edge of the blade against the substrate and not up into the

foot. Quickly and gently lift the foot off the substrate. A

smooth deliberate motion is more effective and"less damaging than

repeated prying.

274
14.6.24.4.5 Assess the reproductive condition of the broodstock

by examining the gonads, located under the right: posterior edge

of the shell. An abalone placed upside down on a flat surface

will soon relax and begin moving the foot tryin9 to right itself.

Take advantage of this movement and use 'the dull blade to bend

the foot away, from'the gonad area for inspection. The female

ovary is jade green, the male testes are cream-colored. Whenthe

gonad fully envelopes the dark blue-gray conical digestive gland

and is bulky along its entire' length, the abalone is ready for

spawning (Hahn, 1989). , Ripe (redrude~cent) spa~mers have a

distinct, color difference between f:h~~ gray digestive gland and

the green or cream-colored gonad. Less developed gonads appear

gray (in females) or brown (in males) .

14.6.24.4.6 Abalone 7 to 10 cm in shell length:are recommended

in broodstock. They are easier to handle than larger ones, and

can be spawned more often (approximately every four months under

suitable culture conditions; Ault, 1985)." Thou9h spawning fewer

eggs than larger abalone, 10 cm abalone will produce over 100,000

eggs at a time (Ault, 1985). Twenty to thirty-five thousand eggs

are needed for a single toxicant test, dependin~J on tes"t design.

For further information of red abalone culture, ,see Ebert and

Houk (1984) or Hahn (1989).

14.6.24.5 Culture Materials

14.6.24.5.1 See Section 4, Quality Assurance Section for a

discussion o~ suitable materials to be used in laboratory culture

of abalone. Be sure all new materials are properly leached in

seawater be'fore use. After use, all culture mat:erials should be

washed in soap and water, then rinsed with seawater before reuse.

14.7 EFFLUENT AND RECEIVING WATER COLLECTION, l?RESERVATION, AND

STORAGE

14.7.1 See Section 8, Effluent and Receiving Water Sampling, I

Sample Handling, and Sample Preparation for Toxicity Tests.

14.8 CALIBRATION AND STANDARDIZATION

14.8.1 See Section 4, Quality Assurance.

14.9 QUALITY CONTROL

275
14.9.1 See Section 4, Quality Assurance.

14.10 TEST PROCEDURES

14.10.1 TEST DESIGN

14.10.1.1 The test consists of at least five effluent

concentrations plus a dilution water control. Tests that use

brine to adjust salinity must also contain five replicates of a

brine control.

14.10.1.2 Effluent concentrations are expressed as percent

effluent.

14.10.2 TEST SOLUTIONS

14.10.2.1 Receiving waters

14.10.2.1.1 The sampling point is determined by the objectives

of the test. At estuarine and marine sitest samples are usually

collected at mid-depth. Receiving water toxicity is determined

with samples used directly as collected or with samples passed

through a 60 ~m NITEX® filter and compared without dilution,

against a control. Using five replicate chambers per test, each

containing 200 mL would require approximately '1 L of sample per

test.

14.10.2.2 Effluents

14.10.2.2.1 The selection of the effluent test concentrations

should be based on the objectives of the study. A dilution

factor of at least 0.5 is commonly used. A dilution factor of

0.5 provides hypothesis test discriminat~on of ± 100%, and

testing of a 16 fold range of concentrations. Hypothesis test

discrimination shows little improvement as dilution factors are

increased beyond 0.5 and declines rapidly if smaller dilution

factors are used. USEPA recommends that one'of the five effluent

treatments must be a concentration of effluent mixed with

dilution water which corresponds to the per.mittee's instream,

waste concentration (IWC). At least two of the effluent

treatments must be of lesser effluent concentration than the IWe,

276
with one being at least one-half the concentrati'on of the IWC.

If 100%0 HSB is used as a diluent" the maximum concentration of

effluent that can be tested will be 66% at 34%0 salinity.

14: . 10 .2.2.2 If the effluent is known or suspect.ed to be highly

toxic, a lower range of effluent concentrations :should be used

(such as 25%, 12.5%, 6.25%, 3.12% and 1.56%).

14.10.2~2.3 The volume in each test chamber is 1200 mL.

14.10.2.2.4 Effluent dilutions should be prepa~ed for all

replicates in each treatment in one container to minimize

variability among the replicates. Dispense into the appropriate

effluent test chambers.

14.10.2.3 Dilution Water

14.10.2.3.1 Dilution water should be uncontaminated l-~m-'

filtered natural seawater or hypersaline .brine f>repared from

uncontaminated natural seawater plus reagent water (see Section

7, Dilution Water). Natural seawater may be unc~ontaminated

receiving water. This water ~s used in all dilution steps and as

the control water.

14.10.2.4 Reference Toxicant Test

14.10.2.4.1 Reference toxicant tests should be conducted as

described in Quality Assurance (see Section 4.7) .

14.10.2.4.2 The preferred reference toxicant f6r red abalone is

zinc sulfate (ZnS04oH20). Reference toxicant. tests provide an

indication of the sensitivity of the test organisms' and the

suitability of the testing laboratory (see Section 4 Quality

Assurance) . Another toxicant may be specified by the

appropriate regulatory agency. Prepare a 10,000 ~g/Lzinc stock,

solution by adding 0.0440 g of zinc sulfate (Zn~304oH20) to one

liter of reagent water in a polyethylene volumel~ric flask ..

Alternatively, certified standard solutions can'be ordered from·.

commercial companies.

14.10.2.4.3 Reference toxicant solutions shoul<:l be five

replicates each of 0 (control), 10, 18, 32, and 56, and 100 ~g/L

total zinc. Prepare one liter of each concentration by adding 0,

277
1.0, 1.8, 3.2, 5.6, and 10.0 mL of stock solution,'respectively,'

to one-liter volumetric flasks and fill with dilution water.

start with control· solutions and progress to the highest

concentration to minimize contamination.

14.10.2.4.4 If the effluent and reference toxicant tests are to

be run concurrently, then the tests must use embryos from the

same spawn. The tests must be handled in the same way 'and test

solutions delivered to the test chambers at the same time.

Reference toxicant tests must be conducted at 34 ± 2~.

14.10.3 COLLECTION OF GAMETES FOR THE TEST

14.10.3.1 Spawning Induction

14.10.3.1.1 Note: Before beginning the.spawning induction

process, be sure that test solutions will be mixed, sampled, and

temperature equilibrated in time to receive the newly fertilized

eggs. Spawning induction generally takes about three hours, but

if embryos are ready before test solutions are at the proper

temperature, the delay may allow embryos to develop past the onecell stage before transfer to the toxicant. Transfer can then

damage the embryos, leading to unacceptable' test results.

14.10.3.1.2 Culture work (spawning, etc.) and toxicant work

should be done in separate laboratory rooms, and care should be

taken to avoid contaminating organisms prior to testing.

14.10.3.1.3 Ripe abalone can be induced to spawn by s~imulating

the sYnthesis of prostoglandin-endoperoxide in the reproductive

tissues (Morse et al., 1977). This can be done in two ways:

addition of hydrogen peroxide to seawater buffered with Tris

(Morse et al., 1977), or irradiation of seawater with ultraviolet

light (Kikuchi and Uki, 1974). The first method is preferable

for small laboratories because it avoids the cost and maintenance

requirements of a UV system. If a UV system is available, this

method may be preferable because it is simple, does not use

chemicals that could accidentally harm larvae,and is considered

to be less likely to force gametes from unripe adults.

14.10.3.1.4 If brood stock are shipped ~o the laboratory by a

supplier, it is important to allow two days or more for

laboratory acclimation before spawning induction; this should

278
increase the probability of achieving a success,ful spawn of

viable gametes. Always bring brood stock up to' acclimation

temperature slowly to avoid premature spawning.:

14.10.3.2 Hydrogen Peroxide Method

14.10.3.2.1 Select four ripe male abalone and :four ripe females.

Clean their shells of any loose debris. Place the males in one

clean polyethylene bucket and the females in another. Cover the

buckets with a tight fitting perforated lid, supply the chambers

with flowing or recirculating (1 liter/minute) ,20~~m-filtered

seawater (15°C), and leave the animals without ~ood for 24 to 48

hours to acclimate and eliminate wastes. If flowing seawater ii

unavailable, keep the spawners in larger (>30 liter) aquaria with

aeration at 15 ± 1°C for 24 hours without food to elimi~ate

wastes. Three hours prior to the desired spawn'ing time, drain .

the buckets, wipe and rinse out· mucus and debris, and refill with

6 liters of 1 ~m-filtered seawater. If abalone have been kept in

larger aquaria, put them in the buckets at this time. Check the

abalone from time to time to make sure they remain underwater.

Add air stones to. the buckets and keep them aer'ated until

spawning begins.

14.10.3.2.1 Dissolve 12.1 g of Tris into 50 mVof reagent water.

When the Tris has dissolved completely, mix the, hydrogen peroxide

(H2 0 2 ) solution in a separate flask by pouring 10 mL of fresh*

refrigerated H20 2 (30%) into 40 mL of ref:r;-igerated reagent water

(1:5 dilution). Pour 25 mL of Tris solution and 25 mL of H20 2

solution into each of the spawning buckets (male and female) .

Stir well. to mix; the final concentration in the spawni'ng buckets

will be approximately 6 roM Tris (pH = 9.1) and 5 m H20 2 • Allow'

the abalone to remain in contact with the chemicals for 2.5 hours

at 15 ± 1°C. The chemical reaction is temperature dependent

(three hours of contact with H2 0 2 would be necessary at 11°C).

Temperatures higher than 15°C are not recommended for spawning.

Maintain constant aeration. Since females often begin spawning

after the males, it may be useful to induce mal:e spawni.ng 15-30

minutes later, however egg quality should not be compromised if

females spawn first (See 14.10.3.3.2 below).

*Note: Hydrogen peroxide.loses potency over time. 'Purchase

reagent or certified grade H2 0 2 in small cqntainers (100

279
mL). Store unopened containers for no more than one year,

and discard open containers after one month. Mark the

purchase date and opening date on all containers, and keep

all containers refrigerated.

14.10.3.2.3 After 2.5 hours, empty the spawning buckets, rinse

them well, and refill them to the top with fresh dilution water

seawater at the same temperature (15 ± 1°C). Keep the containers

clean by siphoning away mucus and debris. Maintain constant

aeration until spawning begins, then remove the air stones. The

abalone begin spawning about three hours after the introduction

of the chemicals (at 15 ± 1°C). Eggs are dark green and are

visible individually to the naked eye, sperm appear as white

clouds emanating from the respiratory pores.

14.10.3.2.4 If spawning begins before the chemicals have been

removed, drain the buckets immediately, discarding any gametes.

Rinse the buckets thoroughly and refill with clean, dilution

water seawater (15 ± 1°C). Use only the gametes subsequently

spawned in clean water for testing.

14.10.3.3 UV Irradiation Method

14.10.3.3.1 Select four ripe male abalone and four ripe females.

Clean their shells of any debris. Place the males in one clean

polyethylene bucket and the females in another. Cover the

buckets with a tight fitting perforated lid, supply the

containers with flowing or recirculating·(l liter/minute) 20-~m~

filtered seawater (15 ± 1°C), and leave the animals without food

for 24 to 48 hours to acclimate and eliminate wastes. .If flowing

seawater is unavailable, keep the spawners in larger (>30 liter)

aquaria with aeration at 15 ± 1°C for 24 hours. Three hours

prior to the desired spawning time, drain the buckets, wipe and

rinse out mucus and debris, and refill with just enough water to

cover the abalone (which should all be placed in the bottom of

the bucket). Begin slowly filling the buckets with dilution

water seawater (15 ± 1°C) that has passed through the UV

sterilization unit. Flow rates to each of the buckets ·should be

150 mL/min. A low total flow rate (300 mL/minute) in the UV unit

is necessary to permit sufficient seawater irradiation. (The

sterilization unit should be cleaned and the UV bulb replaced at

least once annually). Place the buckets.in a water bath at 15 ±

1°C to counter the temperature increase caused by the slow

280
passage of the water past the UV lamp. Check the containers

periodically, and keep them clean by siphoning out any debris.

After three hours (± about 1/2 hour), abalonestfould be'gin

spawning by ej ecting clouds of gametes into the·'water. Eggs are

dark green and are visible individually to the naked eye, sperm

appear as white clouds emanating from the resp~ratory pores.

14.10.3.3.2 Note: If past experience or other factors indicate

difficulties in achieving synchronous spawning, 'it may be helpful

to induce a second group of females about an hour after the

first. This will increase the chances of providing fresh eggs

(less than one hour old) for fertilization if males spawn late

(see below). Senescence of sperm is. seldom a problem because

males' ~ontinue spawning over a longer pe~iod.of time.

14.10.3.4 Pooling Gametes

14.10.3.4.1 Although it is not necessary, it is preferable to

have more than one abalone of each sex spawn. To increase the

probability of multiple spawners without r~sking senescence of

the gametes, allow one-half hour after the first individual of

the second sex begins to spawn before initiating fertilization.

For example, if males spawn first, wait one~half hour after the

first female spawns before fertilizing eggs. In.most qases this

will provide time for more than one of each sex to spawn. More

important than multiple spawning, however, is av()iding delay of

fertilization. Eggs should be fertilized within one hour of

release (Uki and Kikuchi 1974). All sperm shoulcl be pooled, and

all eggs should be 'pooled prior to fertilization. This can be

accomplished by gentle swirling wi,thin the spawning buckets.

Note: Take care to avoid contaminating eggs with sperm prior to

the intended fertilization time. It is important that .

development is ~ynchro?ous among all test embryos:.

14.10.3.5 Fertiliz~tio~

:.'.. 14.10.3.5.1 As the t"emale~ spa~J:l' allow the eggs to settle to

the bottom. If necessary, ge~tly' stir to even,ly ~istribute the

eggs. Siphon out and discard any eggs that appear clumped

together. Eggs are ready to transfer to a third (fertilization)

bucket when either: (1) one-half hour has p~ssed since the first

individual ,of the second sex has spa~ned (2) multiple individuals

of each sex have spawned, or 3) there are too many eggs on the r . •

281
bottom of the bucket to allow evenly distributed eggs to'avoid

each other. Slowly siphon eggs into a third clean polyethylene

bucket containing one or two liters of dilution water seawater

(15 ± 1°C). Siphon carefully to avoid damaging the eggs and to

avoid collecting any debris from the spawning container. Siphon

about 100,000 eggs, enough to make a single even layer on the

container bottom. Each egg should be individually

distinguishable, and not touching other eggs. If excess eggs are

available, siphon them into a second fertilization bucket to be

used as a reserve. Keep all containers at 15 ± 1°C. Make sure

that water temperatures differ by no more than 1°C when

transferring eggs or sperm from one container to another.

14.10.3.5.2 As the males spawn, siphon sperm from directly above

the respiratory pore and collect this in a 500 mL flask with

filtered seawater. Keep the flask at 15 '± 1°C, and use it as a

back-up in case the males stop spawning. If spawning continues

renew this reserve every 15 minutes. Usually the males will

continue spawning, turning the water in the bucket milky white.

As long as the males continue spawning, partially drain and

refill the bucket every 15 minutes, replacing old sperm-laden

water with fresh seawater (15 ± 1°C). Use the freshest sperm

possible for fertilization.

14.10.3.5.3 Make sure eggs are fertilized within one hour of

release (Uki and Kikuchi, 1974, see note after Section 14.8.5.2)

To fertilize the eggs, collect about 200 mL of sperm-laden water

in a small beaker. The sperm concentration in the beaker does

not have to be exact, just enough to give a slightly cloudy

appearance (approximately 1 to 10 X 106 cells/mL in the

fertilization bucket). See Hahn (1989) for further information

on sperm concentrations and the method for fertilization. Pour

the sperm solution into the fertilization bucket containing the

clean isolated eggs. Usin~a hose fitted with a clean' glass

tube, add dilution water seawater to the fertilization bucket,at

a low flow rate «1 liter/min; 15 ± 1°C). Use the water flow to

gently roil the eggs to allow them to mi~ with the sperm and

fertilize. When the bucket is about half-full and eggs are

evenly mixed, stop the water flow and allow the eggs to settle to

the bottom of the bucket (about 15 minutes)., Fertilization is

then complete .

•

282
14.10.3.5.4 Note: Once fertilized eggs .have' se:ttled to the

bottom of the bucket (15 minutes after addition 'of sperm), the

following steps . (rinsing, concentrating, and cou.nting the

embryos) must prbce~d without delay to assure that embryos are

transferred into the test solutions within about one hour.

Embryos must be delivered to the test chambers before the first

cell division takes place. (Multicellular embryos are more

susceptible to damage in handling, and test 'endpoint analysis , , '

assumes that the first cell division takes place in the toxica~t

solution) .

14.10.3.5.5 After embryos have settled, carefully pour or siphon

off the water from above the s~ttled embryos to remove as much of

the sperm laden Water as possible without losing substantial

numbers of embryos. Slowly refill the bucket with dilution water

seawater (15 ± l°e). Allow the embryos to settle, and siphon

them into a tall 1000 mL beaker for counting. Siphon at a slow,'

flow rate, and move the siphon along the bottom of the bucket

quickly to pick up a large number of embryos in the short amount

of time it takes to fill' the beaker. Examine a 13ample of the

embryos at 100X magnification. One to one hundred sperm should

be visible around the circumference of each embryo, 15 sperm per

egg is optimal. If sperm are so dense t~at the embryos appear

fuzzy (»100 sperm/egg), the abalone may develop abnormaliy and

should' not be used.

14.10.4 START OF THE TEST

14.10.4.1 Prior to Beginning the Test

14.20.4.1.1 The test should begin as soon as possible,

preferably within 24 h of sample collection. The maximum holding

time following retrieval of the sample from the sampling device

should not exceed 36 h for off-site toxicitY,tests unless

permission is granted by the permitting authority:. In no case

should the sample be used in a test more than 72 h after sample

collection (see Section 8 Effluent and Receiving Water Sampling,

Sample Handl,ing, and Sample Preparation for Toxicity Test) .

14.10.4.1.2 Just prior to test initiation (approximately 1 h),

the temperature of a sufficient quantity of the sample to make

283
the test solutions should be adjusted to the test temperature (15

± 1°C) and maintained at that temperature' during the addition of

dilution water.

14.10.4.1.3 Increase the temperature of the water bath, room, or

incubator to the required test temperature (15 ±.l°C).

14.10.4.1.4 Randomize the placement of test chambers in the

temperature-controlled water bath, room, or incubator at the

beginning of the test, using a position chart. Assign numbers

for the position of each test chamber us~ng a random numbers or

similar process (see Appendix A, for an example of

randomization). Maintain the chambers in this configuration

throughout the test, using a position chart. Record these

numbers on a separate data sheet together with the concentration

and replicate numbers to which they corr~spond. Identify this

sheet with the date, test organism, test number, laboratory, and

investigator's name, and safely store it away until after the

abalone have been examined at the' end of the test.

14.10.4.1.5 Note: Loss of the randomization sheet would

invalidate the test by making it impossible to analyze the data

afterwards. Make a copy of the randomization sheet and store

separately. Take care to follow the numbering system exactly

while filling chambers with the test solutions.

14.10.4.1.6 Arrange the test chambers randomly in the water bath

or controlled temperature room. Once chambers have been labeled

randomly and filled with test solutions, they can be arranged in

numerical order for convenience, since this will also ensure

random placement of treatments.

14.10.4.2 Estimation of Embryo Density

14.10.4.2.1 Evenly mix the embryos in the 1000 mL beaker by

gentle vertical stirring with a clean perforated plunger. Never

allow embryos to settle densely in the bottom of the beaker, and

take care not to crush embryos while sti~ring. Take. a sample of

the evenly suspended embryos using a 1 mL wide bore graduated

pipet. Hold the pipet up to the light and count the individual

embryos using a hand counter. Alternatively, empty the contents

of the pipet onto a Sedgewick-Rafter slide and count embryos

under low magnification on a compound scope. Discard the sampled

284
embryos after counting. Density of embryos in t',he beaker should

be between 200 and 300 embryos/mL. Dilute if the concentration

is too high, let embryos settle and pour'off'exc'ess water if

concentration is too low. Take themean6f five samples from

this solution to estimate the number of, embryos per milliliter.

14.10.4.3 Delivery of Fertilized Embryos

14.10.4.3.1' Using the estimated embryo density in the 1000 mL

beaker, calculate the volume of water that contains 1000 embryos.-

Remove 1000 embryos {or less for smaller volumes', see Section

14.10.1.3) by drawing the appropriate volume of ,water from the

well-mixed beaker using a 10 mL wide bore pipet., Deliver the

embryos into the test chambers directly from the pipet making

sure not to touch the pipet to the test solution. Stir the

embryo beaker with the plunger pefore'taking aliquots. The

temperature of the embryo suspension tnus~ be within 1°C of the

temperature of the test solution. (As above, all solutions are

kept at 15 ± 1°C). Record the volume of water delivered into the

test chambers with the embryos. Embryos must be delivered into

the test solutions within one hour of fertilization. Immediately

after the embryos have been delivered, take a sample from, the

embryo beaker and examine it under 100X magnification. All

embryos should still be in the one-cell stage; record any

observations to the contrary on the data ·sheet.

14.10.4.4 Incubation

14.10,.4.4.1 Incubate test organisms for 48 hourfl in the test

chambers' at 15 ± 1°C under 'low lighting (approximately 10

fJ-E/m2/s) with 16L:8D photoperiod. Fertilized embryos become

trochophore larvael. hatch, a;nd develop into veligrer larvae in the

test solutions during the exposure period.

14.10.5 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERA~TURE

14.10.5.1 The light quality and intensity should be at ambient

laboratory conditions. Light intensity should be 10-20 fJ-E/m2 /s,

or 50 to 100 foot candles (ft-c), with a.16 h light and 8 h dark

cycle.

14.10.5.2- The water temperature in the test chambers Should be

,maintained at 15 ± 1°C. If a water bath is used 1:0 maintain the

285
test temperature, the water depth surrounding the test cups

should be as deep as possible without floating the chambers.

14.10.5.3 The test salinity should be in the range of 34 ± 2~.

The salinity should vary by no more than ±2~ among the chambers

on a given day. If effluent and receiving water ~ests are

conducted concurrently, the salinities of these tests should be

similar.

14.10.5.4 Rooms or incubators with high volume ventilation

should be used with caution because the volatilization of the

test solutions and evaporation of dilution water may cause wide

fluctuations in salinity. Covering the test chambers with clean

polyethylene plastic may help prevent volatilization and

evaporation of the test solutions.

14.10.6 DISSOLVED OXYGEN (DO) CONCENTRATION

14.10.6.1 Aeration may affect the toxicity of effluent .and

should be used only as a last resort to maintain 'a satisfactory

DO. The DO concentration should be measured on new solutions at

the start of the test (Day 0) . The DO should not fall below 4.0

mg/L (see Section 8, Effluent and Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity Tests). If

it is necessary to aerate, all treatments and the control should

be aerated. The aeration rate should' not exceed that necessary

to maintain a minimum acceptable DO and under no circumstances

should it exceed 100 bubbles/minute, using a pipet with a i-2 mm

orifice, such as a 1 mL KIMAX® serological pipet No. 37033, or

equivalent.

14.10.7 OBSERVATIONS DURING THE TEST

14.10.7.1 Routine Chemical and Physical ·Observations·

14.10.7.1.1 DO is measured at the beginning of the exposure

period in one test chamber at each test concentration and'in the

control.

14.10.7.1.2 Temperature, pH, and salinity are measured at the

beginning of the exposure period in one test chamber at each

concentration and in the control. Temperature should also be

monitored continuously or observed and recorded daily for at

. 286
least two locations in the environmental control system or the

samples. Tempe~ature ·should be measured in a sufficient number

of test chambers at the end of the test to determine temperature

variation in .the environmental chamber.

14.10.7.1.3 Record all the measurements on the I data sheet.

14.10.8 TERMINATION OF THE TEST

14.10.8.1 Ending the Test

14.10.8.1.1 Record the time the test is terminated.

14;10.8.1.2 Temperature, pH, dissolved oxygen, and salinity are

measured at the end of the exposure period in one ·test ·chamber at

each concentration and in the control.

14.10.8.2 Sample Preservation

14.10.8.2.1 After 48 h9urs exposure, the abalone larvae are

fixed in formalin or glutaraldehyde. The two methods for sample

preservation are described. Be sure that samples for

physicochemical measurements have been taken before further

processing of test solutions.

14.10.8.2.2 At the end of the 48-hour incubation period, remove

each test chamber, swirl the solution to'suspend all the larvae,

and pour the entire contents through a 37 tim-mesh screen. The

test solution is discarded and the larvae are retained on the

screen. Using streams of filtered seawater from,a squeeze

bottle, rinse the larvae from the screen through'a funnel into 25

mL screw cap vials. Be careful not to hit the larvae directly

with the streams of water; rough handling during transfer may

cause fragmentation of the larvae, making counting more difficult I

and less accurate. Add enough buffered formalin to preserve

larvae in a 5% solution (some laboratories have l:~uccessfully

preserved larvae with lower formalin concentrations. Underpreserved larvae disintegrate quickly, however, a~nd whole tests

may have to be rejected if larvae have not been a;dequately

fixed). Addition of formalin is more accurate if the vials are

premarked with lines' showing the volume of sample and the volume

287
of formalin to be added. Alternatively, a 0.05% final

glutaraldehyde solution may be substituted. Larvae should be

counted within two weeks.

14.10.8.2.3 Note: Formaldehyde has been identified as a

carcinogen and both glutaraldehyde and ·f~rmaldehyde are

irritating to skin and mucus membranes. Neither should be used

at higher concentrations than needed to achieve morphological

preservation and only under conditions of maximal ventilation and

minimal opportunity for volatilization into room air.

14.10.8.3 Counting

14.10.8.3.1 To count the larvae using a standard compound

microscope, pipet all the larvae from the bottom of the

preservation vial onto a Sedgewick-Rafter counting cell. Examine

100 larvae from each vial under 100X magnification. To best

characterize the sample and to avoid bias, select groups of

larvae one field of vision at a time, moving to the next field

without looking through the lens. Be careful to work acrOS$ the

slide in one direction to avoid recounting the same areas. Count

the number of normal and abnormal larvae using hand counters.

The percent normal larvae is calculated as the number normal

divided by the total number counted. After counting, use a

funnel to return the larvae to the vial for future reference.

14.10.8.4 Endpoint

14.10.8.4.1 Examine the shape of the larval shell to distinguish

normal from abnormal larvae. Count veliger larvae as normal if

they have smoothly curved larval shells that are striated with

calcareous deposits and are somewhat opaque. It is common for

normal larvae to have a slight curved indentation near the

leading edge of the shell. A single indentation is this area is

counted as normal.

14.10.8.4.2 Larvae with both multiple indentations and an

obvious lack of calcification (i.e. clear appearance in at least

part of the shell) are counted as abnormal. The combination of

these two features indicates inhibition of a biological process

(lack of calcification) and actual damage to the organism

(indentations) allowed by the thin shell. Refer to the

accompanying photographs (Figure 1) for classification of

288
marginally deformed larvae. The following typel:! of larvae are

also counted as abnormal: (1) larvae that have arrested

development (from one cell through trochophOre stage) /·(2) larvae

with obvious severe deformations, (3) larvae wit.h broken shells,. - , -

(4) larval shells separated from the rest of the animal, and(S)

larvae found remaining in the egg membrane (howE~ver/ take care to

distinguish these-from larvae that may have come in contact with

loose egg cases). Record all counts and-the test chamber number

on the data sheet.

14.11 SUMMARY OF TEST CONDITIONS AND TEST ACCE:J:lTABILITY CRITERIA

14.14.1 A summary of test conditions and test acceptability

criteria is listed in Table 3.

14.12 ACCEPT~BILITY OF TEST RESULTS

14.12.1 Test results are acceptable only if all the fOllowing

requirements are met:

(1) the ntean larval normality must be at least 80% in the

controls.

(2) the response from 56 fl9/L zinc treatment must be

significantly different from the control response~

(3) the minimum significant difference (~MSD) is <20%

relative to the control for the refereace toxicant.

14.13 DATA ANALYSIS

14.13.1 GENERAL

14.13.1.1 Tabulate and summarize the data. Caldulate the

proportion of larvae with normally developed shells for each

replicate. A sample set of_ test data is listed i.n Table 4.

14.13.1.2 The statistical tests described here miust be used with

a knowledge of the assumptions upon which the tests are

contingent. -The assistance of a statistician is recommended for

analysts who are not proficient in statistics.

289
FIGURE 1. 48-HOUR-OLD ABALONE VELIGER LARVAE

Figures 1A -D Provided by John Hunt, Institute of

Marine Sciences. Photocopied from:

"Marine Bioassay Project Procedures Manual of October, 1990."

California State Water Resources Control Board.

The following three pages show 12 photographs of 48-hour-old

abalone veliger larvae from effluent toxicity tests. All larvae

were taken from intermediate effluent concentrations and were

chosen to represent "borderline" cases (~.e. larvae that were

slightly affected and are therefore, difficult to categorize as

normal or abnormal). In- most cases, larvae from lower and higher

effluent concentrations are more easily categorized than those

shown here; in the lower concentrations they are obviously

without shell abnormalities and in the higher concentrations they

are severely deformed. These photographs are presented as a

visual reference to help standardize test analysis and eliminate

bias in the interpretation of marginally'deformed larvae. All

larvae on the left-hand side of these pages were counted as

\

normal, all larvae on the right-hand side were counted .as

abnormal.

290
A. Normal larva with well calcified

(striated) shell but slight uneven shell

outline,

C. Normal larva with some shell thinning

and mild flattening ofshell curvature near

the leading edge (left side of photograph),

291

B. Obviously abnorniallarva with transparent

shell and numerous shell deformities.

D', Abnormal larva with multiple slight

indentations and transparency near the

leading edge (left side of photograph)
E. Normallarva with well calcified

(striated) shell but uneven shell outline.

G. Normal larva, anterior (rather than

lateral) view. Well striated, smooth

rounded shell outline.

292

F. Abnormal larva with transparent shell and

large indentation.

H. Abnormal larva, anterior (rather than

lateral) view. Transparent irregular shell with

indentations.
I. Normal larva with well calcified shell and

e small indentation at leading edge.

Three normal larvae, all well calcified with

small indentations at the leading edge.

293

J. Abnormal larva with shell

transparencies, indentations, and

irregular shape.

L. Abnormal larva with arrested development

at an early stage:. Any larva found within the

egg membrane, no matter howell developed, is

counted as abnormal.
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY

CRITERIA FOR, HALIOTIS RUFESCENS, LARVAL DEVELOPMENT

TEST WITH EFFLUENTS AND RECEIV~NG WATERS

1. Test type: Static non-renewal

2. Salinity: 34 ± 2%0 ,

3. Temperature: 15 ± 1°C

I

, 4. Light quality: Ambient laboratory light

I Light intensity: fJ.E/m2 , 5. 10 I s

(Ambient laboratory levels)

6. Photoperiod: 16 h light, 8 h darkness

I 7. Test chamber size: 600 mL

8. Test solution volume: 200 mLIn:ipl icate

9. Larvae density per 5-10 per'mL

i chamber:

10. Ne. replicate chambers 5

I per concentration:

11. Dilution water: Uncontaminated 1-fJ.m-filtered

natural seawater or'hypersaline

brine plus reagent water

; 12. Test concentrations: Effluents: Minimum of 5 and a

control

: Receiving waters: 100% I

receiving water and a control

13. Dilution factor: Effluents: ~0.5

I

Receiving waters: None or ~0.5

,

14. Test duration: 48 h

15. Endpoint: Normal shell development

294
16.

17.

18.

Test acceptability

criteria:

Sampling requirements:

Sample volume required:

~80% normal shell!development in

the controls; must have

statistical significant effect

at 56 Jlg/L zinc; must achieve a.

%MSD of <20% I

One sample colled:ed at test

initiation, and preferably used

within 24 h of the time it is

removed from the sampling

device (see Section 8,

Effluent and Receiving Water

Sampling, Sample Handling, and

Sample Preparation for Toxicity

Tests)

2 L per test

14.13.1.3 The endpoints of toxicity tests using t:he red abalone

. are based on the reduction in proportion of norma:L shell

development. The IC25 is calculated using the Linear

Interpolation Method (see Section 9, Chronic Toxicity.Test

Endpoints and Data Analysis). LOEC and'NOEC values for larval

development are obtained using a hypothesis testing approach such

as Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank

Test (Steel, 1959; Miller, 1981) (see Section 9) .' Separate

analyses are performed for the estimation of the l~OEC and NOEC

endpoints and for the estimation of the IC25. See the Appendices

for examples of the manual computations, and examples of data

input and program output. '

14.13.2 EXAMPLE OF ANALYSIS OF RED ABALONE, HAL~~OTUS RUFESCENS,

LARVAL DEVELOPMENT DATA

14.13.2.1 Formal statistical analysis of the larval development

is outlined in Figure 2. The response used in thE! analysis is

the proportion of larvae with normally developed ~hells in each

test or control chamber. Separate analyses are performed for the

estimation of the NOEC and LOEC endpoints and for the estimation

of the IC25 endpoint. Concentrations at .which there is no normal

shell development in any of the test chambers are lexcluded from

statistical.analysis of the NOEC and LOEC, but inc:luded in the

estimation of the IC25.

295
- - -- -- TABLE 4. DATA FROM RED ABALONE, HALIOTUS RUFESCENS,

DEVELOPMENT TEST

Effluent

Concentration No. Larvae Number proportion

(%) Replicate Counted Normal Normal

Brine A 100 100 1. 00

Control B 100 98 0.98

C 100 100 1. 00

D 100 99 0.99

E 100 99 0.99

Dilution A 100 99 0.9.9

Control B 100 99 0.99

C 100 99 0.99

D 100 100 1. 00

0.56 A 100 99 0.99

B 100 99 0.99

C 100 98 0.98

D 100 100 1.00

E 100 100 1. 00

1.00 A 1.00 99 0.99

B 1.00 1.00 1.00

C 1.00 99 0.99

D 1.00 99 0.99

E 100 100 1. 00

1.80 A 100 99 0.99

B 100 99 0.99

C 100 99 0.99

D 100 98 0.98

E 1.00 97 0.97

3.20 A 100 39 0.39

B 100 57 0.57

C 100 61 0.61

D 100 65 0.65

E 100 80 0.80

5.60 A HO 0 0.00

B 100 0 0.00

C 100 0 0.00

D 100 0 0.00

E 100 0 0.00

10.00 A 100 0 0.00

B 1.00 0 0.00

C 100 0 0.00

D 100 0 0.00

E 100 0 0.00

-

296

- ,I
14.13.2.2 For the case of equal numbers of replicates across all

concentrations and the control, the evaluation of the NOEC and

LOEC endpoints is made via a parametric test, Dunnett's

Procedure, or a nonparametric test, Steells Man;(-one Rank Test,

on the arc sine square root transformed data. Underlying

assumptions of Dunnett's Procedure, normality and homogeneity of

variance, are formally tested. The test for normality is the

Shapiro-Wilkls Test, and Bartlett's Testis useel to test for

homogeneity of variance. If either of these tef~ts fails, the

nonparametric test, Steel's Many-one Rank Test, is used to

determine the NOEC and LOEC endpoint's. If the cissumptions of

Dunnett's Procedure are met, the endpoints are ~stimated by the

parametri,c procedure.

14.13.2.3 If unequal numbers of replicates occur among the

concentration levels tested, there are parametri.c and

nonparametric alternative analyses. The parametric analysis ~s a

t test with the Bonferroni adjustment (see Appendix D). The

Wilcoxon Rank Sum Test with the Bonferroni adjusltment is the

nonparametric alternative.

14.13.2.4 Comparison of Brine and Dilution Controls

14.13.2.4.1 This example uses toxi~ity data from a red abalone,

HaliotuB ~ufeBcenB, larval development test performed with

effluent. The response of interest is the proportion of larvae

with norma,liy developed shells, ·thus each replic,ate must first be

transformed by 'the arc sine square root transformation procedure

described in Appendix B. Because the example test was ·run using

both brine and dilution controls, the two controls must first be

tested for significant differences in the ~ormal, shell

development proportions. The raw and transformed data, means and

variances of the transformed observations for the two controls

are listed in Table 5.

14.13.2.4.2 Tests for Normality

14.13.2.4.2.1 In the two sample situation, the distributional

assumption is that each sample comes from a norm':llly distributed

population. Thus in comparing the brine and dill.ltioncontrols,

the data for each concentration must be separately checked'f()r

normality. When the two response groups are tested separately,

it is not necessary to center the'data.

297
STATISTICAL··

LAR\f

POINTESTIMATION

ENDPOINT ESTIMATE

IC25

HOMOGENEOUSVARIANCe

NO

YES

tTESTWlTH

BONFERRONI

ADJUSTMENT

Figure 2. Flowchart for statistical-analysis of red abalone,

Haliotus rufescens, development data.

298
TABLE 5~ RED AB~ONE, HALIOTUS RUFESCENS, LARVAL

DEVELOPMENT DATA FROM BRINE AND DILUTION

CONTROLS

RAW

ARC SINE

SQUARE R.oOT

TRANSFORMED

Mean (Yi)

Sf

i

Replicate

A

B

C

D

E

A

B

C

D

E

Brine

Control

1.00

0.98

1.00

0.99

0.99

1.521

1.429

1.521

1.471

.1.471

1.483

0.00152

1

Dilution

Control

0.99

0.99

0.99

1.00

1.471

1.471

1.471

1.52·1

·1.484

0.000625

:2

14.13.2.4.2.2 Calculate the denominator, D, of· the statistic

for each control group:

n - 2 D .. L(X.-X)

,£.1

Where·: Xi = the ith centered observation

X = the overall mean of the centered observatio~s

n = the total number of centered observations

14.13.2.4.2.3 For the brine control data,

n = 5

299
x = -1- (7.413) = 1.483

5

D = 0.00609

For the dilution control data,

n = 4

x = _1_ (5.934)

4

D = 0.00191

1.484

14.13.2.4.2.4 Order the observations for each control group from

smallest to largest

X (1) :s: X (2 ) :s: ••• :s: X lnl

where Xli) denotes the ith ordered observation. The ordered

observations for the two groups in this example are listed in

Table 6.

TABLE 6. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO~WILK'S

EXAMPLE

Brine. Control Dilution Control

i . X(il i X lil

1 1.429 1 . 1. 471

2 1.471 2 1.471

3 1.471 3 1.471

4 1.521 4 1.521

5 1.521

300
14.13.2.4.2.5 From Table 4, Appendix B, for the number of

observations, n, obtain the coefficients .al , a v .•. a k where k is

n/2 if n is even and (n-'-1)/2 if n is odd. For the datasets in

this example; n = 5 and k = 2 for the brine control group, and n

= 4 andk = 2 for the dilution control group. The ai values are

listed in Table 7.

TABLE 7. COEFFICIENTS AND DIFFERENCES FOR SfmPIRO-WILK'S

EXAMPLE

i ai Xln - i +1 ) - Xli)

Brine Control Group

1 0.6646 0.092 xes) - X(1)

2 0.2413 0.050 X (4 ) - X( 3 )

Dilution Control Group

1 0.6872 0.050 X(4) - X(1)

2 0.1667 0.000 X(3) - X(2) .

14.13.2.4.2.6 Compute the test statistic, W, fOl:- each group as

follows:

k 2

W = ..!:.[La.(x 1n-i •1 )_x(i»))

D i.l 2

The differences, X 1n- i +l ) - XCi), are listed 'in Table 7. For the

data in the brine example:

W = 1

0.00609

(0.07321)2 0.880

For the data in the dilution example:

W = 1 (0.03436)2

0.00191

0.618

14.13.2.4.2.7 The decision rule for this test is to compare W as

301
calculated in Subsection 2.6 to a critical value found in

Table 6, Appendix B. If 'the computed W is less'than the critical

value, conclude that the data are not normally distributed. For

the data in the brine control, the critical value at a

significance level of 0.01 and n = 5 observations is 0.686.

Since W = 0.880 is greater than the critical value, conclude that

the brine control data are'norma~ly distributed. For the data in

the dilution control, the critical value at a significance level

of 0.01 and n = 4 observations is 0.687. Since W = 0.618 is less

than the critical value, conclude that the dilution control data

are not normally distributed.

14.13.2.4.2.8 Since the dilution control data does not meet the

normality assumption, the Wilcoxon Rank Sum Test will be used to

compare the responses in the two control groups.

14.13.2.4.3 Wilcoxon Rank Sum Test

14.13.2.4.3.1 To perform the Wilcoxon Rank Sum test, combine the

data from the two groups and arrange in order from smallest to

largest. Assign the rands (1, 2, .~., 9) to the ordered

observations with a rank of 1 assigned to the smallest

observation, rank of 2 assigned to the next larger observation,

etc. If ties occur when ranking, assign the average rank to each

tied observation. A table of the ranks is given in Table 8.

TABLE 8. ASSIGNING RANKS TO THE BRINE AND DILUTION CONTROLS

FOR THE WILCOXON RANK SUM TEST

Rank

1

4

4

4

4

4

8

8·

8

Transformed

Proportion

Normal

1.429

1.471

1.471

1.471

1.471

1.471

1.521

1.521

1.521

Control Group

Brine

Brine

Brine

Dilution

Dilution

Dilution

Brine

Dilution

Dilution
I

14.13.2.4.3.2 The ranks are·then summed for both of the control

groups. For this data, the sum of the ranks in it-he brine control

group is 25 and the sum of' the ranks in the dilut.ion control

group is 20.

14.13.2.4.3.3 For ·this· situatioIh we wish to o.el:ermine if the

proportions of normally developed larvae 'in the two control

grouPE? are significantly different. To do this, jcompare the rank

sum of the group with the smaller sample size with some "minimum"

or critical· rank sum, at or below which the devlopment in the

controls would be considered significantly different.· At a

significance level of 0.05, the minimum rank sum in a test with

five replicates in one group and and four replica.tes in the· other

is 11 (See Snedecor and Cochran, 198D).

14.13.2.4.3.4 The dilution control sample size is smaller than

the sample size of the brine control group so its rank sum is

compared to the critical value. Since its rank sum of 20 is

greater than the critical value of 11', conclude t;hat the

development proportions for the two control groups are not

significantly different.

14.13.2.5 Example of Analysis of Larval Development Data

14.13.2.5.1 Since the responses in the two cont±'ol groups are

not significantly different, only the dilution control group will

be used in the analysis of the shell development 'responses for

the effluent concentrations. As above, each repl'icate must first

be transformed by the arc sine square root transformat~on

procedure described in Appendix B. The raw and transformed data,

means and variances of the transformed observations at each

effluent concentration and dilution control are Listed in

Table 9. The data are plotted in Figure 3. Since there is 100%

: abnormality in all replicates for the 5.6% and 1Q.0%

concentrations, they are not included in the stat~stical analysis

and are considered qualitative abnormality effects.

14.13.2.6 Test for Normality

14.13.2.6.1 The first step of the test for normality is to

center the observations by subtracting the mean of all

303
observations within a concentration from each observation in that

concentration. The centered observations are summarized in

Table 10.

14.13.2.6.2 Calculate the denominator, D, of the statistic:

Where: Xi = the ith centered observation

X = the overall mean of the centered observations

n = the total number of c~ntered observations

14.13.2.6.3 For this set of data, n = 24

x = __1__ (-0.004) = 0.000

24

D :0 0.1127

14.13.2.6.4 Order the centered observations from smallest to

largest

where XCi) denotes the ith ordered observation. The ordered

observations for this example are listed in Table 11.

14.13.2.6.5 From Table 4, Appendix B, for the number of

observations, n, obtain the coefficients a l , a 2 , ••• ak where k is

n/2 if n is even and (n-1)/2 if nis odd. For the data in this

example, n = 24 and k = 12. The ai values are listed in

Table 12.

14.13.2.5.6 Compute the test statistic, W, as follows:

304
EFFLUENT CONCENTRATION (%)

* * * NMULJIII'IlJl:ATE......

---- CClMCf1;1lt!IoWINWJ,lI!PCRIJlICttCOlC8ll'M'I'I:IN

1.0

0.9

0.8

.....J

« 0.7 :::::

0:::

0 0.6 z

z

0 0.5 -

f0::: LV 0 0.4

a 0-

Ol 0

0::: 0.3 0-

0.2

0.1

0.0

0.00 0.56

- ---------

1.00 1:80

*

*

*

3.20 5.60 10.00

Figure 3. Plot of mean proportion of normally developed red

abalone, Haliotus rufescens larvae.
TABLE 9. RED ABALONE r HALIOTUS RUFESCENSr SHELL DEVELOPMENT

DATA

Effluent Concentration (%)

Dilution

Replicate Control 0.56 1.00· 1.80 3.20 5.6 10.0

A 0.99 0.99 0.99 0.99 0.39 0 0

RAW B 0.99 0.99 1. 00 0.99 0.57 0 0

C 0.99 0.98 0.99 0.99 0.61 0 0

D 1.00 1. 00 0.99 0.98 0.65 0 0

E 1.00 1.00 0.97 0.80 0 0

ARC SINE A 1.471 1.471 1.471 1.471 0.674

SQUARE ROOT B 1.471 1.471 1.521 1.471 0.856

TRANSFORMED C 1.471 1.429 1.471 1.471 0.896

D 1.521 1. 521 1.471 1.429 0.938

E 1.521 1.521 1.397 1.107

Mean (Yi ) 1.484 1.483 1.491 1.448 0.894

Sl 0.000625 0.001523 0.000750 0.001137 0.024288 - i 1 2 3 4 5 6 7

TABLE 10. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S

EXAMPLE

Effluent Concentration (%)

Replicate Control 0.56 1. 00 1. 80 3.20

A -0.013 -0.012 -0.020 0.023 -0.220

B -0.013 -0.012 0.030 0.023 -0.038

C -0.013 -0.054 -0.020 0.023 0.002

D 0.037 0.038 -0.020 -0.019 0.044

E 0.038 0.030 -0.051 0.213

\

306
TABLE· 11. ORDERED CENTERED OBSERVATIONS FOR SHAPIROWILK'S EXAMPLE

i

1

2

3

4

5

6

7

8

. Xli)

-0.220

-0.054

-0.051

-0.038

-0.020

-0.020

-0.020

-0.019

i

13

14

15

16

17

18

19

20

Xli)

-0.012

0.002

0.023

0.023

0.023

0.030

0.030

0.037

TABLE 12. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S

EXAMPLE

i ai x(n-i+1) - Xli)

1 0.4493 0.433 X(24) - X(1)

2 0.3098 0.098 X(231 - X(21

3 0.2554 0.089 X(22) - X(3)

4 0.2145 0.076 X(21) - X(4)

5 0.1807 0.057 . X I2O ) - Xes)

6 0.1512 0.050 X(19) - X(6)

7 0.1245 0.050 Xl 1S1 - X(7)

8 0.0997 0.042 X(17) .;. 'X(S)

The differences, Xln-i+~) - Xli), are listed in Table 12. For the

data in this example:

W = 1

0.1127

(0.2974)2 = 0.7848

307
14.13.2.5.8

normality,

Adjustment

14.13.2.5.7 The decision rule for this test is to compare W as

calculated in 14.13.2.5.6 to a critical ~ralue found in Table 6,

Appendix B. If the computed W is less than the critical value,

conclude that the data are not normally distributed. For the

data in this example, the critical value at a signific~nce level

of 0.01 and n = 24 observations is '0.884. Since W = 0.7848 is

less than the critical value, conclude that the data ar.e not

normally distributed.

Since the data do not meet the assumption of

the Wilcoxon Rank Sum Test with the Bonferroni

will be used to analyze the shell development data.

14.13.2.6 Wilcoxon Rank Sum Test with the Bonferroni ~djustment

14.13.2.6.1 For each control and concentration combination,

combine the data and arrange the observations in order of size

from smallest to largest. Assign the ranks (I, 2, ... , 9) to

the ordered observations with a rank of 1 assigned to the

smallest observation, rank of 2 assigned to the next larger

observation, etc. If ties pccur when ranking, assign the average

rank to each tied observation.

14.13.2.6.2 An example of assigning ranks to the combined data

for the control and 0.56% concentration is given in Table 13.

This ranking procedure is repeated for e~ch control/concentration

combination. The complete set of rankings is summarized in

Table 14. The ranks are then summed for each concentration

level, as shown in Table 15.

14.13.2.6.3 For this example, determine if the survival in any

of the concentrations is significantly lower than the survival in

the control. If this occurs, the rank sum at that concentration

would be significantly lower than the rank sum of the control.

Thus compare the rank sums for the survival at each of 'the

various concentration levels with some "minimum" or cri,tic,al rank

sum, at or below which the survival would be considered

significantly lower than the control. At a significance level of

0.05, the minimum rank sum in a test with four concentrations

(excluding the control), four control replicates and five

concentration replicates is 15 (See Table 5, Appendix F) .

308
TABLE 13. i

ASSIGNING RANKS TO THE CONTROL 'AND 0.56%

CONCENTRATION LEVEL FOR THE WILCOXON RANK SUM

TEST WITH THE BONFERRONI ADJUS1MENT

Transformed

Proportion

Rank Normal Concentration

1 1.429 ,,0.56 %"

4 1.471 0.56 %"

4 1.471 0.56 %.J

4 1.471 Control

4 1.471 Control

4 1.471 Control

8 1.521 0.56 %"

8 1.521 0.56%"

1. 521 Control

TABLE 14. TABLE OF RAN:KS1

Effluent Concentration (%")

Replicate Control 0.56 1. 00 L80 3.20

1 1.471(4,3.5,5.5,7) 1.471 (4.) 1.471(3.5) 1.471(5.5) 0.674(1)

2 1.471(4,3.5,5.5,7) 1.471 (4) 1. 521 (8) 1.471(5.5) 0.856(2)

3 1.471(4,3.5,5.5,7) 1. 429 (1) 1.471 (3.5) 1.471(5.5) 0.896(3)

4 1.521·(8,8,9,9) 1. 521 (8) 1.471(3.5) 1.429(2) 0.938(4)

5 1.521(8) 1.521(8) 1. 397 (1) 1.107 (5)

iControl ranks are given in the order of the concentration with which

they were ranked~

309
TABLE 15.

Concentration

(% Effluent)

0.56

1.00

1. 80

RANKSUM8

Rank Sum

25.0

26.5

19.5

14.13.2.6.4 Since the rank sum for the 3.20% concentration level

is equal to the critical value, the proportion normal in that

concentration is considered significantly less than that in the

control. Since no other rank sum is less than or equal to the

critical value, no other concentration has a significantly lower

proportion normal than the control. Hence, the NOEC and the LOEC

are 1.80% and 3.20%, respectively.

14.13.2.7 Calculation of the ICp

14.13.2.7.1 The shell development data in Table 4 are utilized

in this example. As can be seen from Table 4 and Figure 4, the

observed means are not monotonically non-increasing with respect

to concentration (mean response for each higher concentration is

not less than or equal to the mean response for the previous

concentration and the responses between concentrations do not

follow a linear trends). Therefore, the'means are smoothed prior

to calculating the IC. In the following discussion, the observed

means are represented by'Y i and the smoothed means by Mi.

14.13.2.7.2 Starting with the control mean, Y1 = 0.993 and Y 2 -

0.992, we see that Y1 > Y2 • Set M1 = Y1 • Comparing Y2 to Y 31 Y 2< Y 3·

14.13.2.7.3 Calculate. the smoothed means:

14.13.2.7.4 Since Y 7 = 0 < Y 6 = 0 < Y s = 0.604 < Y4 = 0.984 < Y 3

... 0.993, set M3 = 0.993, M4 = 0.984, Ms = 0.604, M6 =0, and set M7

... o.

310
IS.. 10.00

*

---,..-

*

* * *---

1.00 1.10 UO

EFFLUENTCONCEN'TRATION ('I)

I I -r- I , $

1.0

• .1

•.1

...J

-« 0.7

:::I

0:::

0 0.1 z

z

0 0.1 -

....

0::: W 0 OA .... a. t-& 0

a::

a. •.3

0.2

•.1

0.0 ,..

0.00 0.•

Figure 4. Plot of raw data, observed means, and smoothed means for red

abalone, Haliotus rurescens, development data from Tables 4 and 16.
TABLE 16. RED ABALONE, HALIOTUS RUFESCENS, MEAN SHELL

DEVELOPMENT RESPONSE AFTER SMOOTHING

Response Smoothed

Effluent Means, Y i Means, Mi

Cone. (%) i (proportion) (proportion)

Control 1 0.993 0.993

0.56 2 0.992 0.993

1.00 3 0.994 0.993

1.80 4 0.984 0.984

3.20 5 0.604 0.604

5.60- 6 0.000 0.000

10.00 7 0.000 0.000

14.13.2.7.5 Table 16 contains the response means and smoothed

means and Figure 4 gives a plot of the smoothed response curve.

14.13.2.7.6 An IC25 can be estimated using the Linear

Interpolation Method. A 25% reduction in mean proportion of

fertilized eggs, compared to the controls, would result in a mean

proportion of 0.745, where M1 (1-p/100) = 0.993(1-25/100).

Exa~ining the means and their associated concentrations

(Table 16), the response, 0.745, is bracketed by C4 = 1.80%

effluent and Cs = 3.20% effluent.

14.13.2.7.7 Using the equation from Section 4.2 in Appendix L,

the estimate of the IC25 is calculated as follows:

(C -C) ICp. Cj

+[M1 (l-p/lOO)-Mj J (j-1) j

(MU•

1 )-Hj )

IC25 = 1.8 + [0.993(1 - 25/100) - 0.984] (3.2 1.8i

= 2.68%.

(0.604 - 0.984)

14.13.2.7.8 When the ICPIN progrclm was used to analyze this set

of data, requesting 80 resamples, the estimate of the IC25 was

2.6818%. The empirical 95.0% confidence interval for the true

312
mean was 2.5000% to 3.1262 %. The computer program output for

the IC25 for this data set is shown in Figure 5.

14.14 PRECISION AND ACCURACY

14.14.1 PRECISION

14.14.1.1 Single-Laboratory Precision

14.14.1.1.1 Data on the single laboratory prec1s10n of the

Haliotis rurescens larval development method using zinc ~ulfate

are shown in Table 17. Zinc concentrations were 18, 32, 56, and

100 ~g/L. All tests were conducted at the Marine Pollution

Studies Laboratory. There was good agreement among test ECSOs,

with a coefficient of variation of 8%.

14.14.1.2 Multi-laboratory Precision

14.14.1.2.1 The multi-laboratory data indicate a similar level

of test precision Table 18. Data are presented for four

interlaboratory trials in which either two or three laboratories

tested both split effluent samples and reference toxicants. The

mean coefficient of variation between EC50 values from different

laboratories was 15%.

14.14.2 ACCURACY

14.14.2.1 The accuracy of toxicity tests cannot be determined.

313
Cone. ID 1 2 3 4 5 6 7

Cone. Tested o .56 1 1.8 3.2 5.6 10

Response 1 .99 .99 .99 .99 .:s9 0 0

Response 2 .99 .99 1.00 .99 .57 0 0

Response 3 .99 .98 .99 .9!} 61 0 0

Response 4 1.00 1.00 .99 .98 .65 0 0

Response 5 1.00 1.00 .97 .80 0 0

----------------------------------------------- *** Inhibition Concentration percen'.:agd Estimate "'** Toxicant/Effluent: Effluent

Test Start Date: Test Endin£] Dat :

Test Species: Red Abalone

Test Duration: 48 hours

DATA PILE: abalone. icp

OOTPl1r PILE: abalone. i25

Cone. Humber Concentr;l' .ion Response Std. Pooled

ID Replicates , Means Dev. Response Means

---------------------_._-------~----------------~-----------------------

1 4 0.000 0.993 0.005 0.993

2 5 0.560 0.992 0.008 0.993

3 -5 1.0:>(} 0.994 0.005 0.993

4 5 1.<:11) 0.984 0.009 0.984

5 5 3.20J 0.604 0.148 0.604

6 5 5.500 0.000 0.000 0.000

7 5 10.000 0.000 0.000 0.000

The Linear Interpolation EstiJ~te: 2.6818 Entered P Value: 25

2.7085

Lower:

Iwwer:

0.27

Humber of Resamplings: 80

The Bootstrap Estimates Mean:

original Confidence Limits:

Expanded Confidence Limits:

Resampling time in S~conds:

Standard Deviation: 0.1510

2.5000 upper: 3.1262

2.4091 upper: 3.3484

Random_Seed: -770872716

FigurE; 5. ICPIH program output for the IC25.

314
TABLE 17. SINGLE LABORATORY PRECISION DATA FOR THE RED ABALONE,

HALIOTIS RUFESCENS LARVAL DEVELOPMENT 'rEST WITH ZINC

(ZN ~G/L) SULFATE AS A REFERENCE TOXI~\NT .

Test Date NOEC (jlg/L) EC50 (~tg/L)

March 1990 32 42 1 I

- May 1990 32 391

January 1991 18 341

February 1991 18 402

Mean 38.4

SD 3.0

CV (%) 7.8

1

2

Source:

Source:

Hunt et al., 1991

Anderson et al., 1994

315
TABLE 18. MULTI-LABORATORY PRECISION OF THE RED ABALONE, HALIOTIS

RUFESCENS LARVAL DEVELOPMENT TEST PERFORMED WITH ZINC

(ZN ~G/L) SULFATE AND EFFLUENT (%) AS THE TOXICANTS

Test Date Toxicant Lab NOEC EC50 CV

March 1990 Effluent A >3.2% nc

I

March 1990 Effluent B >3.2% nc

March 1990 Effluent C 0.32% 1.83 nc

March 1990 Zinc A 32 41

March 1990 Zinc B 18 28 ,

March 1990 Zinc C 18 31 20%

! May 1990 Effluent A 3.2% 4.7

May 1990 Effluent D 1.8% 3.5

, May 1990 Effluent C 3.2% 3.8 1.6% i

May 1990 Zinc A 32 39

May 1990 Zinc D 32 46 ('

,

May 1990 zinc C 32 37 1.2%

,

January Effluent A <0.56% 1.5

1991

January Effluent C 1..25% 1.8 1.3%

1991

January Zinc A 1.8 34

1991.

, January Zinc C 32 48 24%

1991

I January Effluent A 1..0% 2.7

1991

January Effluent C 1..8% 2.8 3.0%

1991 -

Mean Interlaboratory CV = 1.5%

CV values could be calculated.

Interlaboratory CV based on 6 tests for which

Source: Hunt et al., 1.991..

nc s indicates that the CV could not be Calculated because only one laboratory

observed a 50% effect and calculated an EC50.

316
APPENDIX I. RED ABALONE TEST: STEP-BY-STEP SUMMARY

PREPARATION OF TEST SOLUTIONS

A. Determine test concentrations and appropria.te. dilution water

based on NPDES permit conditions and guidance from the

appropriate regulatory agency.

B. Prepare effluent test solutions by diluting; well mixed

unfiltered effluent using volumetric flasks. and pipettes;

Use hypersaline brine where necessary to maintain all test

solutions at 34 ± 2~. Include brine controls in tests that

use brine.

C. Prepare a zinc reference toxicant st6ck s6lution (10,000

~g/L) .by adding 0.0440 g of zinc sulfate (ZhS04 0 7H20) to 1

liter of reagent water.

D. Prepare zinc reference toxicant solution of 0 (control) 10,

18, 32, 56 and 100 ~g/L by.adding 0, 1.0 1.8, 3.2, 5.6 and

10.0 mL of stock solution, respectively, to a 1-L volumetric

flask and filling to 1-L with dilution water.

E. Sample effluent and reference toxicant solutions for

physical/chemical analysis. Measure salini·ty, pH and

dissolved oxygen from each test concentration.

F. Randomize numbers for test chambers and record the chamber

numbers with their respective test concentditions on a

randomization data sheet. Store the data sheet safely until

after the test samples have been analyzed.

G. Place test chambers in a water bath or enviJronmental chamber

set to 15°C and allow temperature to equilibrate.

H. Measure the temperature daily in one random replicate (or

separate chamber) of each test concentration. Monitor the

temperature of the water bath or environmental chamber

continuously.

I. At the end of the test, measure salinity, pl1, and dissolved

oxygen concentration from each test concentration.-

317
PREPARATION AND ANALYSIS OF TEST ORGANISMS

A. Obtain test organisms and hold or condition as necessary for

spawning.

B. Induce four male and four female abalone to spawn using

either H202 and Tris or UV irradiated seawater (300 mL/min

flow rate through the UV unit). All solutions should be .

maintained at 15 ± laC.

c. Siphon eggs into a fertilization bucket. Add 200 mL of

sperm-laden water to fertilize the eggs. Wash the

fertilized eggs at least twice by slowly decanting and

refilling the chamber with fresh filtered seawater.

Temperatures should vary by no more than laC between waters

used in mixing and refilling.

D. Suspend the embryos evenly in a 1000 mL beaker and count

five samples in a 1 mL pipet 'to estimate embryo density.

E. Pipet 1000 fertilized embryos into each 200 mL test chamber.

Be sure temperatures in the embryo b~aker and the solutions

are at 15 ± laC. Incubate for 48 h. For smaller-sized

chambers, use proportionately fewer embryos.

F. At the end of the 48 h period, pour-the entire test solution

with larvae through a 37 ~m~meshed screen. Wash larvae from

the screen into 25 mL vials. Add buffered formalin to

preserve the larvae in a 5% solution or glutaraldehyde for a

0.05% solution. Cap the flask and invert gently to mix.

G. Pipet a sample from each vial onto· a Sedgwick-Rafter

counting slide and count 100 larvae.. Return the larvae to

the vials for future reference.

H. Count the number of normal larvae for each replicate and

divide by the total counted.

I. Analyze the data.

J. Include standard reference toxicant point estimate values in

the standard quality control charts.

318
Salinity Adjustment Worksheet for Abalone

Date Sampled:

Date Adjusted:

Batch:

Region:

VS (TS-SS)

(SB -TS)

SS = Salinity of Sample

VS = Volume of Sample

TS = Target Salinity (34±2%)

VB = Volume of Brine

SB = Salinity <>f Brine

VDW = VBL - VES VDW = Volume of Dilution Water (Adjusted to 34±2%)

VBL = Largest Volume of Brine added to adjust salinity I '

VBS = Volut:ne of Brine added to each Sa~ple

Total Volume = VB added +VDW added

(Total volume should be the same for 'all samples)

Imtlals: Double Checked:

, PrecIsIon and

Accuracy for :SIte coae (lU urg H) Imtlal Vol. ot VoI.UIl. Total , l'mal Refractometer or concentration Salinity TS Brine Water Volume Salinity

,34±2

j4±:l

, j4±:l

34±2

34±2

±

±

±

±

j4±:l

34±2

l4±:l

l4±2

± "

±

±

±

l4±2

:;4±:l

34±2

j4±:l

:;4±:l

34±2

j4±:l

j4±:l

:;4±:l

34±2

j4±:l

:;4±:l ..

319
Data Sheet for Mollusc Larval Development Toxicity Test

Test Start Date:

Test End Date:

Reference

Toxicant:Reference

Toxicant:

Start Time:

End Time:

Mollusc Species:

Collection!Arrival Date:

Broodstock Source:

Sample Source:

Sample Type: Effluent Ref Tox Solid Elutriate Pore Water WaterSample Type: Effluent Ref Tox

Solid Elutriate Pore Water Water

Test ~tal1on Arter 4lS hours

Cont. Code or Sample Normal Abnormal Notes

1/ Concentration !D# Larvae Larvae

.

I.:OmpUICr U3la IlloragCl.:omputcr Noles

DalJ:Slorage

Disk:

File:

320
SECTION 15

PURP~E URCHIN, Strongylocentrotus purpuratus

AND SAND DOLLAR, Dendrast(:lr excentricus

LARVAL DEVELOPMENT TEST METHOD:

Adapted from a method developed by

Steven Bay and Darrin Greenstein

Southern California Coastal Water Research Project

Westminster, ·CA 92683

TABLE OF CONTENTS

Appendix' I

~5.~

~5.2

~5.3

~5.4

~5.5

15.6

~5.7

~5.8

~5.9

~5.~O

1.5.11

~5.12

~5.~3

15.14

Scope and Application

Summary of Method

Interferences

Safety

Apparatus and Equipment

Reagents and Supplies

Effluents and Receiving Water Collection,'

Preservation, and Storage

Calibration and Standardization

Quality Control

Test Procedures

Summary of Test Conditions and Test

Acceptability Criteria

Acceptability of Test Results

Data Analysis

Precision and Accuracy

Step-by Step Summary

321
SECTION 15

SEA URCHIN, Strongylocentrotus purpuratus

AND SAND DOLLAR, Dendraster excentricus

LARVAL DEVELOPMENT TEST

15.1 SCOPE AND APPLICATION

15.1.1 This method estimates the chronic toxici~y of, effluents

and receiving waters to the developing embryos of the purple sea

urchin, Strongylocentrotus purpuratus, and the sand dollar,

Dendraster excentricus, during a 72-h static non-renewal

exposure. The effects include the synergistic, antangonistic,

and additive effects of all chemical, physical, and biological

components which adversely affect. the physiological a~d

biochemical functions of the test organisms.

15.1.2 Detection limits of the toxicity of an effluent or

chemical substance are organism dependent.

15.1.3 Brief excursions in toxicity may not be detected using

24-h composite samples. Also, because of thE! long sample

collection period involved in composite sampling and because the

test chambers are not sealed, highly volat:i,.le and higbly

degradable toxicants in the source may not be detected in the

test.

15.1.4 This test is commonly used in one of two forms: (1) a

definitive test, consisting of a minimum-of five effluent

concentrations and a control, and (2) a receiving water test(s),

consisting of one or more receiving water concentrations and a

control.

15.1.5 This method should be restricted to use by, or ,under the

supervision of, professionals experienced in aquatic toxicity

testing. Specific experience with any toxicity test is usually

needed before acceptable results become routine.

15.2 SUMMARY OF METHOD

15.2.1 The method provides the step-by-step instructions for

322
performing a 72-h static non-renewal test using the early

development of the purple sea urchin, StJ;'ongylocentrotus

purpuratus, and the sand dollar, Dendraster excE?ntricus, to

determine the toxicity of substances ·in marine smd estuarine

waters. The test endpoint. is normal larval development and may·

include mortality if modified for total counts a.t test initiation

and termination.

15.3 INTERFERENCES

15.3.1 Toxic substances may be introduced by contaminants in

dilution wat·er, glassware, sample hardware, and testing equipment

(see Section 5, Facilities, Equipment;:; and Supplies) .

15.3.2 Improper effluent sampling and handling may adversely

affect test results (see Section 8, Effluent and, Receiving Water

Sampling and Sample Handling, and Sample Preparation for Toxicity

Tests) .

15.4 SAFETY

15.4.1 See Section 3~ Health and Safety.

15.5 APPARATUS·AND EQUIPMENT

15.5.1 Tanks, trays, or aquaria -- for holding and acclimating

adult sea urchins and sand dollars, e.g., standard salt water

aquarium or Instant Ocean Aquarium (capable of maintaining

seawater at 10-20°C), with appropriate filtration' and aeration

system.

15.5.2 Air pump, air lines, and air stones for aerating water

containing broodstock or for supplying air to tes,t solutions with

low dissolved oxygen.

15.5.3 Constant temperature chambers or water baths -- for

maintaining test solution temperature and keeping dilution water

supply, gametes, and embryo stock suspensions at test temperature

(15°C). prior to the test.

323
15.5.4 Water purification system -- Millipore Super-Q, Deionized

water (DI) or equivalent.

15.5.5 Refractometer

15.5.6 Hydrometer(s)

for determining·salinity.

for calibrating refractometer.

15.5.7 Thermometers, glass or electronic, laboratory grade

for measuring water temperatures.

15.5.8 Thermometer, National Bureau of Standards.Certified (see

USEPA METHOD 170.1, USEPA, 1979) -- to calibrate laboratory

thermometers.

15.5.9 pH and DO meters -- for routine physical and chemical

measurements.

15.5.10 Standard or micro-Winkler apparatus -~ for determining

DO (optional) and calibrating the DO meter.

15.5.11 Winkler bottles -- for dissolved oxygen determinations.

15.5.12 Balance -- Analytical, capable of accurately weighing to

0.0001 g.

15.5.13 Fume hood -- to protect the analyst from effluent or

formaldehyde fumes.

15.5.14 Glass stirring rods for mixing test solutions.

15.5.15 Graduated cylinders Class A, borosilicate glass or

non-toxic plastic labware, 50-1000 mL for making test solutions.

(Note: not to be used interchangeably for gametes or embryos and

test solutions) .

15.5.16 Volumetric flasks -- Class A, borosilicate glass or nontoxic plastic labware, 100-1000 mL for making test solutions.

15.5.17 Pipets, automatic -- adjustable, to cover a range of

delivery volumes from 0.010 to 1.000 mL.

15.5.18 Pipet bulbs and fillers -- PROPIPET@ or equivalent.

324
15.5.19 Wash bottles - - for reagent water,for i:opping off

graduated cylinders, for rinsing small glassware and instrument·

electrode~ and probes.

15.5.20 Wash bottles -- for dilution water.

15.5.21 20-liter cubitainers or polycarbonate welter cooler jugs

-- for making hypersaline bri~e.

15.5.22 Cubitainers, beakers, or similar chambers of non-toxic

composition for hblding, mixing, and dispensing dilution water

and other general non-effluent, non-toxicant 'cont.act uses ..' These'

should be clearly labeled and not used for other 'purposes.

15.5.23 Beakers, 5-10 mL borosilicate glass -- for collecting

sperm from sand dollars.

15.5.24 Beakers, 250 mt borosilicate glass -- for preparation of

test solutions.

15.5.25

support

eggs.

Beakers, lob mt borosilicate glass -- fo~ spawning; to

sea urchins and to collect sea urchin and sand dollar

15.5.26 Beakers, 1,000 mL borosilicate ~lass -- for rinsing and

settling sea urchin eggs.

16.5.27 Vortex mixer -- to mix sea urchin semen. in tubes prior

to sampling.

15.5.28 Compound microscope _~ for examining gametes, counting

sperm cells {200-400x),'eggs and embryos and (100~), and

examining larvae. Dissecting scopes are·sometimes used to count

eggs at a lower magnification. ,One piece of equipment worthy of

a special mention is an inverted microscope. The: use ~f an

inverted scope is not required, but recommended. Its use reduces

the exposure of workers to hazardous fumes (formalin Or

glutarqldehyde) during the counting of larvae and reduces sample

examination time. .Alternatively, a Sedgewick-'Raft:'er cell may. be

used on a regular compound scope.

15.5.29 Counter, two unit, 0-999 -- for recordin~J sperm, egg,

embryo, and larval counts.

325
15.5.30 Sedgwick-Rafter counting chamber -- for counting egg and

embryo stock and examining larval development at the end of the

test.

15.5.31 Centrifuge tubes, test tubes, or vials -- for holding

semen.

15.5.32 Hemacytometers, Neubauer ~- for cou~ting sperm.

15.5.33 Siphon hose (3 mm i.d.) -- for removing wash water from

settled eggs.

15.5.34 Perforated plunger for maintaining homogen~ous

distribution o~ eggs and embryos during sampling and distribution

to test chambers.

15.5.35 Enamel or plastic tray -- for optional spawning

platform.

15.5.36 Nitex@ screening (O.5mm mesh) -- cleaning egg solutions.

15.5.37 60 ~m NITEX® filter -- for filtering receiving waters.

15.6 REAGENTS AND SUPPLIES

15.6.1 Sample containers -- for sample shipment and storage (see

Section 8, Effluent and Receiving Water Sampling, and Sample

Handling, and Sample Preparation for Toxicity Tests) .

15.6.2 Data sheets (one set per test) -- for data recording (see

Figures 1-4).

15.6.3 Tape, colored

containers.

for labelling te.st chambers and

15.6.4 Markers, water-proof -- for marking containers,' etc.

15.6.5 Parafilm -- to cover graduated cylinders and vessels

containing gametes and embryos.

15.6.6 Gloves, disposable -- for personal protection from

contamination.

326
15.6.9 Coverslips -- for microscope slides.

15.6.7 Pipets, serological -- 1-10 mL, graduated.

15.6.8 Pipet tips -- for automatic pipets. ~: pipet tips for

handling semen should be cut off to produce an opening about 1 mm

in diameter; pipet tips for handling eggs should be cut off to

produce an opening about 2 mm in diameter. This is necessary to

provide smooth flow of the viscous semen, accurate sampling of

eggs, and to prevent injury to eggs passing through a restricted

opening. A clean razor blade can be used to trim pipet tips'.

I

I

.

15.6.10 Lens paper - - for cleaning microscope opitics.

15.6.11 Laboratory tissue wipes for 'cleaning and drying

electrodes, microscope slides, etc.

15.6.12 Disposable countertop covering -- for protection of work

surfaces and minimizing spills and contamination. I

I

15.6.13 pH buffers 4, 7, and 10 (or as per instructions of

instrument manufacturer) -- f6r standards and calibration check

(see·USEPA Method 150.1, USEPA, 1979). !

,

15.6.14 Membranes and filling solutions -- for dissolved oxygen

probe (see USEPA Method 360 ..1, USEPA, 1979), or rE~agents for

modified Winkler analysis. !

15.6.15 Laboratory quality assurance samples and:standards

for the above methods.

15.6.16 Test chambers -- 30-mL glass scintillation vials with

polypropylene caps, four chambers per concentration.

,

15.6.17 Formaldehyde,10%, in seawater -'- for preserving larvae.

Note: formaldehyde has been identified as a carcinogen and is

irritating to skin and mucous membranes. It should not. be used

at a concentration higher than necessary to achieve morphological

preservation of larvae for counting and only undei.conditions of

maximal ventilation and minimal opportunity for volatilization

into room air.

15.6.18 Glutaraldehyde, 1% in seawater -- for preserving larvae.

327·
Figure 1. Sample experiment set-up sheet.

SEA URCHIN EMBRYO DEVELOPMENT TEST

EXPERIMENT DESCRIPTION

Bioassay No.------ Investigator _

Start Date __

End Date------

Start time _

End Time'-----

Sample Sample Concentration Other Information

Numbers

•

328
Figure 2. Sample worksheet for urchin spawning information.

SEA URCHIN DEVELOPMENT TEST

SPAWNING WORKSHEET

Bioassay no. _

Spawning

Date

No. Injection Sex Accepted?

time (Cqmments) <

1

2

3

4

5

6 I

7

8

9

10

11

12 ,

Sperm aensity

#sperm counted= _

<mean= _

(mean) x (5 x 106 )= _

Egg dilution

# eggs counted=-------

sperm/mL

mean= _

(mean) x 100= eggs/mL i~'s~ock -------- eggs/mL in stock + 1,000= Egg dilution factor

329
Figure 3. Sample worksheet for sea urchin fertilization

information.

SEA URCHIN DEVELOPMENT TEST

FERTILIZATION WORKSHEET

Bioassay No.

Date--------

_______mL eggs used ~_'mL dilution water used

Fertilization and initiation

mL in egg dilution x 1,000 eggs/mL ------ eggs in dilution -------

eggs in dilution x 500 ~perm/egg

________ ------ sperm needed

-:-:-::--~;----sperm needed . sperm/mL in sperm

dilution= mL sperm dilution needed

Percent fertilized after 10 min

Time of inoculation-------

330
Figure 4. Sample data sheet for embryo microscopic examination.

SEA URCHIN DEVELOPMENT TEST: RESULTS

Bioassay No.--------

Counter--------

Date _

Number Sample Abnormal Normal %Normal Notes

331
~5.6.~9 Acetic acid, 10%, reagent grade,' in filtered (10~)

seawater -- for prepariBg killed sperm dilutions for sperm

counts.

~5.6.20 Haemo-Sol or equivalent cleaner -- for cleaning

hemacytometer and cover slips.

~5.6.2~ 0.5 M KCl solution -- for inducing spawning.

~5.6.22 Syringe, disposable, 3 or 5 mL -- for injecting KCl into

sea urchins and sand dollars to induce spawning.

15.6.23 Needles, 25 gauge -- for injecting KC1.

15.6.24 Pasteur pipets and bulbs -- for.sampling eggs from

spawning beakers.

15.6.25 Hematocrit capillary tubes -- for sampling sperm for

examination and for loading hemacytometers.

~5.6.26 Microscope well-slides -- for pre-test assessment of

sperm activity and egg condition.

~5.6.27 Reference toxicant solutions (see Section 15.10.2.4 and

Section 4, Quality Assurance) .

15.6.28 Reagent water -- defined as distilled or deionized water

that does not contain substances which are toxic to the test

organisms (see Section 5, Facilities, Equipment, and Supplies) .

~5.6.29 Effluent and receiving water -- see Section 8, Effluent

and Surface Water Sampling, and Sample Handling, and S~mple

Preparation for Toxicity Tests.

15.6.30 Dilution water and hypersaline brine -- see Section 7,

Dilution Water and Section 15.6.24, Hypersaline Brines. The

dilution water should be uncontaminated l-~m-filtered natural

seawater. Hypersaline brine should be prepared "from dilution

water.

15.6.31 HYPERSALINE BRINES

15.6.31.1 Most industrial and sewage treatment effluents

332
entering marine and estuarine systems have little measurable

salinity. Exposure of larvae to these effluentf3 will usually

require increasing the salinity of the test solutions. _ It is

important to maintain an essentially constant salinity across all

treatments. In some applications it may be desi.rable to match

the test salinity with that of the receiving water (See Section

7.1). Two salt sources are available to adjust :salinities --

artificial sea salts and hypersaline brine (HSB)' derived from

natural seawater. Use of artificial sea salts is necessary only

when high effluent concentrations preclude salinity adjustment by

HSB alone.

15.6.31.2 Hypersaline brine (HSB) can be made by concentrating

natural seawate~ by freezing or evaporation. HBB should be.made

from high quality, filtered seawater, ,"and can be added to the

effluent or to reagent water to increase salinity. HBB has

several desirable-characteristics for use in eff~uent toxicity

testing.. Brine derived from natural seawater contains -the

necessary trace metals, biogenic colloids, and some of the

microbial components necessary for adequate growth, survival,

and/or reproduction of marine and estuarine organisms, and it can

be stored for prolonged periods without ~ny apparent degradation.

However, even if the maximum salinity HSB (100~) is used as a

diluent, the maximum.concentration of effluent (0%0) that can be

tested is 66% effluent at 34~ salinity (see Tabl~ 1) .

15.6.31.3 High quality (and preferably high salinity) seawater

should be filtered to at least 10 ~m before placing into the

freezer or the brine generator. Water should be collected on an

incoming tide to minimize the possibility of cont:amination.

15.6.31.4 Freeze Preparation of Brine

15.6.31.4.1 A convenient container for making HBB by freezing is

one that has a bottom drain. One liter of brine can be made from

four liters of seawater.. Brine may be collected by partially

freezing seawater at -10 to -20°C until the remaining liquid has.

reached the' target salinity. Freeze for approxin~ately six hours,

then separate the ice (composed mainly of freshwater) from the

remaining liquid (which has now become hypersaline) .

333
TABLE 1. MAXIMUM EFFLUENT CONCENTRATION (%) THAT CAN BE TESTED

AT 34~ WITHOUT THE ADDITION OF DRY SALTS GIVEN THE

INDICATED EFFLUENT AND BRINE SALINITIES.

, Effluent Brine Brine Brine Brine Brine

Salinity 60 70 80 90 100

%0 %0 ~o ~o %'0

0 43.33 51.43 57.50 62.22 66.00

, 1 44.07 52.17 58.23 62.92 66.67

2 44.83 52.94 58.97 63.64 67.35

3 45.61 53.73 59.74 64.37 68.04

i 4 46.43 54.55 60.53 65.12 68.75

5 47.27 55.38 61.33 65.88 69.4.7

! 10 52.00 60.00 65.71 70.00 73.33 I

I 15 57.78 65.45 . 70.77 74.67 77.65

20 65.00 72.00 76.67 80.00 82.50 :

25 74.29 80.00 83.64 86.15 88.00

15.6.31.4.2 It is preferable to monitor the water until the

target salinity is achie~ed rather than allowing total freezing

followed by partial thawing'. Brine salinity should never exceed

100%0. It is advisable not to exceed about 70%0 brine salinity

unless it is necessary to test effluent concentrations greater

than 50%.

15.6.31.4.3 After the required salinity is attained, the HSB

should be filtered through a 1 ~m filter and poured directly into

portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity-and the date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after several months in storage.

15.6.31.5 Heat Preparation of Brine

334
15.6.31.5.1 The ideal container for making briIle using heatassisted evaporation of 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 corning in contact with 'the seawafer 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 is to use a thermostatically c;ontrolled heat

exchanger made from fiberglass. If aeration is needed, use only

oil-free air compressors to prevent contamination.

15.6.31.5.2 Before adding seawater to the brine generator,

thoroughly clean the generator, aeration supply:tube, heater, and

any other materials that will be in direct contact with the

brine. A good quality biodegradable detergent should be used,

followed by several (at least three) thorough reagent water

rinses.

15.6.31.5.3 Seawater should be filtered to at least 10 ~m before

being put into the brine generator. The tempera~ure 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) ~o 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.

15.6.31.5.4 After the required salinity is attained, the HSB

should be filtered through a 1 ~m filter and poured directly.into

portable containers (20-L cubitainers or polycarlbonate 'water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and the. date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been accep.table). HSB is usually

of acceptable quality even after several months in storage.

15.6.31.6 Artificial· Sea Salts

15.6.31.6.1 No data from sea urchin or sand dollar larval tests'

using sea salts or artificial seawater (e.g., GP2) are available'

335
for evaluation at this time, and their use must be considered

provisional.

15.6.31.7 Dilution Water Preparation from Brine

15.6.31.7.1 Although salinity adjustment with brine is the

preferred method, the use of high salinity brines and/or reagent

water has sometimes been associated with discernible adverse

;

effects on test organisms. For this reason, it is recommended

that only the minimum necessary volume of brine and reagent water

be used to offset the low salinity of the effluent, and that

brine controls be included in the test. The remaining 'dilution

water should be natural seawater. Salinity may be adjusted in

one of two ways. First, the salinity of the highest effluent

test concentration may be adjusted to an acceptable salinity, and

then serially diluted. Alternatively, each effluent

concentration can be prepared individualiy with appropriate

volumes of effluent and brine.

15.6.31.7.2 When HSB and reagent water are used, thoroughly mix

together the reagent water and HSB before mixing in the effluent.

Divide the salinity of the HSB by the expected test salinity to

determine the proportion of reagent water to brine. For example,

if the salinity of the brine is 100~ and "the test is to be

conducted at 34~, 100~ divided by 34~ = 2.94. The proportion

is 1 part brine plus 1.94 parts reagent water. To mak~ 1 L of

dilution water at 34~ salinity from a HSB of 100~, 340 mL of

brine and 660 mL of reagent water are required. Verify the

salinity of the resulting mixture using a refractometer.

15.6.31.8 Test Solution.Salinity Adjustment

15.6.31.8.1 Table 2 illustrates the preparation of test

solutions (up to 50% effluent) at 34~ by combining effluent,

HSB, and dilution water. Note: if the highest effluent

concentration does not exceed 50% effluent, it is convenient to

prepare brine so that the sum of the effluent salinity and brine

salinity equals 68~; the required brine ~olume is then always

equal to the effluent volume needed for each effluent

concentration as in the example in Table 2.

15.6.31.8.2 Check the pH of all test solutions and adjust to

within 0.2 units of dilution water pH by adding, dropwise, dilute

336
hydrochloric acid or sodium hydroxide (see Section 8.8.9,

'Effluent and Receiving Water Sampling, Sample Handling, and

Sample Preparation for Toxicity Tests) .

15.6.31.8.3 To calculate the amount of brine to add to each

effluent dilution, determine the following quantities: salinity

of the brine (SB, in ~), the salinity of the effluent (SE, in

%0), and volume of the effluent ~o be added (VE, in mL) . Then

use the following formula to calculate the volume of brine (VB"

in mL) to be added:

VB VE x (34 - SE)/(SB - 34)

15.6.31.8.4 This calculation assumes that dilution wat~r

salinity is 34 ± 2~.

15.6.3l.9 Preparing Test Solutions

15.6.31.9.1 Ten mL of test solution are needed for each test

chamber. To prepare test solutions at low effluent

concentrations (~6%), effluents may be added directly to dilution

water. For example, to prepare 1% effluent, add 1.0 mL of

effluent to a 100-mL volumetric flask using a volumetric pipet or

calibrated automatic pipet. Fill the volumetric flask to the

100-mL mark with dilution water, stopper.it, and shake to mix.

Pour into a (150-250 mL) beaker and stir. Distribute equal

volumes into the replicate test chambers. The remaining test

solution can be used for chemistry.

15.6.31.9.2 To prepare a test solution at highl:r effluent

concentrations, hypersaline brine must usually be used. For

example, to prepare 40% effluent, add'40~ mL of effluent ,to a 1-

liter volumetric flask. Then, assuming an effluent salinity of

2~ and a brine salinity of 66%'0, add 400 mL of bJdne (see

equation ~bove and Table 2) and top off the flal;:;k with, dilution

water. Stopper the flask and shake well. Pour into, a (100-250

mL) beaker and stir. Distribute equal volumes into the' replicate

test chambers. The remaining test, solution can be used for

chemistry.

, I

I

337
TABLE 2. EXAMPLES OF EFFLUENT DILUTION SHOWING VOLUMES OF

EFFLUENT (AT X~), BRINE, AND DILUTION WATER NEEDED FOR

ONE LITER OF EACH TEST SOLUTION.

FIRST STEP:

to achieve a

for dilution

Combine brine with reagent water or natural

brine of 68-x~ and, unless natural seawater

water, also a brine-based dilution water of

seawater

is used

34~.

SERIAL DILUTION:

Step 1. Prepare the highest effluent concentration to be tested by'adding

equal volumes of effluent and brine to the appropriate volume of dilution

water. An example using 40% is shown.

i Effluent Cone. Effluent x%o Brine (68- Dilution Water*

(lIi) x) %0 34%0

I

I 40 800 mL 800 mL 400 mL

. Make ser~al d~lut~ons from the h~ghest test concentrat~on

Effluent Cone. (%) Effluent Source Dilution Water* (34%0)

20 1000 mL of 40% 1000 mL

10 1000 mL of 20% 1000 mL

i

I 5 1000 mL of 10% 1000 mL

2.5 1000 mL of 5% 1000 mL

Control none 1000 mL

Steo 2

INDIVIDUAL PREPARATION'

Effluent Cone. Effluent x%o Brine (68-x) %0 Dilution Water*

(%) 34%0

i 40 400 mL 400 mL 200 mL

20 200 mL 200 mL 600 mL

10 100 mL 100 mL 800 mL

5 50 mL 50 mL . 900 mL

2.5' 25 mL 25 ·mL 950 mL

Control none none 1000 mL

. *May be natural seawater or brine-reagent water equivalent.

338
15.6.31.10 Brine Controls

15.6.31.10.1 Use brine controlf? in all tests where brine is

used. Brine controls contain the same volume of brine as does

the highest effluent concentration using brine, plus the volume

of reagent water needed to reproduce the hyposalinity of the

effluent in the highest concentration, plus dilu1:ion water.

Calculate the amount of reagent water to add to brine controls by

rearranging the above equation, (See, 15.6.31.8.3) setting SE =

0, and solving for VE.

VE = VB x (SB - 34)/(34 - SE)

If .effluent salinity is essentially 0%"0, the reagl~mt water volume

needed in the brine control will equal the effluent volume at the

highest test concentration. However, as effluent salinity and

effluent concentration increase, less reagent wa'\:er volume is

needed.

15.6.32 TEST ORGANISMS, PURPLE URCHINS

15.6.32.1 Sea Urchins, Strongylocentrotus purpuratus

(approximately 6 of each sex per test) .

15.6.32.2 Species Identification

15.6.32.2.1 Although identification of purple sea urchins,

Strogylocentrotus purpuratus, is usually a simple mat~er of

confirming general body color, size, and spine appearance, those

unfamiliar with the species should seek confirma~ion from local

experts.

15.6.32.3 Obtaining Broodstock

15.6.32.3.1 Adult sea urchins (Strongylocentrotus purpuratus)'

can be obtained from commercial,supplJers or collected .from

uncontaminated intertidal areas. State collection permits are

usually required for collectic;m of sea urchins and collection is

prohibited or restricted in some areas. The animals are best

transported "dry," surrounded either by moist se,aweed or paper

towels dampened with seawater. Animals should be kept at

approximately their collection or culture temper'ature to prevent

thermal shock which can prematurely induce spawning.

339
~5.6.32.4 Broodstock Culture and Handling

~5.6.32.4.~ The adult sea urchins are maintained in glass

aquaria or fiberglass tanks. The tanks are supplied continuously

(approximately 5 L/min) with filtered natural seawater, or salt

water prepared from commericial sea salts is recirculated. The

animals are checked daily and any obviously unhealthy animals are

discarded.

~5.6.32.4.2 Although ambient temperature seawater is usually

acceptable, maintaining sea urchins in spawning condition usually

requires holding at a relatively constant temperature. The

culture unit should be capable of maintaining a constant

temperature between 10 and 14°C with a water temperature control

device.

~5.6.32.4.3 Food for sea urchins kelp, recommended, but not

necessarily limited to, Laminaria sp., Hedophyllum sp.,

Nereocystis sp., Macrocystis sp., Egregi~ sp., Alaria sp. or

romaine lettuce. The kelp should be gathered from known

uncontaminated zones or obtained from commerical supply houses

whose kelp comes from known uncontaminated areas, or romaine

lettuce. Fresh food is introduced into the tanks at least

several times a week. Sun dried (12-24 hours) or oven dried

(60°C overnight) kelp, stores well at room temperature or frozen,

rehydrates well and is adequate to' maintain sea urchins for long

periods. Decaying food and fecal pellets are removed as

necessary to prevent fouling.

~5.6.32.4.4 Natural seawater (>30~) is used to maintain the

adult animals and (~32~) as a control water in the tests.

~5.6.32.4.5 Adult male and female (if sexes known) animals used

in field studies are transported in separate or partitioned

insulated boxes or coolers packed with wet kelp or paper

toweling. Upon arrival at the field site, aquaria (or a single

partitioned aquarium) are filled with control water, loosely

covered with a styrofoam sheet and allowed to equilibrate to the

holding temperature ·before animals are added. Healthy animals

will attach to the kelp or aquarium within hours.

~5.6.32.4.6 To successfully maintain about 25 adult animals for

seven days at a field site, 40-L glass aquaria using aerated,

340
recirculating, clean saline water (32%0) and a gravel bed

filtration system, are housed within a water bath, such as an

INSTANT OCEANR Aquarium. The sexes should beheld separately if

possible.

15.6.33 TEST ORGANISMS, SAND DOLLARS

15.6.33.1 Sand Dollars, Dendraster excentricus, (approximately 6

of each sex per test) .

15.6.33.2 Species Identification

15.6.33.2.1 Although identification of sand dollars, Dendraster

excentricus, is usually a simple.matter of confirming general

body appearance, those unfamiliar with the species should seek

confirmation from local experts.

15.6.33.3 Obtaining Broodstock

15.6.33.3.1 Adult sand dollars (Dendraster excentricus)~'can be·

obtained from commercial suppliers or collected.from ~ubtic:ial

zones (most areas) or from intertidal zones of some sheltered

waters (e.g., Puget Sound). S'tate collection permits may be

required for collection of sand dollars and collection· prohibited

or restricted in some areas. The animals are b~st transported

"dry," surrounded either by moist seaweed or paper towels

dampened with seawater. Animals should be kept at approximately

their collection or culture temperature to prevE~nt thermal shock

which can prematurely induce spawning.

15.6.33.4 Broodstock Culture and Handling

·15.6.33.4.1 The adult sand dollars are maintained in glass

aquaria or. fiberglass tanks. The tanks are supplied continuously

(approximately 5 L/min) with filtered natural SE!awater,or

saltwater prepared from commercial sea salts is !recircu.lated.

The animals are checked daily and any obviously unhealthy animals

are discarded. For longer periods than a few days, several

centimeters or more of a sand substrate may be desirable.

15.6.33.4.2 Although ambient temperature seawat.er is usually

acceptable, maintaining sand dollars in spawning[ condition

usually requires holding at a relatively constar.lt temperature.

341
The culture unit should be capable of maintaining a co~stant

temperature between 8 and 12°C with a water temperature control

device.

15.6.33.4.3 Sand dollars will feed on suspended or benthic

materials such as phytoplankton, benthic diatoms, etc. No

reports of laboratory populations being maintained in spawning

condition over several years are known. It is probably most

convenient to obtain sand dollars, use them, and then discard

them after they cease to produce good quality gametes.

15.6.33.4.4 Natural seawater (>30~) is used to maintain the

adult animals and (~32~) as a control water in the tests.

15.6.33.4.5 Adult male and female (if sexes known) animals used

in field studies are transported ,in separate or partitioned

insulated boxes or coolers' packed with wet kelp or paper

toweling. Upon arrival at the field s~te, trays or aquaria (ora

single partitioned aquarium) are filled with control water,

loosely covered with a styrofoam sheet 'and allowed to equilibrate

to the holding temperature before animals are added.

15.6.33.4.6 To successfully maintain about 25 adult a~imals for

seven days at a field site, 40-L glass aquaria using aerated,

recirculating, clean saline water (>30~) are housed within a

water bath, such as an INSTANT OCEANR Aquarium. The sexes should

be held separately if possible.

15.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION AND

STORAGE

15.7.1 See Section 8, Effluent and Receiving Water Sampling and

Sample Handling, and Sampling Preparation for Toxicity Tests.

15.8 CALIBRATION AND STANDARDIZATION

15.8.1 See Section 4, Quality Assurance.

15.9 QUALITY CONTROL

15.9.1 See Section 4, Quality Assurance.

15.10 TEST PROCEDURES

342
15.10.1 TEST DESIGN

I

15.10.1.1 The test consists,of at least four r~plicates of five

effluent concentrations plus a dilution'water control. Tests

that use brine to 'adjust salinity must also cont:ain four

replicates of a brine control.

15.10.1.2 Effluent concentrations are expressed as percent

effluent.

15.10.2 TEST SOLUTIONS

15.10.2.1 Receiving waters

15.10.2.1.1 The sampling point is determined by the objectives

of the test. At estuarine and marine site13, samples are usually

collected at mid-depth. Receiving water toxicit:y is de'termined

with samples used directly as collected or with'samples passed

through a 60 jlm'NITEX® filter and compared without dilution,

against a control. U~Jing four replicate chambers per test, each

containing 10 mL, and 400 mL for chemical analysis, would require

approximately 440 mL of sample per test.'

15.1D.2.2 Effluents

"

15.10.2.2.1 The selection of the effluent test 'concentrations

should be based on the objectives of the study. i A dilution

factor of at least 0.5 is commonly uS,ed. A dilution factor of

0.5 provides hypothesis test discrimination of :c 100%, and

testing of a 16 fold range of concentrations. Hypothesis test

discrimination shows little improvement as dilution factors are

increased beyond 0.5 and declines rapidly if smaller dilution

factors are used. USEPArecommends that one of. the five effluent

treatments must be a concentration of effluent mixed with

dilution water which corresponds to the permittE!e l s instream

waste concentration (IWC). At least two'of the effluent

treatments must be of lesser effluent concentrat:ion than the IWC,

with one being at least one-half the concentration of t.he IWC.

If 100%0 HSBis used as a diluent, the maximum concentration of

effluent that can be tested will be 66% at 34%0 salinity.

,

"

343
15.10.2.2.2 If the effluent is known or suspected to be highly

toxic, a lower range of effluent concentrations should be used

(such as 25%, 12.5%, 6.25%, 3.12% and 1.56%).

15.10.2.2.3 The volume in each test chamber is 10 mL.

15.10.2.2.4 Effluent dilutions should be prepared for all

replicates in each treatment in one container to minimize

variability among the replicates. Dispense into the appropriate

effluent test chambers.

15.10.2.3 Dilution Water

15.10.2.3.1 Dilution water should be uncontaminated l-~mfiltered natural seawater or hypersaline brine prepared from

uncontaminated natural seawater plus reagent water (see Section

7, Dilution Water). Natural seawater may be uncontaminated

receiving water. This water is used in all dilution steps and as

the control water.

15.10.2.4 Reference Toxicant Test

15.10.2.4.1 Reference toxicant tests should be conducted as

described in Quality Assurance (see Section 4.7) .

15.10.2.4.2 The preferred reference toxicant for sea urchins and

sand dollars is copper chloride (CuCI 2oH20). Reference toxicant

tests provide an indication of the sensitivity of the test

organisms and the suitability of the testing laboratory (see

Section 4 Quality Assurance). Another toxicant may be specified

by the appropriate regulatory agency. Prepare a copper reference

toxicant stock solution (2,000 mg/L) by adding 5.366 g ·of copper

chloride (CuC12 02H20) to 1 liter of reagent ~qater. For each

reference toxicant test prepare a copper sub-stock of 3 mg/L by

diluting 1.5 mL of stock to one liter with reagent water.

Alternatively, certified standard solutions can be ordered from

commercial companies.

15.10.2.4.3 Prepare a coptrol (0 ~g/L) plus four replicates each

of at least five consecutive copper reference toxicant solutions

(e.g., from the series 3.0, 4.4, 6.5, 9.5, 13.9, 20.4, and 30.0

~g/L, by adding 0.10, 0.15, 0.22, 0.32, 0.46, 0.68, and 1.00 mL

of sub-stock solution, respectively, to 100-L· volumetric flasks

344
,, ,

,

and filling to 100-mL with dilution water). Alternatively,

certified standard solutions can be ordered fromicommercial

companies. Start with control solutions and pr09ress to the

highest concentration to minimize contamination.

15.10.2.4.4 If the effluent and reference toxic~nt tests are to

be run concurrently, then the tests must use embryos from the

same spawn. The tests must be handled in the same way and test

solutions delivered to the test chambers'at the same time.

Reference toxicant tests must be conducted at 34 ± 2~.

15.10.3 COLLECTION OF GAMETES FOR THE TEST

15.10.3.1 Spawning Induction

15.10.3.1.1 Pour seawater into 100 mL beakers and place in 15°C

. bath or room. Allow to come to temperature. Select a sufficient

nUTl)ber of sea urchins or sand dollars (based UpOIl recent or past

spawning success) so that three of each sex are likely to provide

gametes of acceptable quantity and quality for the test. During

optimal spawning periods this may only require six animals, three

of each sex, when the sexes are known from prior spawning.

During other periods, especially if the sex is not known, many

more animals may be required.

15.10.3.1.2 Care should be exercised when removing sea urchins

from holding tanks so that damage to tube-feet is minimized.

Following removal, sea urchins should be placed into a container

lined with seawater-moistened paper towels to prevent

reattachment.

15.10.3.1.3 Place each sand dollar, oral side up, on a 100 mL

beaker filled with 15°C seawater or each sea urchin ont6 a clean

tray covered with several layers of seawater moistened paper

towels.

15.10.3.1.4 Handle sexes separately onceknowni ,this minimizes

the chance of accidental egg fertilization. Throughout the test

process, it is best if a different worker, diffeJ~ent pipets, etc.

are used for males (semen) and females (eggs). Prequent washing

of hands is a good practice.

15.10.3.1.5 Fill a 3 or 5 mL syringe with 0.5 MKCl and inject

345
0.5 mL through the soft periostomal membrane of each sea urchin

(See Figure 5) or into the oral opening each sand dollar. If

sexes are known, use a separate needle for each sex. If sexes

are not known, rinse the needle with hot tap water between each

injection. This will avoid the accidental injection of sperm

from males into females. Note the time of injection on the data

sheet.

15.10.3.1.6 Spawning of sea urchins is sometimes induced by

holding the injected sea urchin and gently shaking or swirling it

for several seconds. This may provide an additional physical

stimulus, or may aid in distributing the injected KCI.

15.10.3.1.7 Place the sea urchins onto the beakers or tray (oral

side down). Place the sand dollars onto the beakers (oral side

up). Females will release orange (sea urchins) or purple (sand

dollars) eggs and males w~ll release cream~colored semen.

15.10.3.1.8 As gametes begin to be shed, note the time "on the

data sheet and separate the sexes. Place male sand dollars with

the oral side up atop a small (5-10 mL) glass beaker filled with

12°C seawater. Leave spawning sea urchin males on tray or beaker

(oral side down) for semen collection. Female sand dollars and

sea urchins are left to shed eggs into the 100-mL beakers.

15.10.3.1.9 If sufficient quantities of gametes are available,

only collect gametes for the first 15 min after each a~imal

starts releasing. This helps to insure good quality gametes.

As a general guideline, do not collect gametes from any

individual for more than 30 minutes after the first injection.

15.10.3.1.10 If no spawning occurs after 5 or 10 minutes, a

second 0.5 mL injection may be .tried. If animals do not produce

sufficient gametes following injection of 1.0 mL of KCI, they

should probably not be reinjected as this seldom results in

acquisition of good quality gametes and may result in mortality

of adult urchins.

15.10.3.1.11 Sections 15.l0.4.2 and~15.~0.6.4 descr~be

collection and dilution of the sperm and eggs. While some of the

gamete handling needs to 'be in a specific order, parts of the

procedure can be done simultaneously while waiting for 'gametes to

settle.

346
Figure 5. Showing the location and orientation used in the

injection of KCI into sea urchins to stimulate SI?awning.

15.10.3.2 Collection of Sperm

15.10.3.2.1 Sea urchin semen should be collected dry (directly

from the surface of the sea urchin), ,using either a Pasteur

pip~tte or a 0.1 mL autopipette with the end of t:he tip cut off

so that the opening is at least 2 mm. pipette sE~men from each

male into separate 1-15 mL conical test tubes, stored in an ice

water bath.

15.10.3.3 Viability of Sperm

15.10.3~3.1 Early in the spawning process, place a very small

amount of sperm from each male sea urchin or sanel dollar into

347
dilution water on a microscope slide (~ell slides work nicely) .

Examine the sperm for motilitYi use sperm from males with high

sperm motility.

15.10.3.4 Pooling of Sperm

15.10.3.4.1 Pool equal quantities of semen from each of the 'sea

urchin males that has been deemed good. If possible, 0.025 mL

should be pooled from each of those used and a total of at least

0.05 mL of pooled semen should be available. Sperm collected

from good male sand dollars should be pooled after first

decanting off the overlying water (the final sand dollar sperm

density usually is between 2x10 9 and 2x10lo sperm/mL).

15.10.3.5 Storage of Sperm

15.10.3.5.1 Cover each test tube or beaker with a cap or

parafilm, as air exposure of semen may alter its pH through gas

exchange and reduce the viability of the sperm. Keep sperm

covered and on ice or refrigerated «5°C),. The sperm should be

used within 4 h of collection.

15.10.4 PREPARATION OF SPERM DILUTION FOR USE.IN THE TEST

15.10.4.1 Sperm Dilution

15.10.4.1.1 When ready to use ,sperm, mix by agitating the tube

with a vortex mixer. Add about 0.025 mL'of semen to a 100 mL

beaker containing 50 mL of 15°C dilution water. Stir this

solution thoroughly with' a Pasteur pipette. A drop of .egg

solution from each female may be placed on a well slide and a

small amount of sperm solution added to test fertilization. If'

no fertilization membrane forms on eggs from any female, then new

gametes should be collected. Keep the sperm dilution covered and'

at 15°C until ready for use~ This dilution should be used.to

fertilize the eggs within 1.5 hours of being made.

15.10.4.2 Sperm Density Determination

15.10.4.2.1 Take 0.5 mL subsample of the sperm solution and add

it to 5 mLof 10% acetic acid in a 50 mL graduated cylinder, to

kill the sperm. Bring the volume to 50 mL with dilution water.

Mix by inversion and place one drop 'of the killed sperm solution

348
onto ~ach side of a hemocytometer. Let sperm settle for about 15

minutes. Count the number of sperm in 80 small ,squares on each

side of the hemocytometer. If the counts for each side' are

within 80% of one another,then take the mean of those two

counts. If the counts are not that close, then.refilI the

hemocytometer, recount and take the mean of the four counts. Use

the following equations to determine sperm density and record the'

results on the spawning worksheet (Figure 2).

#sperm/mL-(dilution) (count) (hemacytometer conversion factor) (mm3/mL)

number of squares counted

dilution=100

conversion factor=4000

mm3 /mL=1000

number of squares=80

Therefore:

#sperm/mL= (count) (5 x 106 ) (Equation 2A)

15.10.5.2

15.10.5 PREPARATION OF EGG SUSPENSION FOR USE IN THE TEST

15.10.5.1 Acceptability of Eggs

15.10.5.1.1 Place a small sample of eggs from each female in the

counting chamber and examine eggs with the micro:pcope. . Look for

the presence of significant quantities of immatu:~e or abnormal

appearing eggs (germinal vesicle present, unusually large or

small or irregularly shaped). Do not use the eggs from females

having more than 10% abnormal eggs or from femalE:s whose eggs did

not fertilize during the test in Section·15.10.5.1.

Pooling of Eggs

15.10.5.2.1

Decant some

not to pour

Allow eggs to settle in the collection beakers.

of the water from the collection beakers taking care

off many eggs. Pour the remaining sea urchin eggs

349
through the Nitex@ screen (to remove fecal material 'and other

debris) into a 1 liter beaker. Repeat with each of the' 11 good 11

females. Bring the volume up to about 600 mL with dilution

water. Allow the eggs to settle to the bottom again. Siphon off

about 400 mL of the overlying water and then bring back up to 600

mL with dilution water. Do not allow the temperature to rise

above the 15°C test temperature; somewhat cooler temperatures for

holding would be acceptable.

15.10.5.2.2 Pooled sand dollar eggs should be treated gently and

no additional screening or rinsing step is recommended. Mix well

once just before subsampling for egg stock calculations. This is

best done in a large graduated cylinder appropriate for the

number of eggs available. Cover with parafilrn and invert gently

several times.

15.10.5.3 Density of Eggs

15.10.5.3.1 Using a plunger, mix the sea urchin egg suspension

well. While continuing to mix, remove a 10 mL sample and place

in a 1 liter graduated cylinder. Bring the volume up to 1 liter

with dilution water. Mix this dilution well.and remove a 1 mL .

sample to a counting cell. Count all the eggs in the 1 mL

sample. Repeat the process and take the mean of the two counts.

Calculate the number of eggs per mL in the stock solution using

Equation 3 and record the results.

# of eggs in count x 100= # eggs/mL in stock

15.10.5.4 Dilution of Eggs

(Equation 3)

15.10.5.4.1 When using scintillation vials as the test chamber,

the final concentration of eggs in the diluted stock m~st be 250

eggs/0.25 mL, which is equal to 1,000 eggs/mL. To calculate the

dilution factor for the eggs, use Equation 4. (If larger test

chambers are used, the total number of eggs used will be greater

and the stock solution density may be adjusted, but the final

concentration of eggs in the test solutions must remain 25

eggs/mL) .

# of eggs/rnL in stock 1,000= Dilution factor (Equation 4)

15.10.5.4.2 The dilution factor must be greater than one. If

350
not, concentrate the eggs and recount (starting at Section

1.5.4.5.3 The dilution factor minus 1. equals thl= number of parts

of water that go with one part of eggs in the final dilution.

For example: if the dilution factor were 5.3, then 4.3 parts of

water would be. used with 1. part eggs.

1.5.1.0.5.4.3 Make a dilution of the egg stock so that there is.

more than -enough volume to perform the bioassay.

1.5.1.0.5.5 Fertilization of Egg$

1.5.1.0.5.5.1. . The recommended initial sperm to e~Jg ratio for

fertilization of the .eggs is 500:1.. Calculate ehe volume of

sperm dilution (Section 1.5.1.0.5.1.) to add to the egg dilution, by

using the following equations and record the results (Figure 3).

volume of egg dilution x 1,000 eggs/mL= total # of eggs in ,dilution (Equation

SA)

total # of eggs in dilution x 500 sperm/egg= # of sperm needed

(Equation 58)

# of sperm needed

(Equation SC)

+ # sperm/mL in sperm dilution= mL of sperm solution

,

I •

1.5.1.0.5.5.2 Add this volume of the sp~rm dilution tb the egg

. dilution and mix gently with a plunger; Wait 1.0 min, then check

for fertilization. If fertilization is not at least 90%, ad~ a

second volume of the sperm dilution. Wait 10. min and recheck.

If fertilization is still not 90%, then. the test: must be

restarted with different gametes.

15.10.5.5.3 The test should be initiated within 1.hour of

fertilization being achieved.

15.10.6 START OF THE TEST

15.10.6.1 Prior to Beginning the Test

i

.,

1.5.10.6.1.1 The test should begin as soon as po~sible, .

preferably within 24 h of sample collection. The maximum holding

time following retrieval of the sample from.the sampling device

should not exceed 36 h for off-site toxicity tests unless

permission is granted by the permitting authority. In no case

351
should the sample be used in a test more than 72 h after sample

collection (see Section 8 Effluent and Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity Test) .

15.10.6.1.2 Just prior to test initiation (approximately 1 h),

the temperature of a sufficient quantity of the sample to make

the test solutions should be adjusted to the test temperature (15

± IDe) and maintained at that temperature during the addition of

dilution water.

15.10.6.1.3 Increase the temperature of the water bath, room, or

incubator to the required test temperature (15 ± IDe) .

15.10.6.1.4 Randomize the placement of test chambers in the

temperature-controlled water bath, room, or incubator at the

beginning of the test, using a position chart. Assign numbers

for the position of each test chamber using a random numbers or

similar process (see Appendix A, for an example of

randomization). Maintain the chambers in this configuration

throughout the test, using a position chart. Record these

numbers on a separate data sheet together with the con~entration

and replicate numbers to which they correspond. Identify this

sheet with the date, test organism, test number, laboratory, and

investigator's name, and safely store it away until after the sea

urchins or sand dollars have been examined at the end of the

test.

15.10.6.1.5 Note: Loss of the randomization sheet would

invalidate the test by making it impossible to analyze 'the data

afterwards. Make a copy of the randomization sheet and store

separately. Take care to follow the numbering system exactly

while filling chambers with the test solutions.

15.10.6.1.6 Arrange the test chambers randomly in the water bath

or controlled temperature room. Once chambers have been labeled

randomly and filled with test solutions, they can be arranged in

numerical order for convenience, since this will also ensure

random placement of treatments.

15.10.6.1.6 If mortality is to be included as an endpoint, at

least 5 extra control chambers should be 'set up and identified on

the randomization sheet as initial count chambers.

352
15.10.6.2 Delivery of Fertilized Eggs

15.10.6.2.1 Gently mix the solution of fertili:~ed eggs. Deliver

0.25 mL of egg solution to each vial, using an automatic pipette

with the tip cut off to provide at least.a 0.5 tnm opening.

Deliver the embryos into the test chambers directly from the

pipette, taking care not to touch the p~pette to the test

solution. The egg solution temperature'must be within ·l°e of the

test solutions. Keep the eggs well mixed durin~J the delivery

procedure.

15.10.6.3 Incubation

15.10.6.3.1 The embryos are incubated for 72 hours in the test

chambers at 15 ± 1°C at ambient light level.

15.10.6.3.2 The optional extra control chambers for initial

counts should be counted as soon as possible aft,er test

initiation. If they are sampled and counted ina non-destructive

manner they may be returned to the test but used only as a check

for larval developmental rate. They must not be: used for for

routine control counts at the end of the test.

15.10.7 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE

15.10.7.1 The light quality and intensity should be at ambient

laboratory cdnditions. Light intensi~y ~hould b~ 10-20 ~E/~/s,

or 50 to 100 foot candles (ft-c), with a 16 h light and 8 h dark

cycle.

15.10.7.2 The water temperature in the test chambers should be

maintained at 15 ± 1°C. If a water bath is used to maintain the

test temperature, the water depth surrounding th~ test cups

should be as deep as possible without·floating tne chambers. . ! .

15.10.7.3 The test salinity should be in the range of 34 ± 2~.

The salinity should vary by no more than ±2~ among the .chambers

on a given day. If effluent and receiving water tests are

conducted concurrently, the salinities of these 'tests should be

similar.

15.10.7.4 Rooms or incubators with high'volume ventilation

should be used with caution because the volatilization of the

353
test solutions and evaporation of dilution water may cause wide

fluctuations in salinity. Covering the test chambers with clean

polyethylene plastic may help prevent volatilization and

evaporation of the test solutions.

~S.~0.8 DISSOLVED OXYGEN (DO) CONCENTRATION

~5.~0.8.1 Aeration may affect the toxicity of effluent and

should be used only as a last resort to maintain a satisfactory

DO. The DO concentration should be measured on new solutions at

the start of the test (Day O). The DO should not fall below 4.0

mg/L (see Section 8, Effluent and Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity Tests). If

it is necessary to aerate, all treatments and the control should

be aerated. The aeration rate should not exceed that necessary

to maintain a minimum acceptable DO and under no circumstances

should it exceed 100 bubbles/minute, using a pipet with a 1-2 mm

orifice, such as a 1 mL KIMAX® serological pipet No. 37033, or

equivalent.

~5.10.9 OBSERVATIONS DURING THE TEST

~5.~0.9.1 Routine Chemical and Physical Observations

~5.~0.9.~.1 The DO should be measured in each test solution at

the beginning of the exposure period.

~5.~0.9.1.2 The temperature, pH, and salinity should be measured

in all each test solution at the beginning of the exposure

period. Temperature should also be monitored continuo~sly or

observed and recorded daily for at least two ·locations in the

environmental control system or the samples. Temperature should

be measured in a sufficient number of test >chambers at the end of

the test to determine temperature variation· in th~ environmental

chamber.

~S.10.9.1.3 Record all the measurements on the data sheet.

15.10.9.2 Routine Biological Observations

15.10.9.2.1 Developing embryos do not need to be monitored

during the test under normal circumstanc~s.

354
15.10.10. TERMINATION OF THE TEST

15.10.10.1 Ending the Test

15.10.10.1.1 Record the time the test is terminated.

15.10.10.1.2 Temperature, pH, dissolved oxygen, and salinity are

measured at the end of the exposure period in one test chamber at

each concentration and in the control(s). !.

15.10.10.2 Sample Preservation

15.10.10.2.1 To terminate the test, add 1.0 mI~ of,37%

(concentrated~ buffered formalin to each sample to give a final

formalin concentration of 4%. As an alternate fixative, 0.5 mL

of 1.0% glutaraldehyde may be used, in each test chambe,r.

Tightly cap and gently mix each chamber and store for later

evaluation. (If the test is performed in largElr chambers, a 10

mL subsample of well mixed test solution is to be taken from each

chamber and preserved) .

15.10.10.} Counting

15.10.10.3.1 It is recommended that the embryos be examined

within one week of preservation. Longer storag1e ,times may also

be used, but run the risk of sample degradation due, to improper

preservation. Larvae can be counted directly fn the

scintillation vials using an inverted microscope. If an inverted

scope is not available, then samples should be loaded into a

Sedgewick-Rafter cell, as follows. The embryos should first be

allowed to settle to the bottom of the sample chamber.. All but

about 1 mL of the overlying, liquid should then be removed. All

of the remaining liquid containing the embryos should then be

transferred to the counting chamber. Whichever scope is used,

the embryos should be examined at about 100x power. The first

100 embryos encountered are counted using a multi-unit

handcounter to track normal versus abnormal larvae. Record the

data by sample number on a data sheet (Figure 4) .

15.10.10.3.2 Mortality can be determined only if: (1) all

surviving larvae are counted (either in the test vials with an

inverted microscope or by total transfer to a counting chamber);

or (2) the test solution is stirred with·a plunger and

355
quantitative subsampling is conducted followed by total larval

counts on the subsample. The latter procedure requires

homogeneous distribution of larvae in the test solution,

quantitative transfer of larvae (without adherence to transfer

hardware or test chambers), and accurate volume measurements.

Mortality is most important to consider with point estimates

(e.g., EC25) or when mortality occurs at the NOEC for normal

development.

15.10.10.4 Endpoint

15.10.10.4.1 Normal Larvae

15.10.10.4.1.1 Normally developed pluteus larvae have several

distinctive characteristics:

(1) The larvae should have a pyramid shape with a pair of

skeletal rods that extend at least half the iength of

the. long axis of the larvae (Figure 6D) .

(2) The gut should be differentiated into three parts

(Figure 6E). If the gut appears lobed and constricts

distally in specimens with an obstructed view (e.g.,

Figure 6D), then normal gut development may be

inferred.

(3) Development of post-oral arms has begun.

15.10.10.4.2 Abnormal Larvae

15.10.10.4.2.1 Larvae need only be scored. as abnormal or normal

to conduct the test, but the categories of abnormalities may be

tracked as well: Abnormal larvae should fit into one of the

following categories:

(1) Pathological prehatched: Embryos at. the single or

multi-cell stage with the fertilization membrane still

visible.

(2) Pathological hatched:. larvae that have no

fertilization membrane and demonstrate an extensive

356
!

degree of malformation, or necrosis. Most of these

larvae appear as dark balls of cells or dissociated,

'blobs of cells.

i

(3) Inhibited: ,larvae at the blastula or: gastrula stage

that have no gut differentiation or h,3.ve no or

underdeveloped skeleton. These larvae appear 'to be

developing regularly, but are at a stage earlier than

attained by control organisms (e. g., lrigure 6A-C) .

I'

(4) Gut abnormalities: larvae whose overall appearance is

normal, but have guts that are lackin~r,

undifferentiated, abnormally shaped or project outside

of the larvae (exogastrulated). '

(5) Skeletal abnormalities: larvae whose overall

appearance is normal, but have missing spicules,

extraneous spicules or rods growing in abnormal

directions. Note: Some larvae may e:>l:hibit a ,

separation of the rods at the apex. This may be caused

by preservation 'and should not-be termed abnormal.

SinGe the test is started with already fertil,ized eggs,

any unfertilized eggs that are encountered should not

be counted as either' normal or abnorma,l, but should be

ignored.

15.11 SUMMARY OF TEST CONDITIONS

15.11.1 A summary of test conditions and test ,acceptability

criteria is ltsted in Table 3.

15.12 ACCEPTABILIT~ OF TEST RESULTS

15.12.1 Test results are acceptable only if all: the following

requirements are met:

(1) larval normality must ,be at least 80% in the' controls.

{2} the minimum significant difference (%MSD) is ':!:20%

relative to the controls.

15.13 DATA ANALYSIS

15.13.1 ,GENERAL

,15.13.1.1 Tabulate and summarize the data. Calculate the

357
A B

a/.arm sid

o

Figure 6. Stages of sea urchin emb~yo development

(modified from Kume and Dan 1957). A. blastula; B.

gastrula; C. prism; D. pluteus (frontal view); E.

pluteus (lateral view). ai.arm: anterior lateral'

arm, e: esophagus, i: intestine,

st: stomach, std: stomodaeum.

358
TABLE 3. SUMMARY OF TEST CONDITIONS AND "TEST ACCEPTABILITY

CRITERIA FOR THE PURPLE URCHIN, STRONGYLOCENTROTUS

PURPURATUS, AND SAND DOLLAR" DENDRASTER EXCENTRICUS

EMBRYO DEVELOPMENT TEST WITH EFFLUENTS AND RECEIVING

WATERS I

I. Test type: Static non-renewal

2. Salinity: 34 + 2%0

3. Temperature: 15 + 1°C

4. Light quality: Ambient laboratory:

illumination

5. Light intensity: 10-20 fJ-E/m2 / s (Ambient

laboratory levels)

6. Photoperiod: 16 h light~ 8 h darkness ,

7. Test chamber size: 30 mL

8 . Test solution volume: 10 mL

9. No. replicate chambers 4 i

I

per concentration:

10. Dilution water: Uncontaminated 1-fJ-rn-filtered

natural seawater O:r:i

hypersaline brine f!repared

from natural seawater

1I. Test concent.rations: Effluents: Minimum of 5 and a

control I

Receiving waters: 100%

receiving water and a control

12. Dilution factor: Effluents: ~0.5 i

Receiving waters: 100%

receiving water arid a control

13. Test duration: 72 ± 2 hr

14. Endpoint: Normal development ;,1

mortality can be in,cluded

15. Test acceptability ~80% normal shell development

criteria: in the controls; must achieve

a %MSD of <25%

359
16. Sampling requirements: One sample collected at test

initiation', and preferably

used within 24 h of the time

it is removed from the

sampling device (see Section

8, Effluent and Receiving

Water Sampling, Sample

Handling, and Sample I

Preparation for Toxicity

Tests)

17. Sample volume 1 L per test

required: _.

proportion of normally developed larvae for each replicate. A

sample set of test data is listed in Table 4.

15.13.1.2 The statistical tests described here must be used with

a knowledge of the assumptions upon which the tests are

contingent. The assistance of a statistician is recommended for

analysts who are not proficient in statistics.

15.13.1.3 The endpoints of toxicity tests_using the purple sea

urchin are based on the reduction in proportion of normally

developed larvae. The IC25 is calculated using the Linear

Interpolation Method (see Sectiop 9, Chronic Toxicity Test

Endpoints and Data Analysis). LOEC and NOEC values for

development are obtained using a hypothesis testing approach such

as Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank

Test (Steel, 1959; Miller, 1981) (see Section 9). Separate

analyses are performed for the estimation of the LOEC and NOEC

endpoints and for the estimation of the IC2~. See the Appendices

for examples of the manual computations, and examples of data

input and program output.

15.13.2 EXAMPLE OF ANALYSIS OF PURPLE SEA URCHIN,

STRONGYLOCENTROTUS PURPURATUS, DEVELOPMENT DATA

15.13.2.1 Formal statistical analysis of the larval development

data is outlined in Figure 7. The response used in th~ analysis

is the proportion of normally developed larvae in each test or

control chamber. Separate analyses are performed for the

estimation of the NOEC and LOEC endpoints and for the estimation

of the IC25 endpoint. Concentrations at 'which there are no

normally developed larvae in any of the test chambers are

excluded from" statistical analysis of the NOEC and LOEC, but

included in the estimation of the IC25.

360
TABLE 4. DATA FROM PURPLE SEA URCHIN,

STRONGYLOCENTROTUS PURPURATUS, DEVELOPMENT

TEST

Copper No. Larvae

Concentration No. Larvae Normally Proportion

(jlg/L) Replicate Exposed Develpped Normal

Control A 1.00 87 0.87

B 1.00 89 0.89

C 1.00 81. 0.81.

D 1.01. 89 0.88

E 74 62 0.84

3.2 A 1.1.0 98 0.89

B 1.00 82 0.82

C 1.00 91. 0.91.

D 1.00 83 0.83

E 1.00 89 0.89

5.6 A 1.02 86, 0.8.4

B 1.00 89 0.89

C 1.00 85 0.85

D 1.07 90 0.84

E 1.00 85 0.85

10.0 A 1.00 70 0.70

B 1.00 71.: 0.71.

C 100 77 0.77

D 1.00 74 0.74

E 1.00 87 0.87

1.8.0 A 1.00 7 . 0.07

B 100 1.2 0.12

C 100 14 " 0.14

D 100 1.6 0.16

E 1.00 1.0 ! 0.10

361
NO

.',

tTl:STWtTH

BONFERRdNI

ADJUSTMENT

ENDPOINTeSTIMATE

IC25

:,1,1,HOMOGENEOUS I VARIANCE

Figure 7. Flowchart for statistical analysis of sea urchin,

Strongylocentrotus pur,puratus, development test.

362
15.13.2.2 For the case of equal numbers of replicates across all

concentrations and the control, the evaluation of the NOEC and

LOEC endpoints is made via a parametric test, Dunnett's

Procedure, or a nonparametric test, Steel's Many-one Rank Test,

on the arc sine square root transformed data. lJnderlying

assumptions of Dunnett's Procedure, normality and homogeneity of

variance, are formally tested. The test for normality is the

Shapiro-Wilk's Test, and Bartlett's Test is used to test for

homogeneity of variance. If either of these tests fail's, the

nonparametric test, Steel's Many-one Rank Test, is used to

determine the NOEC and LOEC endpoints. If the assumptions of

Dunnett's Procedure are met, the endpoints are estimated by the

parametric procedure.

15.13.2.3 If· unequal numbers of replicates occur among the

concentration levels tested, there are parametric'and

nonparametric alternative analyses. The parametric analysis is a

t test with the Bonferroni adjustment (see Appendix D) The

Wilcoxon Rank Sum Test with the Bonferroni adjustment is the

nonparametric alternative.

15.13.2.4 Example of Analysis of Development Data

15.13.2.4.1 This example uses toxicity data from a purple sea

urchin, Strongylocentrotus purpuratus, development test performed

with copper. The response of interest is the proportio'n of

normally developed larvae, thus each replicate rnust first be

transformed by the arc sine square root transformation procedure

described .in Appendix B. The raw and transformE~d data, means and

variances of the transformed observations at ea(~h copper

concentration and control are lfsted in Table 5.. The data are

plotted in Figure 8. Because there is zero normal development in

all five replicates of the 32.0 Jlg/L copper concentration, it was

not included in the statistical analysis and is considered a

qualitative development effect.

15.13.2.5 Test for Normality

15.13.2.5.1 The first step of the test for normality is to

center the observations by subtracting the mean of all

observations within a concentration from each observation in that

concentration. The centered observations are s\lmmariz~d in

Table 6.

15.13.2.5.2 Calculate the denominator, D, of the ~tatistic:

n 2

D=L(X.-X)

1.1 ,

I

I

i ,

363
Where: Xi = the ith centered observation

X = the overall mean of the centered observations

n = the total number bf centered observations

15.13.2.5.3 For this set of data, n = 25

X = -l- (-0.001) = 0.000

25

D 0.0680

TABLE 5. SEA URCHIN, STRONGYLOCENTROTUS PURPURATUS,

DEVELOPMENT DATA

Copper Concentration (/lg/L)

Replicate Control 3.2 5.6 10.0 18.0

A 0.87 0.89 0.84 0.70 0;07

B 0.89 0.82 0.89 0.71 0.12

RAW C 0.81 0.91 0.85 0.77 0.14

D 0.88 0.83 0.84 0.74 0.16

E 0.84 0.89 0.85 0.87 0.10

A 2.202 2.234 2.159 0.991 0.268

ARC SINE B 2.234 2.133 2.234 2.002 . 0.354

SQUARE ROOT C 1.120 2.266 1.173 1.071 0.383

TRANSFORMED D 1.217 2. 146 1:. 159 2. 036 0.412

E 2. 159 2.234 1.173 2.202 0.322

Mean (Yi ) 1.186 2.203 2.180 2. 060 0.348

Si 0.00215 0.00351 0.00097 0.00725 0.00311

i 1 2 3 4 5

364
,FigureS. Plot o~ proportion'of normally developed sea urchin,

Strongylocentrotus purpuratus, larvae,
15.13.2.5.4 Order the centered observations from smallest to

largest

where Xli) denotes the ith ordered observation. The ordered

observations for this example are listed in Table 7.

TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S

EXAMPLE

Copper Concentration (~g/L)

Replicate Control 3.2 5.6 10.0 18.0

A 0.016 0.031 -0.0:21- -0.:069 -0.080

B 0.048 -0.070 0.054 -0.058 0.006

C -0.066 0.063 -0.007 0.011 0.035

D 0.031 -0.057 -0.021 -0.024 0.064

E -0.027 0.031 -0.00·7 0.142 -0.026

15.13.2.5.5 From Table 4, Appendix B, for the number of

observations, n, obtain the coefficients a l , a 2, ... a k where k is

n/2 if n is even and {n-1} /2 if n is odd.. For the data in this

example, n = 25 and k = 12. The ai values. are listed in Table 8.

15.13.2.5.6 Compute the test statistic, W, as follows:·

k 2

W = ~.[ La. (X (n-i.l) -x (i)) ]

D i.l

The differences, x(n-i+l} - X(i), are listed in Table 8. For the

data in this example:

W :=; 1

0.0680

(0.2545}2 :=; 0.953

366
,

TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S

EXAMPLE

i Xli} i Xli}

1 -0.080 14 CI.006

2 -0.070 15 CI.011

3 -0.069 16 Cl.016

4 -0.066 17 Cl.031

5 .-0.058 18 0.031

6 -0.057 19 0.031

7 -0.027 20 0.035

8 -0.026 21 0.048

9 -0.024 22 0.054

10 -0.021 23 01.063

11 -0.021 24 0.064

12 -0.007 2·5 0.142

13 -0.007

TABLE 8 . COEFFICIENTS AND DIFFERENCES FOR SHA.PIRO-WILK'S

EXAMPLE

i ai x(n-i+1.) - Xli)

1 0.4450 0.222 X(2S} - X(1.}

2 0.3069 0 .. 134 X(24} - X(2}

3 0.2543 0.132 X(23) - X 13 )

4 0.2148 0.120 X(22} - X(4}

5 0.1822 0.106 X(21.) - Xes)

6 0.1539 0.092 X(20} - X (6 )

7 0.1283 0.058 X (1.9) - X(7}

8 0.1046 0.057 X(1.S} - X(S}

9 0.0823 0.055 :k(17} X(9}

10 0.0610 0.037 ;[(16) - X(10)

11 0.0403 0.032 X(15} - X(1.1.}

12 0.0200 0.013 :jC (H) - X(12)

15.13.2.5.7 The decision rule for this test is to compare W as

calculated in Subsection 5.6 to a critical value found in

Table 6, Appendix B. If the computed W is less tl~an the critical

value, conclude that the data are not normally di~3tributed. For

367
the data in this example, the critical value at a significance

level of 0.01 and n = 25 observations is .0.888. Since W = 0.953

is greater than the critical value, conclude that the data are

normally distributed.

15.13.2.6 Test for Homogeneity of Variance

15.13.2.6.1 The test used to examine whether the variation in

the proportion of normally developed larvae is the same across

all copper concentrations including the control, is Bartlett's

Test (Snedecor and Cochran, 1980). The test'statistic is as

follows:

p p

[ ( LV.) In 52 - LV. In s:]

i·1 i.1

B = ------------ c

Where: Vi = degrees of freedom for each concentration and

control,

p = number of concentration levels including the control

ni = the number of replicates for' concentration i.

In = loge

i = 1,2, ... , p where p is the number of concentration!=!

including the control

p p

C = 1+[3(p,.1)]-1[Ll/V.-(LV.)-1]

i.1 i.1 .

15.13.2.6.2 For the data in this example (see Table 5)', all

concentrations including the control have the same number of

replicates (ni = 5 for all i). Thus, Vi = 4 for all i.

368
15.13.~.6.3 Bartlett's statistic is, therefore:

p

B= [(20)ln(0.00340) - 4Lln(S;)] /1.1.00

i.l . . .

[20(-5.6840) - 4(-29.4325)]/1.lQO

4.050/1.100

= 3.6818

15.13.2.6.4 B is approximately distributed as chi-square with

p-1 degrees of freedom, when the variances are in fact the same.

Therefore, the appropriate critical value for this test·, at a

significance level of 0.01 with 4 degrees of freedom, is 13.28.

Since B = 3.6818 is less than the critical value of 13.28,

conclude that the variances are not different.

15.13.2.7 Dunnett's Procedure

15.13.2.7.1 To obtain an estimate of the pooled variance for the

Dunnett's Procedure, construct an ANOVA table as described ~n

Table 9.

Where: p number of concentration levels including the

control

TABLE 9. 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)

2

Sa SSB/ (p-l)

2

Sw SSW/ (N-p)

N = total number of observations n 1 + n2 ••• + n p

.369
ni = number of observations in concentration i

p

SSB • :E T~/ n ._G 2/N

1.1

p n,L

SST. :E:E Y;j_G 2/N

1.1j.1

SSW. SST-SSB

Between Sum of Squares

Total Sum of Squares

Within Sum of Squares

G = the ~rand total of all sample observations,

G = ET.

1.1

Ti = the total of the replicate measurements for

concentration i

the jth observation for concentration i

(represents the proportion of normal larvae for

concentration i in test chamber j)

15.13.2.7.2 For the data' in this exampl~:

N = 25

T1 Yll + Y12 + Y13 + Y14 + Y1S 5.932

T2 Y21 + Y22 + Y23 + Y24 + Y2S = 6.013

T3 Y31 + Y32 + Y33 + Y3"4 + Y3S = 5.898

T4 = Y41 + Y42 + Y43 + Y44 + Y4S = 5.302

Ts = YS1 +. YS2 + YS3 + YS4 + Yss = 1. 739

p

SSB = E T~/ni-G2/N

.fd. {137.267)/5 - (24;.884)2/25

p n,L

SST = E:EY~.-G2/N

.t=.j.~ 't: 5 21 - (24. 884 ) 2/25 = 2. 752

2.685

SSW = SST-SSB = 2.752 - 2.685 = 0.067

SI = SSB!(p-1) = 2.685/(5-1) = 0.6713-

S~ = SSW/(N-p) = 0.067/(25-5) = 0.0034

370
15.13.2.7.3 Summarize these calculations in the ANOVA table

(Table 10).

TABLE 10. ANOVA TABLE FOR DUNNETT'S PROCEDURE

EXAMPLE

Source

Between

Within

Total

df

4

20

24

Sum of Squares

(S5)

2.685

0.067

2.752

t'l.ean Square (MS)

(SS/df)

0.671.3

0.0034

15.13.2.7.4 To perform the individual comparisOlls, calculate the

t statistic for each concentration, and control combination as

follows:

Where: Yi = mean proportion normal larvae for concentration i

Yl = mean proportion normal larvae ·for the Control

Sw square root of the.within mean square

n l = number of replicates for the contro.l

ni = number of replicates for concentration i.

Since we are looking for a decreased response from the 'control in

the pr~portion of normally developed larvae, the concentration

mean is subtracted from the control mean.

371
~5.~3.2.7.5 Table 11 includes 'the calculated t values for each

concentration and control combination. In this example,

comparing the 3.2 ~g/L copper" concentration with the control the

calculation is as.follows:

t 3 (1.186 - 1. 203 ) 3 -0.461

2 0. 0583 V(1/5)+(1/5)

TABLE II. CALCULATED t VALUES

Copper Concentration (/lg/L) i t i

3.2 2 -0.461

5.6 3 0.163

10.0 4 3.417

18.0 5 22.727

15.13.2.7.6 Since the purpose of this test is to detect a

significant decrease in the proportion of normally developed

larvae, a one-sided test is appropriate. The critical yalue'for

this one-sided test is found in Table 5, Appendix C. For an

overall alpha level of 0.05, 20 degrees of freedom for error and

four concentrations (excluding the control) the critical value is

2.30. The mean proportion of normally developed larvae for

concentration i is considered significantly less than the mean

proportion of normally developed larvae for the control if t i is

greater than the critical value. Therefore, the 10.0 ~g/L and

18.0 ~g/L concentrations have a significantly lower mean

proportion of normally developed larvae than the control. Hence

the NOEC is 5.6 ~g/L copper and the LOEC is 10.0 ~g/L copper.

15.~3.2.7.7 To quantify the sensitivity of the test, the minimum

significant difference (MSD) that can be statistically detected

may be calculated:

Where: d ::: the critical value for Dunnett's Procedure

Sw::: the square root of the within mean square

372
n = ,the common number of replicates' at each, , '

concentration (this assumes equal replication at

each concentration) I

,I

n'l = the number of replicates in the control.

15.13.2.7.8 In this example,

MSD = 2.30 (0.0583) V(l/S)'+ (liS)

2.30 (0.0583) (0.632!3)

I

0.085

15.13.2'.7.9 The MSD '( 0 . 085) is in transformed units. To

determine the MSD in terms of proportion ,of normally developed

larvae, carry out the following conversion:

1. Subtract the MSD from the transformed control mean.

1.186 - 0.085 = 1.101 I

,

2. Obtain the untransformed values for the control mean and,

the difference calculated, in step' 1. of 13.2.7.9.

[ Sine (1..1.86)]2 0.859

[ Sine (1.101) ] 2 =0.7950

3. The untransformed MSD . (MSDu ) is determined by subt:r:acting

the uritransformed values from step 2 in 14:2.7.9.'

MSDu = O. 859 - O. 795 0.064

15.13.2.7.10 Therefore, for this set of data, the minimum

difference in mean proportion of normally developed larvae

between the control and any copper concentrat.ion' that can be

detected as statistically significant is 0.064.

15.13.2.7.11 This represents a 7.5% decrease in:the proportion

of normally developed larvae from the control.

15.13.2.8 Calculation of the ICp

15.13.2.8.1 The development data in Table 4 are utilized in this

example. As can be seen from Figure 9, the observed means are

not monotonically non-increasing with re~pect to concentration.

373
1.01 * * * 1CMIUIl&.~'--.-r ---~'ftW-.......

* ------ ~_~_WILID

0 0.91- * * --

* lLJ * a..

0 0.8 -'

lLJ

>

lLJ 0.7 0

>- 0.6 -'

-'

«

::;: 0.5 0::

0

z 0.4

W z -..J 0

IJ:lo -

I- 0.3 0::

0

0.. 0.2 0

0::

a.. 0.1

0.0

0.0 3.2 5.6 10.0 18.0 32.0

COPPERCONCENTRATION (ugII.)

•

Figure 9. Plot of raw datal observed means, and

urchinl Strogylocentrotus purpuratus,

Tables 4 and 12.

•.......

smoothed means for the sea

larval development data from

•
Therefore, the means must be smoothed prior to palculating the

IC. i , ,

15.13.2.8.2 Starting with the observed control mean, Y1 = 0.858,

and the observed mean for the lowest copper concentration, Y2 =

0.868, we see that Y1 is less than Y2 •

15.13.2.8.3 Calculate the smoothed means:

15.13.2.8.4 Since Y3 = 0.854 > Y4 = 0.758 > Ys ,- 0.118 > Y6 =

0.0, set M3 = 0.854, M4 = 0.758, Ms = 0.118, and M6 = 0.0.

Table 12 contains the smoothed means and Figure 8 gives a plot of

the smoothed means and the interpolated response curve.

15.13.2.8.5 An IC25 can be estimated using the Linear

Interpolation Method. A 25% reduction in mean proportion of

normally developed larvae, compared to the controls, would result

in a mean proportion of 0.647, where M1 (1-p/100) = 0.863(1-

25/100). Examining the means and their associated concentrations

(Table 12), the response, 0.647, is bracketed by C4 = 10.0 ~g/L

copper and Cs = 18,.0 ~g/L copper. '

TABLE 12. SEA URCHIN, STRONYLOCENTROTUS PURPURA~rus,

MEAN PROPORTION OF NORMALLY DEVELOPED LARVAE

Copper Response Smoothed

Cone. Means, Yi Mea:ns, Mi

(/lg/L) i (proportion) (pr<?portion)

Control 1 0.858 0.863

0.05 2 0.868 0.863

0.10 3 0.854 q.854

0.15 4 0.758 1?758

0.20 5 0.118 0.118

0.40 6 0.000 q.OOO

15.13.2.8.6 Using the equation from Section 4.2 in Appendix L,

the estimate of the IC25 is calculated as follows:

375
Cone. ID

Cone. Tested

Response J.

Response 2

Response 3

Response 4

Response 5

J.

o

.87

.89

.8J.

.88

.84

2

3.2

.89

.82

.9J.

.83

.89

3

5.6

.84

.89

.85

.84

.85

4

J.O

.70

.7J.

.77

.74

.87

5

J.8

.07

.J.2

.J.4

.J.6

.J.O

6

32

o

o

o

o

o

*** Inhibition Concentration Percentage Estimate *** Toxicant/Effluent: Copper Chloride

Test Start Date: Test Ending Date:

Test Species: Purple Sea Urchin, Strongylocentrotuspurpuratus

Test Duration: 72 hours

DATA FILE: urch_dev.icp

OUTPUT FILE: urch_dev.i25

Cone.

ID

Number

Replicates

Concentration

ug/L

Response

Means

Std.

Dev.

Pooled

Response Means

J. 5 0.000 0.858 0.033 0.863

2 5 3.200 0.868 0.040 0.863

3 5 5.600 0.854 0.02J. 0.854

4 5 J.O.OOO· 0.758 0.068 0.758

5 5 J.8.000 0.J.J.8 0.035 0.J.J.8.

6 5 32.000 0.000 0.000· 0.000

The Linear Interpolation Estimate: J.1. 3844 Entered P Value: 25

J.1.3702

Lower:

Lower:

0.J.6

Number of Resamplings: 80

The Bootstrap Estimates Mean:

Original Confidence Limits:

Expanded Confidence Limits:

Resampl~ng time in Seconds:

Standard Deviation: 0.2898

J.0.7785 Upper: J.J..9375

J.0.4756 Upper: J.2.2J.4J.

Random Seed: 8376J.380

Figure J.O. ICPIN program output for the IC25.

376
IC25 10.0 + [0.863(1 - 25/100) - 0.758] (18.0 - 10.0)

(0.118 - 0.758)

= 11.38 Jlg/L.

15.13.2.8.7 When the ICPIN program was used to ~nalyze this set

of data, requesting 80 resamples, the estimate of' the IC25 was

11.3844 Jlg/L. The empirical 95.0% confidence interval for the

true mean was 10.7785 Jlg/L to 11.9375 Jlg/L. The computer program

output for the IC25 for this data set is shown in Figure 10.

15.14 PRECISION AND ACCURACY

15.14.1 PRECISION

15.14.1.1 Single Laboratory Precision

15.14.1.1.1 Data on the single-laboratory precis:ion of the

development test using copper as a reference toxicant is provided

in Table 13. The. NOEC varied by only one concent.ration interval

indicating good precision. The coefficient of va:riation for the

EC50 and EC25 were 22% and 21% indicating accepta.ble precision.

15.14.1.2 MUlti-Laboratory Precision

15.14.1.2.1 Data on the multi-laboratory precision of the

development test using copper as a reference toxi.cant is provided

in Table 14. The NOEC for laboratory's A and B ~rere identical.

The difference in NOEC observed for lab C was prQbably due the

wide range of concentrations used (See Footnote 4) . The

coefficient of variation for the EC5Q was 39%, indicating

acceptable interlaboratory precision.

15.14.2 ACCURACY

15.14.2.1 The accuracy of tox1icity tests cannot, be determined.

377
Table 13. SINGLE-LABORATORY PRECISION OF THE PURPLE SEA URCHIN,

STRONGYLOCENTROTUS PURPURATUB, DEVELOPMENT TEST WITH

COPPER (CU ~G/L) SULFATE AS A REFERENCE TOXICANT1.

Test Number NOEC (~g/L) EC50 (~g/L) EC25 (~g/L)

1 10. O· 19.4 15.1

2 10.0 18.3 15.4

3 5.6 10.8 9.0

14 5.6 14.3 11. 0

5 5.6 16.8 12.9 I

Mean 15.9 12.7

CV(%) 22.0 21.0

1 Tests performed by Marine Poilution Studies Laboratory,

Granite Canyon, Monterey California.

TABLE 14. MULTI-LABORATORY.PRECISION OF THE PURPLE SEA URCHIN,

STRONGYLOCENTROTUS PURPURATUS, .DEVELOPMENT TEST WITH

COPPER (CD ~G/L) SULFATE AS A REFERENCE TOXICANT. 1

Lab NOEC (~g/L) EC50 (/1,g/L)

A2 10.0 22.5 - B3 10.0 15.2

C4 1.8 10.1

Mean 15.9

CV(%) 39.0

lData from labs A and B are from an interlaboratory study using split

reference toxicant samples and dilution water. Test performed in August,

1993. Test duration was 72 hr. Concentrations were 3.2, 5.6, 10, 18 and 32

f,lg/L.

2Test performed by Southern California Coastal Water Research Project,

Westminster, CA.

3Test performed by Marine Pollution Studies Laboratory, Granite Canyon,

Monterey California.

4 Test performed by MEC Analytical Systems, Inc., Tiburon, CA. Test

performed in April, 1994. Test duration was 96 hr. Concentrations were 0.1,

0.32, 1.8, 18 and 56 IJ.g/L. .

378
APPENDIX I. SEA URCHIN DEVELOPMENT: STEP-BY""'STElP SUMMARY

PREPARATION OF TEST SOLUTIONS i , I' '" r '

A. Determine test concentrations and appropriate dilution water

based on NPDES permit conditions and guidance from'the

appropriate regulatory agency.

B. Prepare effluent test solutions by diluting well mixed

unfiltered effluent using volumetric flasks and pipettes.

Use hypersaline brine where necessary to maintain all test

solutions at 34 ± 2%0. Include brine controls in tests that'

use brine.

C. Prepare a copper reference toxicant s~ock solution.

D. Prepare a copper reference toxicant series.. Add 1·0 mL of

test solution each vial.

E.

F ..

G.

H.

1.

Sample effluent and reference toxican~ solutions for

physical/chemical analysis. Measure 'salinity, pH and

dissolved oxygen of each test concentration.

Randomize numbers for test chambers and record the chamber

numbers with their respective testconcentr,~tions·ona . randomization data sheet. Store the data sheet Safely,until

after the test samples have been analyzed. ' j

Place test chambers in a water bath or envi:r-onmental chamber

set to 15°C and allow te.mperature to, equilibrate.

Measure the temperature daily in one random replicate (or

separate chamber) of each test concentratioJi. Monitor, the

temperature of the water ba'th or environmental chamber

continuously.

At the end of the test, measure salinity; pH, and dissolved

oxygen concentration from each test concentlration.

PREPARTION AND ANALYSIS OF TEST ORGANISMS

A. Obtain test organisms and hold or condition as necessary for

spawning.

B. Place six 100 mL beakers of dilution water in 15°C water

bath or room. Select 6-8 sea urchins and pJlace on tray

covered with seawater moistened paper towels. Induce

379
spawning by injecting each sea urchin with 0.5 mL 'of 0.5 M

KCI. Place animals back orito tray, oral, side down.

c. When spawning begins,note time that each animal begins

spawning. Leave males on tray for semen collection. 'Place

spawning females oral side up on 100 mL beakers. Do not

collect gametes more than 15 min after spawning begins.

D. Collect semen using either a Pasteur pipette' or a 100 J,.lL

autopipette. Pipette semen from each male 'into a separate 5

mL conical test tube, stored in an ice water 'bath.

E. Check for the motility of sperm from each male.

F. Pool semen by pipetting equal amounts from each "good" male

to another centrifuge tube. At least 0.025 mL should be

taken from each male and a total of at least 0.05mL should

be available. Cover the tube and store in a refrigerator

until ready for use. '

G. Finish collecting eggs before diluting semen.

H. Mix pooled semen by agitating on a vortex mixer. Add about

0.025 mL of semen to a 100 mL beaker containing 50 mL of ~5°C dilution water. Stir thoroughly with a Pasteur

pipette. Test eggs from each female to petermine if they

can be fertilized.

I. Take 0.5 mL subsample of sperm dilution and add to 5 mL of ~O% acetic acid in a 50 mL graduated cylinder. Bring.to 50-

mL with dilution water. Mix well by inversion and load a

drop into each side of hemocytometer. Count ,the sperm in 80

small squares. Calculate the sperm density·using ,Equation 2A. '

J. Examine sample of eggs from each female. Do not use the

eggs from any female whose eggs appear abnormal or that did

not fertilize in Section G.

K. Decant water from eggs of each usable female and pour

through Nitex@ screen into a 1 liter beaker. Bring volume

up to about 600 mL with dilution water. Allow to 'resettle,

siphon about 400 mL of overlying water and bring back to 600

mL with dilution water.

L. Mix egg solution well and make an accurate 100x dilution

using at least 10 mL of the egg solution. Mix the dilution

well and count two different 1 mL subsamples in a counting

380
cell. Use' the mean of ,the two counts in Equation 3 to

determineth;t?" dens~ty of the egg stock.

M. 'Use Equation 4 to determine the 'egg dilution factor and make

dilution of eggs with ,dilution water. .

N. Use Equations 5 A-C to determine the volume of the sperm

dilution that is necessary to fertiiize the egg dilution.

Add the appropriate volume of sperm and after 10 minutes,

check fertilization success.

O. Gently mix the fertilized egg solution with a plunger and

deliver 0.25 mL of egg solution to each vial. Make sure

that the pipette tip is cut off to provide;at least a 0.5 mm

opening. Keep egg solution well mixed during addition

period.

P. Incubate the embryos for 72 hours at 15±1.°C.

Q. Test, termination and analysis

R. Perform wat,er quality measurements as at the start.

S. After 72 hours, add 1.0 mL of 37% buffered 'formalin or 0.5

rnL of 1.0% glutaraldehyde to each test chamber. Tightly cap

and gently 'mix each 'vial.

T. Examine'each sample with a microscope and &et~rmine the

percentage of normally developed embryos.

U. Analyze the data.

V. Include standard reference toxicant ',point estimate values in

the standard quality control charts. "

381
APPENDIX II. USING THE NEUBAUER HEMACYTOMETER TO ENUMERATE SEA.

URCHIN SPERM

The Neubauer hemacytometer is a specialized microscope slide with

two counting grids-and a coverslip.

TOP VIEW:

COVERSLIP

~- SUPPORT

COUNTING GRIDS

(size exaggerated)

(see,detail next page)

382
Together, the total area of ',each grid (1 mm2) and the vertical

distance between the grid and the covers~ip (0.1 mm), provide

space for a specific microvolume of aqueous sample (0.1 mm3 ) •

SIDE VIEW:

Counting Ala / Coverslip

'---- ---:-~_~;__~Well

Loading "

Notch

END VIEW THROUGH ...·UD'"CROSS SECTION:

. ' " Coverslip ,,"

.', j .," 'Counting i'counting

Loading Area.l J.~Area Loading

NOleh-C ' r '';;::r-Noteh

, "" Overflow Well '

383
This volume of liquid and the cells suspended therein (e.g.,

blood cells or sperm cells) represent 1/10,OOOth of theliqu1d

volume and cell numbers of a full milliliter (cm3 ) of the sampled

material.

NEUBAUER

HEMACYTOMETER

GRID OF 400 SQUARES

If the full 400-squares of each grid are counted, this represents

the number of sperm in 0.1 mm3 • Multiplying this value" times 10

yields the sperm per mm3 (and is the source of the hemacytometer

factor of 4,000 squares/mm3 ). If this product is multiplied by

1,000 mm3 /cm3 , the answer is the number of sperm in one

milliliter of the sample. If the counte~ sample represents a

dilution of a more concentrated original sample, the ab9ve answer

is multiplied by the dilution factor to yield the cell density in

the original sample. If the cells are sufficiently dense, it is

not necessary to count the entire 400-square field, and the final

calculation takes into account the number of squares "actually

counted:

cells/mL:: (dilution) (4,000 squares/mmJ) (1,000 mmJ/cmJ) (cell count)

(number of squares counted)

Thus, with a dilution of 4000 (0.025 mL of semen in 100 mL of

dilution water), 80 squares counted, and a count of laO, the

calculation becomes:

cells/mL:: (4,000) (4,000) (1.000) (100)

80

:: 20,000,000,000 cells/mL

384
There are several procedures that are necessary :Eor counts to be

consistent within and between laboratories. These include mixing

the sample, loading and emptying the hematocrit itube, cleaning

the hemacytometer and cover slip, and actual counting procedures.

Obviously, if the sample is not homogeneous, subsamples can vary

in sperm density. A few extra seconds in mixing can save a lot

of wasted minutes in subsequent counting procedures. A full

hematocrit tube empties more easily than one witl~ just a little

liquid, so withdraw a full sample. This can be l:xpedited by

tipping the sample vial.

Because, the sperm are killed prior to ~ampling, 1:hey will slowly

settle. For this reason, the sample in the hemal:ocr:\.t tube

should be loaded onto the hemacytometer as rapidly as possible.

Two replicate samples are withdrawn in fresh hematocrit tubes and

loaded onto opposite sides of a hemacytometer.

Coverslip

Counting 1 Counting

Area Area Lo:ading

r::;:::;::::;::5=='====!===9c:::r======<=i=db_ -- - Notch i . Overflow Well

385
The, loaded hemacytometer is left for 15 minutes:to allow the

sperm to settle onto the counting field. If the coverslip is

moved after the samples are loaded, the hemacytometer should be

rinsed and refilled with fresh sample. After·15'minutesj the

hemacytometer is placed under a microscope and the cou~tihg grid

located at 100x. Once the grid is properlypo'sitioned,' the

microscope is adjusted to 200x or 400x, and one of the corner

squares fs positioned for counting (anyone of the four corners

is appropriate). For consistency, use the same,procedure each

time (Many prefer to start in the upper'left corner of the'

optical field, and this procedure will be'used in the examples

given below). Examine the first large square in the selected

corner. If no sperm are visible, or if the sperm are so dense or

clumped to preclude accurat,e' counting,' count a sample with a more

appropriate dilution.

In making counts of sperm~ it is necessary to adopt a consistent

method of scanning the smaller squares and counting sperm that

fall upon the lines separating the squares. Count the sperm in

the small squares by beginning in the upper left hand corner

(square 1) and proceding right to squa~e 4, ~own to square 5,

left to square 8~ etc. until all 16 squares are counted. .'

.,

.. • 0 P :

p

1 C\,2 OJ 3 go 0 , . p 4

• .. C\,

p

p p •

a" " • 7 .. 6 5

0 " • cf

" • , .

9 cf io 11 " J o 12 a

• a " cf g..

" q

16· 15 " 14 • 13 u

386
Because sperm that appear.Qll lines mignt be counte~as being. in

either square, it is important to avoid double counting or no~­

counti~g. For this reason a convention is decided,uponand used

consistently:, pare:tphrasing the instructions recE;dved with one, "

(H~usser Scientific) 'counting· chamber, "to avoid ,counting' (sperm),

twice, the best practice, is to count. all touching the "top and

left, and none touching the low~r and right, bOl.:Lndary l·ines."

Whatever convention is chosen, it must be adhereld to., The

example below shows' a. sperm count, based upon a slelected.

convention of counting sperm that fallon the uf~per and left

lines, but not on the lower or right l;ines:

27

28

0=

Not

Counted

54

0)2 0 13

1 03 .p8 12

9 pU

4q. 5 10) g. .~ . 60 ' 10 ,11p, . q. 25. ,20 lOG .. is<'>

24 22 P16,

.P 021 ,C II

'026 '017 23 OJ 19 O'

G29 '032 ' 33 36 ;p 37

.liD!

30'0 034

039 0', (

D

</' 31 35... 38

" 0 41 42d

52 ' . . ..~ 46 , 0 ''11, </' '51 49 8- 44 I, 48 , 47 '043 OJ

053 500 045

r-...

\

'0 .. '-' '

"

In the above illustration, sperm falling on the lower and right

lines are not counted.~ The count 'begins at the upper left as

illustrated in the preceding'figure. A typical (rOunt sequence is

demonstrated by the numbers next to each sperm i]~lustrated.

Sperm identified as numbers ~, 5', 13,20,'27,28; 33, 5,1 and 54

touch lines and are counted as being in the square below them or

to their right. The circled sperm are not: counte~d as being in

this field of 16 small squares (but they would bel included in any

counts of adjacent squares' in which they would be! on upper or

left' hand lines) .

387
Once these counting conventions have been selected, it is

advisable to follow another strict protocol outlining the number

and sequence of large squares to be counted. Because the sperm

may not be randomly distributed across the counting grid, it is

recommended to count an array of squares covering the entire

grid. The follo~ing procedure is recommended:

Count the number of sperm in the first large square.

1. If the number is less than'lO, count all 25 squares using

the same scanning pattern outlined above (left to right

through squares 1 to 5, down to square 6, left through

square 10, down to 11, etc.). See pattern no. 3.

2. If the number is between 10 and 19, count 9 large squares

using pattern no. 2.

3. If the number is 20 or greater, count 5 large squares using

pattern no. 1.

1 2

3

4 5

Pattern no. 1

1 2

4 3

5

7 6

8 9

Pattern no. 2

1 2 3 4 5

10 9 8 7 6

11 12 13 14 15

20 19. 18 17 16

21 22 23 24 25

Pattern no. 3

The final consideration in achieving good replicate counts is

keeping the ~emacytometers and 'coverslip~ clean. They should be

rinsed in distilled water soon after use. The coverslips should

be stored in a good biocleanser such as hemasol. For an hour or

so prior to use, the hemacytometer slides should also be soaked

in the solution. Both slides and coverslips should then be

rinsed off with reagent water, blotted dry with a lint-free

tissue, and wiped with lens paper.

388
SECTION 16

" 1

PURPLE, URCHIN, Strongylocentrotus pUJ::,puratus,

AND- SAND DOLLAR, Dendraster excentricus

FERTILIZATION ,TEST METHOD

Adapted from a method developed ,by

Gary A': Chapman, U': S. EPA, ORD Newport, OR 97365'

,. and Debra L. Denton, U.S. EPA, Region IX, CA 94105

TABLE OF CO~TENT$

Appendix I

16.1

16.2

16.3

16.4

16.5

16.6

16.7

16.8

16.9

16.10

16.11

16.12

16.13

16.14

Scope and ApplicatIon

Summary of Method

Interferences

Safety

Apparatus and Equipment

Reagents and Supp,lies ,

Effltlents'and R~ceivirig Water Collection,

Preservation, and Storage

Calibrat,~6n and Standardization

Quality Control'

Test Proc'edures . .

Summary of ~est Conditions and Test

Acceptability· Criteria

Acceptability of Test Results

Data Analysis

Precision and Accuracy

Step-by Step Summary

389
.SECTION 16

SEA URCHIN, Strongylocentrotus purpuratus

AND SAND DOLLAR, Dendraster excentricus

FERTILIZATION TEST

16.1 SCOPE AND APPLICATION

16.1.1 This method estimates the chronic toxicity of effluents

and receiving waters to.the gametes of sea urchins,

(Strongylocentrotus purpuratus) , or sand dollars (Dendraster

excentricus) during a static non-renewal 20 minute sperm exposure

and a subsequent 20 minute exposure period following the addition

of eggs for measuring the fertilizing capacity of. the sperm. The

effects include the synergistic, antagonistic, and additive

effects of all chemical, physical, and biological components

which adversely affect the physiological 'and biochemical

functions of the test organisms.

16.1.2 The purpose of the test is to determine the

concentrations of a test substance that reduce egg fertilization

by exposed sperm relative to that attained by sperm in control

solutions. Concentrations of materials advers.~ly affecting egg

fertilization under the conditions of this test are· usually

acutely and chronically toxic to one or more of several common

marine test species and, by extension, are presumably acutely and

chronically toxic to other of the many untested marine species.

16.1.3 Detection limits of the toxicity of an effluent or

chemical substance are organism dependent.

16.1.4 Brief excursions in toxicity may not be detected using

24-h composite samples. Also, because of the long sample

collection period involved in composite sampling and because the

test chambers are not sealed, highly volatile and highly

degradable toxicants in the source may not be detected in the

test.

16.1.5 This test is commonly used in one of. two forms: (1) a

definitive test, consisting of a minimum of five effluent

concentrations and a control, and (2) a receiving water test(s),

390
consisting of one or more receiving water concentrations and a

control.

16.1.6 This method should be restricted·to use by, or under the

supervision of, professionals experienced in aquatic toxicity

testing. Specific experience with any toxicity test is usually

needed before acceptable r~sults become routine.

16.2 SUMMARY OF METHOD

16.2.1 The method provides the step-by-step insjtructions fOr

exposing sperm suspensions (appropriate sperm delJ.sity may first

be determined in a trial fertilization test) to effluents or

receiving waters for 20 minutes. Eggs are then added t·o the

sperm suspensions and, twenty minutes after the i:!ggs are' added,

the test is terminated by the addition of a preservative. The

percent fertilization is determined by microscopic examination of

100 eggs in an aliquot of eggs from each.treatment. The test

endpoint is normal egg fertilization.

16.3 INTERFERENCES

16.3.1 Toxic substances "may be introduced by contaminants in

dilutio~ water, glassware, sample hardware, and testing equipment

(see Section 5, Facilities, Equipment, and Supplies) .

16.3.2 Improper effluent sampling and handling may adversely

affect test results (see Section 8, Effluent and Recei~ing Water

Sampling and Sample Handling, and Sample Prepara~ion for Toxicity

T.ests) .

16.4 SAFETY

16.4.1 See Section 3, Health and Safety

16.5 APPARATUS AND EQUIPMENT

16.5.1 Tanks, trays, or aquaria - - "for holding and acclimating

adult sea urchins and sand dollars, e.g., standard salt water

aquarium or Instant Ocean Aquarium (capable of ma'intaining

seawater at 10-20°C), with appropriate filtration and aeration

system.

391
~6.5.2 Air pump, air lines, and air stones -- for aerating water

containing broodstock or for supplying air to test solutions with

low dissolved oxygen.

~6.5.3 Constant temperature chambers or water baths -- for

maintaining test solution temperature and keeping dilution water

supply, gametes, and embryo stock suspensions at test t~mperature

(~2°C) prior to the test. (Incubators are usually unsatisfactory

because test tubes must be removed for addition of sperm and eggs

and the small test volumes can rapidly change temperature at

normal room temperatures.)

~6.5.4 Water purification system -- Millipore Super-Q, Deionized

water (DI) or equivalent.

~6.5.5 Refractometer

~6.5.6 Hydrometer(s)

for determining salinity.

for calibrating refractometer.

~6.5.7 Thermometers, glass or electronic, laboratory grade

,for measuring water temperatures.

~6.5.8 Thermometer, National Bureau of Standards Certified (see

USEPA METHOD 170.1, USEPA, 1979) -- to calibrate laboratory

thermometers.

16.5.9 pH and DO meters -- for routine physical and chemical

measurements.

16.5.10 Standard or micro-Winkler apparatus -- for determining

DO (optional) and calibrating the DO meter.

16.5.11 Winkler bottles -- for dissolved oxygen determinations.

16.5.12 Balance Analytical, capable of accurately weighing to

0.0001 g.

16.5.13 Fume hood to protect the analyst from effluent or

formaldehyde fumes.

16.5.14 Glass stirring rods -- for mixing test solu~ions.

392
16.5.15 Graduated cylinders -- Class A, borosilicate glass or

non-toxic plastic labware, 50-1000 mL for making test solutions.

(Note: not to be used interchangeably for game:tes or embryos and

test solutions).

16.5..16 Volumetric flasks -- Class A, borosilicate glass or nontoxic plastic labware, 10-1000 mL for making test solutio~s.

16.5.17 Pipets, automatic ,- - adj ustable, to cover a range of

delivery volumes from 0.010 to 1.000 mL.

16.5.18 Pipet bulbs and fillers -- PROPIPET@ or equivalent.

,16.5.19 Wash bottles -- for reagent water, for topping off

graduated cylinders, for rinsing small glassware and instrument

electrodes and probes.

16.5.20 Wash bottles -- for dilution water.

16.5.21 20-liter cubitainers or polycar~onate 1~ater cooler jugs

-- for making hypersaline brine.

16.5.22 Cubitainers, beakers, or similar chamb(=rs of non-toxic

composition for holding, mixing, and dispensing dilution water

and other general non-effluent, non-toxicant contact uses. These

should be clearly labeled and not used· for othelr purposes.

Strong solutions of NaOH and formaldehyde should not be held for

several month periods in Cubitainers: interaction or leaching

into solutions of 0.1 N or 1 N NaOH used for pH adjustment of

dilution water has caused poor egg fertilization; forrn~ldehyde

similarly stored has induced abberant partial mE~mbrane elevation

in eggs.

16.5.23 Beakers, 5-10 mL borosilicate glass -~ for collecting

sperm from sand dollars.

16.5.24 Beakers, 100 mL borosilicate glass -- for spawning; to

support sea urchins and to collect sea Urchin and sand dollar

eggs.

16.5.25 Beakers, 1,000 mL borosilicate glass

settling sea urchin eggs.

393

for rinsing and
16.5.26 Vortex mixer -- to mix sea-urchin semen in tubes prior

to sampling.

16.5.27 Compound microscope -- for examining gametes, counting

sperm cells (200-400x) and eggs (lOOx), and examining fertilized

eggs. Dissecting scopes are sometimes used to count eggs at a

lower magnification.

16.5.28 Counter; two unit, 0-999 -- for recording sperm and egg

counts.

16.5.29 Sedgwick-Rafter counting chamber -- for counting egg

stock and examining eggs for fertilization at the end of the

test.

16.5.30 Hemacytometers, Neubauer for counting sperm.

16.5.31 Siphon hose (3 mm i.d.) -- for removing wash water from

settled eggs.

16.5.32 Centrifuge tubes, test tubes, or vials -- for holding

semen.

16.5.33 Perforated plunger -- for maintaining homogeneous

distribution of eggs during sampling and distribution co test

tubes.

16.5.34 60 ~m NITEX® filter

16.6 REAGENTS AND SUPPLIES

for filtering receiving water.

16.6.1 Sample containers -- for sample shipment and storage (see

Section 8, Effluent and Receiving Water Sampling, and Sample

Handling, and Sample Preparation for Toxicity Tests) . .

16.6.2 Data sheets (one set per test)

Figures 1 and 2) .

for data recording (see

16.6.3 Tape, colored -- for labelling test chambers a~d

containers.

16.6.4 Markers, water-proof -- for marking containers, etc.

394
16.6.5 Parafilm .-- to cover graduated cylinders,a~d vessel$

containing gametes. '

16.6. 6 Glove~, gisposable.

contaminat;i.on.

for personal prot,ection f,rom

'16.6.7 Pipets, serological -- 1-10 mL, graduatl~d"

16.6. 8 ~ipe:t tips, ,- -.fQr automatid pipets. No1~: pipet tips for

handling semen should be cut off to· produce an 6pening about 1 mm

in diameter; pipet tips 'for handling eggs should be cut off to

produce an opening about 2·· mm.. in. diameter. This is necessary to

provide smooth .. flow, of 1:he .viscous semen, accurate samplj.ng of.

eggs, and to prevent injury to eggs passing through a restricted

opening. A clean razor blade can be used to trim pipet tips.

16.6.9 Coverslips -- for microscope slides.

16 6 .10 Lens paper - - for cleaning microscope optics.':'"

16.6,,11 Laboratory tissue wipes - - for cleaning: and drying

electrodes, microscope slides, 'etc.

16.6.12 Disposable countertop covering for protection of work

surfaces and. miniplizing spills and. contamination..

16.6.13 pH buffers 4, 7, and 10 (or as per instructions of

instrum~nt.manUfacturer) -- ·for standards and.calibration check·

(see USEPA Method 150.1, USEPA, 1979).

16.6.14 Membranes and filling solutions '-- for dissolved oxygen

probe .(see USEP,A Metho<;:i 360.• 1,- U:SEPA, 1979), or reagents for

modified Winkler analysis. .

16.6.15 Laboratory quality assurance samples and standards

for the abov~ methods.

16.6.16 Test chambers -- test tubes, borosilicate glass, 16 x

100 mm or 16 x 125 mm, .with :caps f.or conducting the test four

chambers per concentration.

395
Figure 1. Sample data sheet for spawning record.

Animal

NQ.

1

2

3

4

5

6

7

8

9

10

11

12

Time

Injected Spawn Comments

Pooled eggs from female nos.

Pooled ( mL) of sperm each from male nos.

396
Figure 2. Sample data sheet for egg and sperm counts.

EGG COUNTS

Sample Dilution Count Eg9s / mL

For 100 mL egg suspension at 2,240 eggs/mL use:

100 mL x 2,240 eggs/mL / {counted eggs/mL) '=. mLdfegg s.tock

224,000 eggs / eggs/mL = mL

If required stock >100 mL, concentrate egg stock by settling the

eggs and decanting off sufficient overlying water to retain:

SPERM COUNTS -

Sample

SPERM/mL

eggs/mL / ~,240 eggs/mL) x 100 =

Dilution Squares

<nIL, FACT,) (COUNT) (4,000) (l000)

(NO. SQUARES COUNTED)

397

% volume
16.6.~7 Formaldehyde, ~O%, in seawater -- for preserving eggs.

Note: formaldehyde has been identified as a carcinogen and is

irritating to skin and mucous membranes. r't should not be used

at a concentration higher than necessary to achieve morphological

preservation of larvae for counting and only under conditions of

maximal ventilation and minimal opportunity for volatilization

into room air~

16.6.~8

~6.6.~3

~6.6.~9

seawater

counts.

Glutaraldehyde, 1% in seawater -- for preserving eggs.

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

Acetic acid, ~O%, reagent grade, in filtered (~O~)

-- for preparing killed sperm dilutions for sperm

~6.6.20 Haemo-Sol or equivalent cleaner -- for cleaning

hemacytometer and cover slips.

~6.6.2~ 0.5 M KCl solution -- for inducing spawning.

~6.6.22 Syringe, disposable, 3 or 5 mL-- for injectiI1;g KCl into

sea urchins and sand dollars to.induce spawning.

~6.6.23 Needles, 25 gauge --' for injecting KCl.

~6.6.24 Pasteur pipets and bulps -- for.sampling eggs from

spawning beakers.

~6.6.25 Hematocrit capillary tubes -- for sampling sperm for

examination and for loading hemacytometers.

~6.6.26 Microscope well-slides -- for pre-test assessment of

sperm activity and egg condition.

16.6.27 Reference toxicant solutions (see 16.10.2.4 and Section

4, Quality Assurance) .

16.6.28 Reagent water -- defined as distilled or deionized water

that does not contain substances which are toxic to the test

organisms (see Section 5, Facilities, .Equipment, and Supplies and

Section 7, Dilution Water) .

398
16.6.29 Effluent and receiving, water -- see Section 8,· Effluent

and Surface Water Sampling, and Sample Handling, and Sample

Preparation for Toxicity Tests.

16.6.30 Dilution water and hypersaline brine·-- see Section 7,'

Dilution Water and Section 16.6.24, Hypersaline :Brines. The

dilution water should be uncontaminated 1-J.Lm-filtered .natural

seawater. Hypersaline brine should be prepared from dilution

water.

16.6.31 HYPERSALINE BRINES

16.6.31.1 Most industrial and sewage treatment effluents

entering marine and estuarine systems have littH: measurable

salinity. Exposure of larvae to these effluentsiwill usually

require increasing the salinity of the test solu·tions.. It is

important to maintain an essentially constant salinity across all

treatments. In some applications it may be desirable to match

the test salinity with that of the receiving wat~r (See Section

7.1). Two salt sources are available to.adjust salinities --

artificial sea salts and hypersaline brine (HSB) derived from

natural seawater. Use of artificial sea salts is necessary only

when high effluent concentrations preclude salinity adjustment by

HSB alone.

16.6.31.2 Hypersaline brine (HSB) can be made by concentrating .'

natural seawater by freezing or evaporation. HS13 should be made

from high quality, filtered seawater, and can be added to the

effluent or to reagent water to increase sali,nity.. · HSB has

several desirable characteristics for use in effluent to~icity

testing. Brine derived from natural seawater contains the

necessary trace· metals, biogenic colloids, and s()me of the . .

microbial components necessary for adequate growth, survival,

and/or reproduction of marine and estuarine organisms, and it can

be stored for prolonged periods without any apparent degradation.

However, even if the maximum salinity HSB (100~) ,is used as a

diluent, the maximum concentration of effluent «()~) that can be

tested is 66% effluent at 34~ salinity (see Table 1).

16.6.31.3 High quality (and preferably high salinity) sea·water

should be filtered to at least 10 J.Lm before placiJ;1g into che

freezer or the brine generator. Water should be~collected on an

incoming tide to minimize the possibility of contamination.

399
~6.6.3~.4 Freeze Preparation of Brine

~6.6.3~.4.~ A convenient container for making HSB by freezing is

one that has a bottom drain. One liter ,of brine can be made from

four liters of seawater.. Brine may be collected.by partially

freezing seawater at -10 to -20°C until the rem~ining liquid has

reached the target salinity. Freeze for.approximately six' hours,

then separate the ice (composed mainly of freshwater) from the

remaining liquid (which has now become hypersaline) .

~6.6.31.4.2 It is preferable to monitor the water until the

target salinity is achieved rather than allowing total freezing

followed by partial thawing. Bri~e salinity should never exceed

~OO~ •. It is advisable not to exceed abo~t 70~ brine salinity

unless it is necessary to test effluent concentrations greater

than 50%.

~6.6.3~.4.3 After the required salinity is attained, the HSB

should be filtered through a 1 ~m filter and poured directly into

portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity'and the date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been acceptable). HSB i~ usually

of acceptable quality even after several months in storage.

~6.6.32.5 Heat Preparation of Brine

~6.6.3~.5.1 The ideal container for making brine using heatassisted evaporation of 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 seaw~ter 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 contaminqte the brine. One

successful method is to use a thermostatically controlled heat

exchanger made from fiberglass. If aeration is applied, use only

oil-free air compressors to prevent contamination.

~6.6.3~.5.2 Before adding seawater to the brine generator,

thoroughly clean the generator, aeration supply tube, heater, and

any other materials that will be in direct contact with the

400
brine. A good quality biodegradable det~rgent should'be used,

followed by several (at least three) thorough reagent water

rinses.

16.6.31.5.3 Seawater should be filtered to at IE~ast- 10 Jlm be'fore

being put into the brine generator. The temperatmre 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%0 and that the temperature does not, exceed 4QoC.

Additional seawater may be added to the brine to obtain the

volume of brine required.

16.6.31. 5.4 After the reql.lired s~linity is attained, the HSB

should be filtered through a 1 Jlm filter-and poured directly into

portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitable). The brine st()rage cOI.ltainers should

ge capped and labelled with the salinity and 'the date the brine

was generated. Containers of HSB should be stOrE~d in tp.e dark at

4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after several months in storage.

16.6.31.6 Artificial Sea Salts

16.6.31.6.1 No data from sea urchin or sand do~lar fertilization

tests using sea salts are available for evaluation at this time,

and their use- should be considered provisional. The use of GP2

artificial seawater '(Table 2) has been found to provide control

fertilization equal to that of natural s~awater..

16.6.31.6.2 The GP2 reagent grade chemicals (Table 2) should, he

mixed with deionized (DI) water or its equivalen1: in a -single

batch, never by test concentration or replicate.' The reagent'

water used for hydration should be between 21-26"C. The

artificial seawater must be conditioned (aerated) for 24 h before

use as the testing medium. If the solution is to beautoclaved,

sodium bicarbonate is added after the' solution'has cooled. A

stock solution of sodium bicarbonate is made up by'~issolving

33.6 g NaHC03 in 500 mL of reagent wate:r:. :Add 2.1

5 mL o~ this

stock solution for each liter of the,GP2 artificial seawater.

16.6.31.7 Dilution Water Preparation from Brine

4;01
~6.6.3~.7.~ Although salinity adjustmen~ with brine is the

preferred method, the use of high salinity brines and/or reagent

water has sometimes been associated with discernible adverse

effects on test organisms. For this reason, it is recommended

that only the minimum necessary volume of brine and reagent water

be used to offset the low salinity of the effluent, and that

brine controls be included in the 'test. The remaining dllution

water should be natural seawater. Salinity may be adjusted in

one of two ways. First, the salinity of the highest 'effluent

test concentration may be adjusted to an acceptable salinity, and

then serially diluted. Alternatively, each effluept

concentration can be prepared individually with appropriate

volumes of effluent and brine.

~6.6.3~.7.2 When HSB and reagent water are used, thoroughly mix

together the reagent water and HSB before mixing in the effluent.

Divide the salinity of the HSB by the expected test salinity to

determine the proportion of reagent water to brine. For example,

if the salinity of the brine is ~oo~ and the test is to be

conducted at 34§o, ~oo~ divided by 34~ = 2.94. rhu!3,the

proportion is one part brine plus'1.94 parts reagent: water). To

make L of dilu~ion water at 34§o salini~y from a HSB of 100~,

340 mL of brine and 660 mL of reagent water are. required. Verify

the salinity of the resulting mixture using a refractometer.

~6.6.3~.8 Test Solution Salinity Adjustment

~6. 6. 3~. 8. Table 3 illustrat.es the preparation of test

solutions (up to 50% effluent) at 34~ by combining effluent,·

HSB, and dilution water. Note: if the highest effluent

concentration does not exceed 50% effluent, it is convenient to

prepare brine so that the sum of the ef.fluent salinity .and brine

salinity equals 68§oj the required brine vol~me is then always

equal to the effluent volume needed for each effluent

concentration as in the example in Table 3.

~6.6.3~.8.2 Check the pH of all brine mixtures and adjust to

within 0.2 units of dilution water pH by adding, dropwise, dilute

hydrochloric acid or sodium hydroxide.

402
TABLE 1. MAXIMUM EFFLUENT CONCENTRATION (%) THAT CAN BE TESTED

AT. 34%0 WITHOUT THE ADDITION OF DRY SALTS GIVEN THE

INDICATED EFFLUENT AND BRINE SALINITIES.

Effluent Brine Brine Brine Brine Brine

Salinity 60 70 80 90 100'

%0 %0 %0 %0 %0 %0

0 43.33 51.43 57.50. 62.22 66.00

1 44.07 52.17 58.23 62.92 66.67

2 44.83 52.94 58.97 63.64 . 67.35

3 45.61 53.'73 59.74 64.37 . 68.0.4 ,

4 46.43 54.55 60.53 65.12 . 68.75

5 47.27 55.38 61.33 65.88 69.4.7

10 52.00 60.00 65.71 70.00 73.33

15 57.78 ; 65.45 70.77 74. 67 77.65

20 65.00 72.00 76.67 ,80.00 82.50

25 74.29 80.00 83.64 86.15 88.00

:

16.6.31.8.3 To calculate the amount' of 'brine to add to each

effluent dilution, 'determine the following quan1:it'ies: salinity

of the brine (SB, in %0), the salinity of th.e effluent (SE, in

%0), and volume of the effluent to be added (VE.~ in mL') • Then

use the following formula to calculate the volurneof brine (VB',

inmL) to be added:

VB = VB x (34 - SE) / (SB -. 3~t)

16.6.31.8.4 This calculation assumes'that dilution water

salinity is 34 ± 2%0.

16.6.31.9 Preparing Test Solutions

403
TABLE 2. 'REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2

~TIFIG~AL pE~WATER FOR THE PURPLE URCHIN

STRONGYLOCENTROTUS PURPURATUS, AND SAND DOLLAR

DENDRASTER EXCENTRICUS TOXICITYTEST1 ,2

Compound Concentration

(giL)

Amount (g)

Required for

20 L

NaCl 23.90

Na2S04 4.00

KCl 0.698

KBr 0.100

Na2B40 7 • 10 H2O 0.039

MgCl2 • 6 H2O 10.80

CaCl2 • 2 H2O 1.50

SrCl2 • 6 H2O 0.025

NaHC03 0.193

478.0

80.0

13.96

2.00

0.78

216.0

30.0

0.490

3.86

lModified GP2 from Spotte et al. (1984)

2The constituent salts and concentrations were taken from USEPA

(1990b). The salinity is 34.0 giL.

16.6.31.9.1 Five mL of test solution are needed for each test

chamber. To prepare test solutions at low effluent

concentrations «6~), effluents may be added. directly to dilution

water. For example; to prepare 1~ effluent, add 1.0 mL of

effluent 'to a 100-mL volumetric flask using a volumetric pipet or

calibrated automatic pipet. Fill the volumetric flask to the

100-mL mark with dilution water, stopper it, and shake "to mix.

Pour into a (150-250 mL) beaker and stir. Distribute equal

volumes into the replicate test chambers. The remaining test

solution can be used for chemistry.

16.6.31.9.2 To prepare a test solution at higher effluent

concentrations, hypersaline brine must usually be used. For

404
example, to prepare 40% effluent, add 400 mL of :effluent to a 1-,

liter volumetric flask.' 'Then, assuming an effluent salinity of

2%0 and a brine salinity of 66%0, add 400 mL of brine (see,

equation above and Table 3) and top off the flask with dilution

water. Stopper the flask and shake well. Pour into a (100-250

mL) beaker and stir. Distribute equal volumes i.nto the replicate

test chambers. The remaining test solution can ,be used for '

chemistry.

16.6.31.10 Brine Controls

16.6.31.10.1 Use brine controls in all tests, where brine is

used. Brine controls contain the same volume of; brine as does

the highest effluent concentration using brine, plus the volume

of reagent water needed to reproduce the hyposalinity of the

effluent in the highest concentration, plus dilultion water.

Calculate the amount of reagent water to add to ,brine controls by,

rearranging the above equation, (See, 16. 6 . 33 . 8 .! 3) sett ing SE = ! '

0, and solving for VE.

VE = VB x (SB - 34)/(34 - SE:)

If effluent salinity is essentially 0%'0, the reag'ent wat'er volume

needed in the brine control will equal the effluent volume at the

highest test concentration. However, as effluent salinity and

effluent concentration increase, less reagent watervolti.me'is"'"

needed.

16.6.32 TEST ORGANISMS, PURPLE URCHINS

16.6.32.1 Sea Urchins, Strongylocentrotus purpuratus

(approximately 6, of each sex per test).

16.6.32.2 Adult sea urchins (Strongylocentrotus purpuratus) can

be obtained from commercial suppliers or'collectedfrom

uncontaminated intertidal or subtidal areas. State collection

permits are usually required for collection of sea urchins and,

collection is prohibited or restricted in ,some areas. The,

animals are best transported "dry," surrounded either by'moistseaweed or paper towels dampened with seawater. Animals should

be kept at approximately their collect,ion or cul~ture temperature

to prevent thermal shock which can prematurely induce spawning.

405
TABLE 3. EXAMPLES OF EFFLUENT DILUTION SHOWING> VOLUMES OF

EFFLUENT (AT X~), BRINE, AND DILUTION WATER ~EEDED FOR

ONE LITER OF EACH TEST SOLUTION.

FIRST STEP:

to achieve a

for dilution

Combine brine with reagent water or natural

brine of 68-x~ and, unless natural seawater

water, also a brine-based dilution water of

seawater

is used

34~.

SERIAL DILUTION:

Step 1. Prepare the highest effluent concentration to be tested by adding

equal volumes of efflue~t and brine to the appropriate volume of dilution

water. An example using 40%' is shown.

, Effluent Cone. Effluent xg;, Brine (68- Dilution Water'll"

(%) x)g;, 34g;, , .. .. 40 800 mL 800 mL 400 tT\L

Step 2. Use either serially prepared dilutions of the highest test

concentration or individual dilutions of lClO%' effluent.

Effluent Cone. (%') . Effluent.Source Dilution, Water-* (34g;,)

20 1000 mL of 40%' 1000 mL ..

I 10 1000 mL of 20%' 1000 mL

I 5 1000 mL of 10%' 1000 dmL ' "

., ,

I 2.5 1000 mL of 5%' 1000 mL

Control none 1000 mL

INDIVIDUAL PREPARATION'

Effluent Cone. Effluent xg;, Brine (68-x)t. Dilution Water*

(%) >, 34g;, ,>

40 400 mL 400 mL 20Cl mL

>' 20 200 mL 200 mL 6,00 mL

10 100 mL 100 mL .. 800 mL ....

i

5 50 mL 50 mL .. 900 mL

2.5 25 mL " 25 mL .." 950 'mL

Control none none 1000 mL

*May be natural seawater or brine-reagent water equivalent.

406
16.6.32.3 The adult sea urchins are maintained in glass aquaria

or fiberglass tanks. ,The tanks are supplied ,continuously

(approximately 5 L/min) with filtered natural seawater, or salt

water prepared from commericial sea salts is recirculated. The

anitllals are checked daily and any obviously unhealthy animals are

discarded.

16.6.32.4 Although ambient temperature seawater is usually

acceptable, maintaining sea urchins in spawning condition usually

requires holding at a relatively constant temperature. The

culture unit should be capa~le of maintaining a constant

temperature between 10 and ,~4°C, with a water teI~perature control

device.

16.6.32.5 Food for sea urchins -- kelp, 'recommended, but not

necessarily limited, to, Laminaria sp., HedophylJum sp.,

Nereocystis sp., Macrocystis sp., Egregia sp., ,Alaria sp. or

romaine lettuce. The kelp should b~ gathered frbm known,

uncontaminated zonesbr' ,obtained from commerical supply house's

whose kelp com~s from known uIicqntaminated,areas, or romaine

lettuce. Fresh food ,is introduced into the tan~s at least

several times a week. ' Sun dried (12-24 hours) or oven dried

. (60°C overnight) kelp, stores well at room temperature or frozen,

rehydrates well and is adequate to maintain sea' urchins for long

periods. Decaying food and fecal pellets are removed as

necessary to prevent fouling.

16.6.32.6 Natural seawater (>30%0) 'is used to maintain the adult

animals. and (~32%o) as a <;::ontrQI,water in ,the tests,.

16.6.32.7 Adult male and female (if sexes known) animals used in

field studies are transported in separate or partitioned

insulated boxes or coolers' packed with wet kelp or paper

,toweling. Upon arrival at the,;Eield site, aquaria (or a single

partitioned aquarium) are filled with control water, loosely

,covered with a styrofoam sheet and allowed to equilibrate to the

holding'temperature before, animals are added. ~ealthy animals

will attach ,to the kelp or, aquarium within hour:s.

16.6.32.8 To successfully maintain about 25 adult animals for

seven days at a field site, 40-L glass aquaria. using aerated,

recirculating, clean saline water (32%0) and a gravel bed

filtration system, are housed within a water bath, such as an

407
INSTANT OCEANR Aquarium. 'The sexes should be held separately if

possible.

~6.6.33 TEST ORGANISMS, SAND DOLLARS

~6.6.33.~ Sand Dollars, Dendraster excentricus, (approximately 6

of each sex per test) .

~6.6.33.2 Adult sand dollars (Dendraster excentricus) can be

obtained from commercial suppliers or collected from subtidal

zones (most areas) or from intertidal zones of some sheltered

waters (e.g., Puget Sound). State collection permits may be

required for collection of sand dollars and collection prohibited

or restricted in some areas. The animals are best transported

"dry,1t surJ;ounded either by moist seaweed or paper towels

dampened with seawater. Animals should be kept at approximately

their collection or culture temperature to prevent thermal shock

which can prematurely induce spawning.

~6.6.33.3 The adult sand dollars are maintained in glass aquaria

or fiberglass tanks. The tanks are supplied continuously

(approximately 5 L/min) with filtered natural seawater, or

saltwater prepared from commercial sea s~lts is recirculated.

The animals are checked daily and any obviously unhealthy animals

are discarded. For longer periods than a few days, several

centimeters or more of a sand substrate may be desirable.

~6.6.33.4 Although ambient temperature seawater'is usually

acceptable, maintaining sand dollars in spawning condition

usually requires holding at a relatively constant temperature.

The culture unit should be capable of maintaining a constant

temperature between 8 and 12°C with a water temperature coptrol

device.

16.6.33.5 Sand dollars will feed on suspended or benthic

materials such as phytoplankton, benthic diatoms, etc. No

reports of laboratory populations being maintained in spawning

condition over several years are known. ·It is probably most

convenient to obtain sand dollars, use them, and then discard

them after they cease to produce good quality gametes.

~6.6.33.6 Natural seawater (>30~) is used to maintain the adult

animals and (~32~) as a control water in the tests.

408
16.6.33.7 Adult male and female (if sexes known) animals used in

field studies are transported in separate or partitioned

insulated boxes or coolers packed with wet kelp or paper

toweling. Upon arrival at the field site, trays or aquaria (or a

single partitioned aquarium) are filled with cont.rol water,

loosely covered with a styrofoam sheet and allowed to equilibrate

to the holding temperature before animals are addled.

16.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION AND

STORAGE

16.7.1 Section 8, Effluent and Receiving Water Siampling and

Sample Handling, and Sampling Preparation for Tmdcity Tests.

16.8 CALIBRATION AND STANDARDIZATION

16.8.1 See Section 4, Quality Assurance:

16.9 QUALITY CONTROL

16.9.1 See Section 4, Quality Assurance.

16.10 TEST PROCEDURES

16.10.1 TEST DESIGN

16.10.1.1 The test consists of at least four effluent

concentrations plus a dilution water control. Tests that use

brine to adjust salinity must also contain four replicates of a

brine control. In addition, four extra controls iare prepared for

egg controls.

16.10.1.2 Effluent concentrations are expressed as percent

effluent.

16.10.2 TEST SOLUTIONS

16.10.2.1 Receivin~ waters

16.10.2.1.1

of t,he test.

collected at

with samples

The .sampling point is determined by the objectives

At estuarine and marine sites, samples ar.e usually

mid-depth. Receiving water toxicity is determined

used directly as collected or with samples passed

409
through a 60 p.m NITEX@ filter and compared without dilution,

against a control. Using four replicate chambers per test, each

containing 5 mL, and 400 mL for chemical analysis, would require

approximately 420 mL or more of sample per test.

~6.~0.2.2 Effluents

~6.~0.2.2.~ The selection of the effluent test concentrations

should be based on the objectives of the study. A dilution

factor of at least 0.5 is commonly used. A dilution factor of

0.5 provides hypothesis test discrimination of ± 100%, and

testing of a 16 fold range of concentrations. Hypothesis test

discrimination shows little improvement as dilution factors are

increased beyond 0.5 and declines rapidly if smaller dilution

factors are used. USEPA recommends that one of the five effluent

treatments must be a concentration of effluent mixed with

dilution water which corresponds to the permittee's instream

waste concentration (INC). At least two.of the effluent

treatments must be of lesser effluent concentration than the IWC,

with one being at least one-half the concentration of the IWC.

If ~OO~ HSB is used as a diluent, the maximum concentration of

effluent that can be tested will be 66% at 34~ salinity.

16.10.2.2.2 If the effluent is known or suspected to be highly

toxic, a lower range of effluent concentrations should be used

(such as 25%, 12.5%, 6.25%, 3.12% and 1.56%).

~6.~0.2.2.3 The volume in each test chamber is 5 mL.

~6.10.2.2.4 Effluent dilutions should be prepared for all

replicates in each treatment in one container to minimize.

variability among the replicates. Dispense into the appropriate

effluent test chambers.

~6.~0.2.3 Dilution Water

~6.10.2.3.1 Dilution water should be uncontaminated 1-p.mfiltered natural seawater, or hypersaline brine prepared from

uncontaminated natural seawater plus reagent water; or sea salts

(see Section 7, Dilution Water). Natural seawater may be

uncontaminated receiving water. This water is used in all

dilution steps and as ·the control water.

410
16.10.2.4 Reference Toxicant Test

16.10.2.4.1 Reference toxicant tests should be conducted as

described in Quality Assurance (see Section 4.7).

16.10.2.4.2 The preferred reference toxicant for sea.uchins and

sand dollar is copper chloride (CuCI 2 oH20). RefE~rence toxicant

tests provide an indication o'f the sensitivity of the t'est

organisms and the suitability of the testing laboratory (see

Section 4 Quality Assurance). Another tox~cant ~may be specified

by the appropriate regulatory agency. Prepare a copper reference

toxicant stock solution (2,000 mg/L) by adding 5.366 9 of copper

chloride (CuC12 02H20) to 1 liter of reagent water. For each

reference toxicant test prepare a copper sub-stock of 3 mg/L by'

diluting 1.5 mL of stock to one liter with reagent water.

Alternatively, certified standard solutions can be ordered from

commercial companies.

16.10.2;4.3 Prepare a control (0 ~g/L) plus fou~ replicates each

of at least five cOI1:secutive copper reference tOfCicantsolutions

(e.g., from the series 3.0, 4.4,·6.5, 9.5, 13.9, 20.4, and 30.0

~g/L, by adding 0.10, 0.15, 0.22, 0.32, 0.46, 0.?8, and 1.00mL

of .sub-stock solution, respectively, to 100-L volumetric flasks

and filling tb 100-mL with dilution water). Start with control

solutions and progress to the highest concentratlon to minimize

contamination.

16.10.2.4.4 If the effluent and reference toxicant tests are to

be run concurrently, then the tests must use embryos from the

same spawn. The tests must be handled in the same way 'and test

solutions delivered to the test chambers at the same time.

Reference toxicant tests must be conducted at 34± 2~.

16.10.3 COLLECTION OF GAMETES FOR THE TEST

16.10.3.1 Spawning Induction

16 .10 3.1.1 Pour seawater into 100 mL beakersaIld place in 12°C

bath or room. Allow to come to temperature. Select a sufficient

number of sea urchins or sand dollars {based UpOIl recent or past

spawning success} so that three of each ~ex areJ,ikely to provide

gametes of acceptable quantity. and quality for the test .. During'

optimal spawning periods this may only require si.x animals, three

411
of each sex, when the. sexes are known from prior spawning.

During other periods, especially if the sex is not known, many

more animals may be required.

~6.~0.3.~.2 Care should be exercised when removing sea urchins

from holding tanks so that damage to tube,-feet is minimized.

Following removal, sea urchins should be placed into a container

lined with seawater-moistened paper towels to prevent

reattachment.

~6.~0.3.1.3 Place each sand dollar, oral side up, on a 100 mL

beaker filled with 12°C seawater or each sea urchin onto a clean

tray covered with several layers of seawater moistened paper

towels.

~6.~0.3.~.4 Handle sexes separately once known; this minimizes

the chance of accidental egg fertilization. ~hroughout the test

process, it is best if a different worker, different pipets, etc.

are used for males (semen) and females (eggs). Frequent washing

of hands is a good practice.

~6.~0.3.~.5 Fill a 3 or 5 mL syringe with 0.5 M KCI and inject

0.5 mL through the soft periostomal membrane of each sea urchin

(See Figure 3) or into the oral opening each sand dollar. If

sexes are known, use a separate needle for each sex. If sexes

are not known, rinse the needle with hot tap water between each

injection. This will avoid the accidental injection. of sperm

from males into females. Note the time of injection (sample data

sheet, Figure 1) .

~6.~0.3.~.6 Spawning of sea urchins is sometimes induced by

holding the injected sea urchin and gently shaking or swirling it

for several seconds.' This may provide an additional physical

stimulus, or may aid in distributingthe.injected KCI.

~6.~0.3.~.7 Place the sea urchins onto the beakers or tray (oral

side down). Place the sand dollars onto the beakers (oral side

up). Females will release orange (sea urchins) or purple (sand

dollars) eggs and males will release cream-colored semen.

~6.~0.3.~.8 As gametes begin to be shed, note the time on the

data sheet and separate the sexes. Plac~ male sand dollars with

the oral side up atop a small (5-10 mL) glass beaker filled with

~2°C seawater. Leave spawning sea urchin males on tray or beaker

412
(oral side down) for semen collection. Female sand dollars and

sea urchins are left to shed eggs into the 100-mL' beakers.

16.10.3.1.9 If sufficient quantities of gametes are available,

only collect gametes for the first 15 min after each animal

starts releasing. This helps to insure good quality gametes.

As a general guideline, do not' collect gametes from any

individual for more than 30 minutes after the first injection.

16.10.3.1.10 If no spawning occurs after 5 or 10 minut·es, a

second 0.5 mL injection maybe tried. If animals do not produce

sufficient gametes following injection of 1.0 mL of KCl, they

should probably not be reinjected as this seldom results in

acquisition of good quality gametes and may result in mortality

of adult urchins.

16.10.3.1.11 Collect the undiluted semen from each male sea

urchin, using a 0.1 mL automatic pipet. Store the sperm from

each male in a separate, labelled, conical, glas~ centrifuge

tube, covered with a cap or parafilm, on ice. Ai~ exposure of

semen may alter its pH through gas exchange and reduce the

viability of the sperm. Note: undiluted·semen from

Strongylocentrotus purpuratus typically contains 'about 4 x 1010

sperm/mL.

16.10.3.1.12 Sections 15.10.4.2 and 15.10.6.4 de'scribe

collection and dilution of the sperm and eggs. Wpile some of. the

gamete handling needs to be in a specific. order, parts of th~

procedure can be done simultaneously whi~e waiting for gametes to

settle.

16.10.3.2 Collection of Sperm'

16.10.3.2.1 Sea urchin semen should be collected dry (directly

from the surface of the sea urchin), using either, a Pasteur

pipette or a 0.1 mL autopipette with the end of the tip cut off

so that the opening is at least 2 mm. Pipette se~en from each

male into separate 1-15 mL conical test tubes, stbred in an ice

water bath.

16.10.3.3 Viability of Sperm

16.10.3.3.1 Early in the spawning process, place a very small

amount of sperm from each male sea urchin or sand dollar into

.413
16.10.4.2

dilution water on a microscope slide (well slides work nicely) .

Examine the sperm for motilitYi use sperm from males wi'th high

sperm motility. It is more important to use high quality sperm

than it is to use a pooled population of sperm.

16.10.3.4 Pooling of Sperm

16.10.3.4.1 Pool equal quantities of semen from each o~ the sea

urchin males that has been deemed good. If possible, ~.02S mL

should be pooled from each of those used and a total of. at least

0.05 mL of pooled semen should be available. Sperm collected

from good male sand dollars should be pooled after first

decanting off the overlying water (the final sand dollar sperm

density usually is between 2x109 and 2x1010 sperm!mL) ;

16.10.3.5 Storage of Sperm

16.10.3.5.1 Cover each test tube or beaker with a cap or

parafilm, as air exposure of semen may alter its pH through gas

exchange and reduce the viability 'of the sperm. Keep sperm

covered and on ice or refrigerated «SOC),. The sperm should be

used in a toxicity test within 4'h of co~lection.

16.10.4 PREPARATION OF EGG SUSPENSION FOR USE IN THE TEST

16.10.4.1 Acceptability of Eggs

16.10.4.1.1 Prior to pooling, a small sample of the eggs from

each female should be examined for the presence of significant

quantities of poor eggs (vacuolated, smail, or irregularly

shaped) and mixed with good sperm to determine extent of

fertilization. If good quality eggs are available from one or

more females, questionable eggs should not be used for the test.

It is more important to use high quality eggs than it is to use a

pooled population of eggs.

Pooling of Eggs

16.10.4.2.1

Decant some

not to pour

1 L beaker,

water. The

Allow eggs to settle in the collection be~kers.

of the water from the collection beakers taking care

off many eggs. The sea urchin eggs are pooled into a

and the volume brought to 600 mL with 12°C dilution

eggs are suspended by swirling and the eggs allowed

414
to settle for 15 minutes at 12°C. About 500 mL of the overlying

water are siphoned off, the volume brought back to 600 mL with

more 12°C dilution water, and the eggs resuspended and allowed to

settle for a second 15 minute period.. .After aga:in siphoning off

the overlying 500 mL, the rinsed eggs are gently transferred to

either a 100 or a 250 mL graduated cylinder and brought to volume

with 12°C dilution water. Eggs are stored at 12°C throughout the

pre -,test period.

16.10.4.2.2 Pooled sand dollar eggs should be treated gently and

no additional rinsing step is recommended. Mix well once just

before subsampling for egg stock calculations. This is best done.

in a large graduated cylinder appropriate for the number of eggs

available. Cover with parafilm and. invert gently several times.

16.10.4.3 Density of Eggs

16.10.4.3.1 Subsamples of the egg stock are then taken for

determining egg density. Place 9 mL of dilution water 'in each of

two 22 mL liquid scintillation vials.. . Label A and B. -Place 1 mL

of well~mixed egg stock intovial.A. Mix well. (The remaining

egg stock is covered with parafilm and stored at' 12°C.). Transfer.

1 mL of egg suspension from vial A to vial B. Mix contents of

vial B and transfer 1 mL of egg suspension B into a SedgewickRafter counting chamber. Count eggs under a compound microscope.

If count is <30, count a 1 mL sample from vial A (see sample data

sheet, Figure 2) .

16.10.4.3.2 Prepare 100 mL of egg stock.in dilu~ionwater at the

final target concentration of 2,240 eggs/mL(224,000 eggs' in .100

mL). If the egg stock is >2,240 eggs/mL'(A >224,or B >30

eggs/mL), dilute the egg stock by transferring:

224,000 eggs / ____~D eggs/mL = mL

of well-mixed egg stock to a 100 mL graduated cylinder and bring

the total volume to 100 mL with dilution water where:

D.= (Count A) x io or (Count B) x 100.

If the egg stock is <2,240 eggs/mL (A <224 eggs/dIL), co'ncentrate

the eggs by allowing them to settle and then decant enough water

to retain the following percent of the original volume:

415
D eggs/mL / 2,240) x 100 = % volume.

16.10.4.3.3 Check the egg stock density.' Place 9 mL of dilution

water into a 22 mL scintillation vial; add 1 mL of the final egg

stock. Mix well and transfer 1 mL into a Sedgewick-Rafter

counting chamber. The egg count should be between 200 'and 245.

Adjust egg stock volume and recheck counts if necessary to obtain

counts within this range. Because some eggs (especially sand

dollar eggs) may be sensitive to handling, it is advisable to

separately prepare egg stocks for the fertilization trial and the

definitive test (but use the same pooled batch of eggs) .

16.10.5 PREPARATION OF SPERM DILUTION FOR USE IN THE ('OPTIONAL)

TRIAL FOR ESTIMATING APPROPRIATE SPERM DENSITY FOR TEST

16.10.5.1 A trial fertilization is recommended to reduce the

likelihood of a failed test due to inadequate control

fertilization or exceeding the maximum acceptable sperm density.

However, two other alternative approaches are acceptable:

1) Conduct the test at a low enough sperm density that

oversperming does not create test insensitivity. This

can be met by using a confirmed sperm stock density of

~5.6x106/mL (this is equivalent to a sperm:egg ratio of

~500:1 at 200 eggs/mL); or

2) Conduct the test, but include two extra ~ets of

controls, one set receiving only 0.050 mL of the sperm

stock and the other receiving 0.2 mL of the sperm

stock. The control fertilization in the 0.050 mL sperm

stock controls must be at least 5% lower than that in

the 0.2 mL sperm stock controls or the test is

unacceptable. Confirm that the sperm stock density did

not exceed the maximum acceptable density of 3.36 x 107

sperm/mL.

16.10.5.2 Fertilization trial is conducted to determine the

sperm density that will provide about 80-100 percent control egg

fertilization while avoiding significant, ,"oversperming" that can

reduce test sensitivity. Although usually expressed as a

sperm:egg ratio (e.g., 1,000:1), because egg density is held

constant at 200/mL, the sperm:egg ratio is also a measure of '

sperm density.

416
16.10.5.3. It is unacceptable to conduct a definitive toxicity

test if the sperm:egg ratio exceeds 3,000:1. This is a cut-off

based on gradual loss of test sensitivity at higher sperm

densities, even in cases where control fertilization is

considerably below 100 percent.

16.10.5.4 It is unnecessary to conduct trials for definitive

toxicity tests at sperm:egg ratios below 500:1, because this

ratio should never cause significant "oversperming."

16.10.5.5 Sperm denE!ity of sea urchin semen or :sand dollar sperm

suspension is checked by hemocytometer counts and a replicated

series of nominal S:E ratios set up (3,000, 1288, 550, 234, and

100:1) based upon appropriate dilution calculations.

16.10.5.6 For sea urchins and sand dollars, prepare a killed

sperm preparation for determining the dilution rl=quired to obtain

a sperm stock (3.36 x 107 sperm/mL) for the maxirrmm sperm density

(6 x 105 sperm/200 eggs/mL--3,000:1) need~d for the trial. A

sperm density of about 1 x 107 is convenient to count. If the

approximate sperm density is k~own, the dilution.procedures

outlined in Table 4 can be followed without init:Lal sperm counts;

the actual trial sperm density must still be determined by

subsequent counts. For example (Table 4), if expected sperm

density is ca. 5 x 108 dilute 0.2 mL of sperm to 10 mL, if ca. 5

x 109 dilute 0.2 mL of sperm to 100 mL (or 0.025 mL of sperm to

10 mL), if ca. 5 x 1010 dilute 0.040 mL to 200 mIJ. Table 4 is

provided for guidance as a quick reference for dilution volumes

if sperm density of pooled semen is can be reasonably e~timated,

and as a check for mathematical accuracy of formula calculations

for sperm dilution.

16.10.5.7 Mix. the pooled sea urchin semen (16.10.3.8) by

agitating the centrifuge tube for about 5 seconds using a vortex

mixer. Very slowly withdraw a subsample of semen using an

automatic pipet, wipe off the outside of the pipet tip ~ith

tissue, and empty the pipet contents into an Erlenmeyer flask

containing the appropriate volume (Table 4) of a. sperm killing

solution of 1% glacial acetic acid in c:iilution Walter (e.g., 10 mL

of 10% glacial acetic acid plus 90 mL of .dilutiori water) .

417
Figure 3. Showing the location and orientation used in the

injection of KCI into sea urchins to stimulate spawning.

Repeatedly rinse the residual semen from·the pipet tip by filling

and emptying until no further cloudy solution is expelled from

the pipet (this may require several dozen rinses) . Cover the

flask with parafilm and mix thoroughly by repeated inversion.

26.20.5.8 Mix the chilled suspension of pooled sand dollar sperm

(26.20.5.6) using a stirring rod. Pipet the appropriate volume

of sperm suspension (Table 4) into an Erlenmeyer flask containing

the appropriate volume (Table 4) of a sperm killing solution of

2% glacial acetic acid i~ dilution water ('e.g., 10 mL of 10%

glacial acetic acid plus 90 mL of dilution water) .

26.10.5.9 Transfer samples of well-mixed sperm suspension to both

sides of two Neubauer hemacytometers. Let the sperm settle 15

min.

16.10.5.10 Count the sperm on one hemacytometer following

procedures outlined in Appendix II. If the lower count. is at

418
least 80% of the higher count use the mean count: to estimate

sperm density in semen and the required dilutioIl volume for the

test stock. If the two counts do not agree within 20%, count the

two fields on the other hemacytometer. Calculat:e the sperm

density in the semen or sperm suspension using the mean of ' all

four counts unless one count can be eliminated as an obvious

outlier.

16.10.5.11 Calculate the volume of dilution wat:er necessary to

dilute the sea urchin semen or the sand dollar sperm suspension

to the sperm density (sperm/mL)reguiredfor tp,e, sperm stock for

the trial. See Table 5 for recommended dilution procedures; it

also provides a quick reference for dilution volumes once Sperm

density of pooled semen is known, or a check for mathematical

accuracy of 'formula calculations for sperm dilution. Note: table

values for sperm densities from lxl08 to 9xl09 'a~e for volume

(mL) of sperm stock for total volume of 100 mL; :tab1e values for

sperm densities ~lx101o are for dilution water volumes for, 0.025·

'mL of semen. Table 5 is used as fol10ws~ given :a sperm d~nsity

in the semen stock (e.g., 4'.7xl09 ) 'find the row c~ontaining the

integer (characteristic) and the'exponent (4x10 9 ) in the left

hand column, then read across to the column coresponding to the

mantissa (0.7). The value at the intersection o:f the row and

column {0.71 mL} is the volume of semen per 100mL needed for

sperm stock to achieve a 3000:1 sperm:egg ratio ~n the trial.

16.10.5.12 For the approximat;.e sperm:egg ratios,: dilute the

3000:1 stock as follows:

1288:1 5 mL 3000:1 stock with 6.6 niL dilution water

550:1 :2 mL 3000:1 stock with 9.9 m:L dilution water

234:1 1 mL 30,00: 1 stock with 11.8 l:nL dilution water

100:1 0.5 'mL 3000:1 stock with 16.5 ,mL dilution water

16.10.6 SPERM DENSITY TRIAL

16.10.6.1 The series of trial sperm: egg ratios /3hould include

3,000:1 and several lower ratios. The ratios 100:1, 234:1,

550: 1, 1288: 1 and 3,000: 1 are recommended becausE3 they evenly

divide the log sperm:egg ratio. Fertilization appears to be a

linear function of the log of sperm density (Fi~~re 4) .

Recommended sperm dilution procedures are given in Table 5.

419
~6.~0.6.4 Quantitive evaluation of the sperm density trial

should be obtained by counting 100 eggs from each tube until a

suitable sperm density can be qetermined for the definitive test.

Examples of sperm density selection are given in Table 6.

Percent fertilization may be lower in the test than in the trial

because the viability of the stored sperm may decrease during the

period of the trial. If the sperm have very good viability

(e.g., cases 1 and 2, Table 6), this loss of viability should be

small. On the other hand, if viability is inherently poorer

(cases 3, 4 and 5, Table 6), the los$ of viability could be

greater and probably should be taken into account in selecting

the sperm density for the test. Case 6 (Table 6) represents a

special case in which egg viability may affect the percent

fertilization; in this case the asymptote of the fertilization

curve is assumed to represent 100% fertilization for purposes of

selection of sperm density for the test.

~6.~0.6.5 Prepare killed sperm preparations of the trial sperm

stock suspensions to provide confirmation of the nominal

sperm:egg ratios. It saves tim~ if thes~ can be prepared and

loaded onto hemacytometers while the trial is being conducted.

Alternatively, once ~he trial has been evaluated, the selected

nominal sperm density can be confirmed by direct hemacytometer

count.

~6.10.6.6 Record all the counts made, select a target sperm:egg

ratio for the test, and calculate the dilution of the stored

sperm stock needed to provide the necessary sperm density for the

definitive test.

16.~0.6.7 Table 5 can be used for deriving the volumes needed

for preparing the final sperm stock. For a pooled sperm

suspension density of 4x109 and a target sperm:egg ratio of

500:1, simply read the dilution for the 3000:1 sperm:egg ratio

from Figure 5 (0.84 mL / 100 mL) and reduce the sperm volume by

3,000 / 500 = 6. In this case 0.84 / 6 = 0.14 mL; the dilution

factor checks out (100 / 0.14 = 714).

420
TABLE 4. Dilution volume guide for initial count of sperm

density to achieve recommended counting density of 1 x

l07/triL. '

Initial mL/10mL mL/100mL ' mL/200mL

Sperm/mL

1 x 10·, 1.000

2 x 10· 0.500

3 x 10· 0.333

4 x 10· 0.250

5 x 10· 0.200

6 x 10· 0.167

7 x 10· 0.143

8 x 10· 0.125

9 x 10· 0.111

1 X 10' 0.100 1.000

2 x 10' 0.050 0.500 " 1.000

3 X 109 0.033 0.333 0.667

4 x 10' 0.025 0.250 I 0.500

5 x 10' 0.200· 0.400

6 x :\.09 0.167 0.333

7 x 10' 0.143 0.286

8 x 10' 0.125 0.250

9 x 10' 0.111 0.222

1 x 10lO 0.100 0.200

2 x 10lo 0.050' 0.100

3 x 10lo 0.033 0.067

4 x 10'0 0.025 O. 050'

5 x 10lo 0.040

6 x 10'0 0.033

7 x 10lo , 0.029

8 x 10lo 0.025

I

9 x 10'0 I 0.022

I

~: to obtain quantitatively repeatable samples of semen it is important 'that: (1) the

pipet tip have an opening of at least 1 mm; (2) samples be withdrawn slowly to avoid

cavitation and entrainment of air in the semen sample; (3) samples not include fragments

of broken spines ~which usually settle to the test tube bottom upon vortexing); and (4)

wiping semen from the pipet tip' with tissue be done with care to ~vOid wicking semen from

within the pipet tip.

421
Bring the indicated volume of sper.m stock to 100 mL

Density 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1.00e+08 33.60 30.55 28.00 25.85 24.00 22.40 21.00 19.76 18.67 17.68

2.00e+08 16.80 16.00 15.27 14.61 14.00 13.44 12.92 12.44 12.00 11.59

3.00e+08 11.20 10.84 10.50 10.18 9.88 9.60 9:33 9.08 8.84 8.62

4.00e+08 8.40 8.20 8.00 7.81 7.64 7.47 7.30 7.15 7.00 6.86

5.00e+08 6.72 6.59 6.46 6.34 6.22 6.11 6.00 5.89 5.79 5.69

6.00e+08 5.60 5.51 5.42 5.33 5.25 5.17 5.09 5.01 4.94 4.87

7.00e+08 4.80 4.73 4.67 4.60 4.54 4.48 4.42 4.36 4.31 4.25

8.00e+08 4.20 4.15 4.10 4.05 4.00 3.95 3.91 3.86 3.82 3.78

9.00e+08 3.73 3.69 3.65 3.61 3.57 3'.54 3.50 3.46 3.43 3.39

1.00e+09 3.36 3.05 2.80 2.58 2.40 2.24 2.10 1.98 1.87 1.77

2.00e+09 1.68 1.60 1.53 1.46 1.40 1.34 1.29 1.24 1.20 1.16

3.00e+09 1.12 1.08 1.05 1.02 0.99 0.96 0.93 0.91 0.88. 0.86

4.00e+09 0.84 0.82 0.80 0.78 0.76 0.75 0.73 0.71 0.70 0.69

5.00e+09 0.67 0.66 0.65 0.63 0.62 0.. 61 0.60 0.59 0.58 0.57

6.00e+09 0.56 0.55 0.54 0.53 0.53 0.52 0.51 0.50 0.49 0.49

7.00e+09 0.48 0.47 0.47 0.46 0.45 0.45 0.44 0.44 ' 0.43 0.43

8.00e+09 0.42 0.41 0.41 0.40 0.40 0.40 0.39 0.39 0.38 0.38

9.00e+09 0.37 0.37 0.37 0.36 0.36 0.35 0.35 0.35 0.34 0.34

To dilute dense semen: add 0.025 mL of semen into these volumes (mL) of dilution water

Density 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1.00e+l0 7.44 8.44 9.44 10.44 11.44 12.44 13.44 14.44 15.44 16.44

2.00e+l0 14.88 15.88 16.88 17.88 18.88 19.88 20.88 21.88 22.88 23.88

3.00e+10 22.32 23.32 24.32 25.32 26.32 27.32 28.32 29.32 30.32 31.32

4.00e+10 29.76 30.76 31.76 32.76 33.76 34.76 35.76 36.76 37.76 38.76

5.00e+l0 37.20 38.20 39.20 40.20 41.20 42.. 20 43.20 44.20 45.20 46.20

6.00e+10 44.64 45.64 46.64 47.64 48.64 49.64 50.64 51.64 52.64 53.64

7.00e+10 52.08 53.08 54.08 55.08 56.08 57.08 58.08 59.08 60.08 61.08

8.00e+10 59.52 60.52 61.52 62.52 63.52 64.52 65.52 66.52 67.52 68.52

9.00e+l0 66.96 67.96 68.96 69.96 70.96 71.96 72.96 73.96 74.96 75.96

TABLE 5. DILUTION VOLUMES OF SPERM STOCK OF INDICATED DENSITY (1.OX10B TO 9.9X10'O ) TO

ACHIEVE THE SPERM STOCK DENSITY (3.36X10 7 ) FOR A 3000:1 SPERM:EGG RATIO.

422
100

90

10

70

80

50

40

30

20

10

o

1

I

1

1

.1••

" •• . I

10

, ·1

, .::1 :1

100

Sperm to Egg RatIo

101110

"'I

I '

10000

Figure 4. Relationship betwe~n sea urchin sperm:egg ration' and

percent control fertilization from 21 trials conducted by EPA

(Feb-May 1991) .

16.10.7 OBSERVATIONS DURING THE TEST

16.10.7.1 It is recommended that all observations be made on

extra test solution remaining after the test tubes have been

filled.

16.10.7.2 DO, pH, and salinity are measured at t:he begfnning of

the test. Due to the short duration of the test,. no additional

measurements of these parameters are required. 1~empeiature_is

measured several times during the test as outlinEld in 16.1.0.7.

\.

16.10.7.3 Record all measurements on the data sheet.

16.10.8 START OF THE DEFINITIVE TEST

16.10.8.1 Prior to Beginning the Test

produced good fertility, or if some produced good fertility.

423
TABLE 6. EXAMPLES OF RESULTS OF TRIAL FERTILIZATION TESTS WITH

SPECIFIED SPERM DENSITIES AND TARGET SPERM DENSITY

SELECTION (SPERM:EGG RATIO) FOR THE DEFINITIVE TEST.

sperm: egg case 1 case 2 case 3 case 4 case 5 case 6

100:1 100* 95* 85 70 40 70

234:1 100 98 95* 80 64 85*

550:1 100 100 98 98* 82 89

1288:1 100 100 100 100 84 90

3000:1 100 100 100 100 88* 90

* recommended selection (interpolation to intermediate sperm:egg

ratios may be used if found desirable)

1. If all trials exceed 90% fertilization, select 100:1 (case 1 and

case 2) .

2. If not all trials exceed 90% fertilization select the lowest

sperm:egg ratio that does exceed 90% fertilization (case 3 and

case 4) .

3. If no trials exceed 90% fertilization, select the highest

sperm:egg ratio (case 5) unless fertilization appears to become

asymptotic below 100% (case 6).

4. If even the highest sperm:egg ratio fails to achieve 70%

fertilization it is probable that an acceptable test cannot be

conducted with these gametes.

11,200 x target S:E ratio = target density; e.g., if target S:E

500:1, target density = 11,200 x 500 = 5,600,000 sperm!mL.

(11,2~0 = (1,120 eggs/tube) (0.1 mL of sperm stock/tube)).

(stock sperm/mL)/(target sperm/mL) = dilution; e.g., if stock

sperm has 4x109 sperm/roL, then dilution = 4x109 / 5.6x106 = 714

16.10.8.1.1 The test should begin as soon as possibie,.

preferably within 24 h of sample collection. The maximum holding

time following retrieval of the sample from the sampling device

should not exceed 36 h for off-site toxicity tests unless

424
permission is granted by the permitting authori.ty. In no case

should the sample be used in a test more.than 72 h after sample

collection (see Section 8 Effluent a,nd Receivin.g Water Sampling, .

Sample Handling, and'Sample Preparation for Toxicity Test).

16.10.8.1.2 Just prior to test initiation (approximately 1 h),

the temperature of a sufficient quantity of the sample to make

the test solutions should be adjusted to the test temperature (12

±l°C) and maintained at that temperature durin9' the addition of

dilution water.

16.10.8.1.3 Increase the temperature of the wa'l:er bath, room, or

incubator to the required test temperature, (12 :± 1°C).

16.10.8.1.4 Randomize the placement of test chambers in the

temperature-controlled water bath, room, or in9ubator at the

beginning of the test, using a position chart. Assign numbers

for the position of each test chamber using a random numbers or

similar process (see Appendix A, for an example of

randomization). Maintain the chambers in this configuration

throughout the test, using a position chart. Record these

numbers on a separate data sheet together with the concentration

and replicate numbers to which they correspond. Identify this

sheet with the date., test organism, test .number, laboratory, and

investigator's'name, and safely store it away until after the sea

urchins and sand dollars have been examined at t~e end of the

test.

16.10.8.1.5 Note: Loss of the randomization sheet would

invalidate the test by making it impossible to analyze the data

afterwards. Make a c~py of the randomiz~ti6n sheet and store

separately. Take care to follow the numbering system exactly

while filling chambers with the test solutions.

16.10.8.1.6 Arrange the test chambers randomly in the water bath

or controlled temperature room. Once chambers have been labeled

randomly, they can be arranged in numerical order for

convenience, since this will also ensure random placement of

treatments.

16.10.9.2 Sperm Exposure

425
16.10.9.2.1 Mix the iced sea urchin semen or sand dollar sperm

suspension as described in 16.10.5.7 and 16.10.5.8 (do not kill

the sperm). combine the required volumes of sperm and dilution

water and mix this sperm stock well by repeated inversion of the

graduate cylinder or beaker. Begin test within 5 minutes. Table

5 (for 3000:1 sperm:egg ratio) can be used to aid in calculating

appropriate volumes by reducing the sperm volume or increasing

the dilution water volume by the factor:

f = 3000:1 / target sperm:egg ratio

16.10.9.2.2 The test tubes containing 5.0 mL of the various test

solutions should have been equilibrated in a 12°C waterbath.

Into each test tube, inject 0.1'00 mL of the sperm stock (except

see 16.7.4 and 16.11.4) and note the time of first and last

injection. It is important that the injection be performed with

care that the entire volume goes directly into the test solution

and not onto the side of ~he test tube. ,Similarly, the pipet tip

should not touch the test solution or the side of the test tube,

risking transfer of traces of test solution(s} into the sperm

stock. Using repeated single 0.100 mL refill and injection,

about 12 tubes per minute is a reasonable injection rate. More

rapid rates of injection can be attained with repeating (single

fill, multiple injection) pipets. Sperm injection rate

(tubes/min) should not exceed that possible for egg injection.

16.10.9.2.3 Unless the test tubes are totally randomized,

injection of sperm should be performed by replicate, i.,e., the

first set of'replicates should receive sperm, then the second

set, then the third set, etc. The sperm stock solution should be

mixed frequently to maintain a homogeneous sperm stock.

16.10.9.2.4 Confirm the sperm density. 'Pipet 9 mL of sperm

stock solution into a vial or test tube containing 1mL of 10%

acetic acid. Fill both sides of a hemacytometer with this

dilution after mixing well. Let stand for 15 minutes. Count

both sides of the hemacytometer using counting pattern no. 1

outlined in Appendix II and take the average count. For a .

sperm:egg ratio of 500:1 the stock sperm density will be

5,600,000 sperm/mL. (For counting pattern no. 1, this amounts to

a total count of 102 sperm for the five large squares.)

Calculate the sperm density in the sperm stock. If either: (1)

the stock sperm density is greater than 33,600,000 sperm/mL (S:E

426
>3 / 000:1), or (2) the sperm density is more than 2x the target

density, the test must be restarted with freshly diluted semen.

16.10.9.2.5 Check the temperature of the test solutions several

times during the sperm exposure by including a temperature blank

test tube containing 5 mL of dilution water with a th~rmometer.

16.10.9.3 Adding Eggs to the Test

16.10.9.3.1 Exactly 20 mihutes after the sperm addition to the

test was begun, begin to add the eggs 1 with every tube (including

egg blanks - 11.7.4) receiving 0.5 mL of egg stock. Follow the

same pattern of introduction for the eggs as use(::I. with the sperm

so that each test tube has a sperm, incubation period of. 20

minutes. Note the time of start and finish of e!jg addition.

This duration should be within one minute of that used for the

sperm. I

I

16.10.9.3.2 In order to maintain the same sperm:egg ratio in

'each test tube, the ~ggs must be maintained in a uniform

distribution in the water column of the egg stock. Slow, gentle

agitation of the egg stock in a beaker using a perforat'ed plunger

appears 'to be the best method of achieving a uniform

di~tribution. Frequent inversion and mixing of egg stock in

either a graduated cylinder or a multiple injection pipet may be

acceptable.

16.10.9.,3.3 The eggs should be inj ected using a. pipet with an

opening of at least 2 mm in order to avoid damaging the eggs and

to provide sufficient flow to obtain a representative sample.

16.10.9.3.4 Two pair of egg blanks should be included'in the

test design, one at the beginning of the injection sequence

(effluent blank) and one at the end of the injection sequence

(egg blank). These tubes receive no sperm. The effluent blank

cohtains the highest concentration of effluent and the ,egg blank

contains dilution water.. Examination of the effluent blank will

indicate if the effluent induces a false fertili2:ation membrane

(a possible event, but probably rare) thus maskirlg toxicity.

Examination of the egg blank will indicate if accidentally

fertilized eggs were used in the test (this is a minor factor

unless a significant portion of the eggs were accidentally

fertilized; it can indicate poor laboratory techniques). These

427
'blanks are kept capped until the eggs are added in order to avoid'

contamination by sperm.

16.10.10 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE

16.10.10.1 The echinoderm fertilization test can be conducted in

the dark or at ambient laboratory light levels. Due to its short

duration, the fertilization test requires no photoperiod.

16.10.10.2 The water temperature in the test chambers should be

maintained at 12 ± 1°C. If a water bath is used to maintain the

test temperature, the water depth surrounding the test cups

should be as deep as possible without floating the chambers. A

sensor placed in a temperature blank vial with standard volume of

test solution can provide a direct measure of test solution

temperature, one which may be more stable than the temperature in

the air or water in the medium surrounding the test vials. Do

not measure temperatures directly in a test vial, but prepare and

handle the temperature blank(s) exactly as the normal control

vials. Record the temperature several times between the

beginning and the end of the test.'

16.10.10.3 The test salinity should be in the range of 34 ± 2~.

The salinity should vary by no more than ±2~ among the chambers

on a given day. If effluent and receiving water tests are

conducted concurrently, the salinities of, these tests should be

similar.

16.10.10.4 Rooms or incubators with high volume ventilation

should be used with caution because the volatilization 'of the

test solutions and evaporation of dilution water may cause wide

fluctuations in salinity.

16.10.11 DISSOLVED OXYGEN (DO) CONCENTRATION

16.10.11.1 Aeration may affect the toxicity of effluent and

should be used only as a last resort to maintain a satisfactory

DO. The DO concentration should be measured on new solutions at

the start of the test (Day 0). The DO should not fall below 4.0

mg/L (see Section 8, Effluent and Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity Tests). If

it is necessary to aerate, all treatments and the control' should

be aerated. The aeration rate should not exceed that necessary

428
to maintain a minimum acceptable DO and. under no circumstances

should it exceed 100 bubbles/minute, using a pipet with a 1-2 mm

orifice, such as a 1 mL KIMAX® serological pipe1: No. 37033, or

equivalent.

16.10.12 OBSERVATIONS DURING THE TEST

16.10.12.1 Routine Chemical and Physical Observations·

16.10.12.1.1 DO is measured at the, beginning of the exposure

period in one test chamber at each test concentration and in the

control.

16.10.12.1.2 Temperature, pH, and salinity are measured at the

beginning of the exposure period in one test chamber at each

concentration and in the control. Temperature should also be

monitored continuously or observed and recorded daily for'at

least two locations in the environmental control! system or the

samples. Temperature should be measured in a sufficient number

of test chambers at the end of the test t,o determine temperature

variation in the environmental chamber.

16.10.12.1.3 Record all the measurements on the data sheet.

16.io.13 TERMINATION OF THE TEST

16.10.13.1 Ending the Test

16.10.13.1.1 Record the time the test is termirt~ted.

16.10.13.1.2 Because of the short test duration water quality

measurements are not necessary at the end.

16.10.13.2 Sample Preservation

16.10.13.2.3. Exactly 20 minutes after the egg addition, the test

should be stopped by the addition of a fixative 'to kill the sperm

and eggs (both unfertilized and fertilized [zyg01tes]) and to

preserve the eggs for examination. Again, the time allotted to

fixative addition should be about the same as th(~t for sperm and

egg addition and the sequence of addition the saI'ne as for the

introduction of the gametes.

429
16.10.13.2.2 The choice of formaldehyde or glutaraldehyde is up

to the individual laboratory. There are at least two acceptable

procedures: (1) the EPA Arbacia method of ~dding 10% formaldehyde

in dilution water at the rate of 2 mL to each test tube; or (2)

the addition of 1% glutaraldehyde (vol/vol) in clean seawater at

the rate of 0.5 mL to each test tube. Glutaraldehyde should be

made up fresh each day. Because concentrated glutaraldehyde is

commonly only 25% strength, 1% glutaraldehyde is obtained by

diluting the concentrate by 25x (e.g., 4, mL + 96 mL seawater).

16.10.13.2.3 It must be noted that formaldehyde has been

identified as a carcinogen and that both glutaraldehyde and

formaldehyde are irritating to skin and mucous membranes.

Neither should be used at higher concentrations than needed to

achieve morphological preservation of eggs for counting and only

under conditions of maximal ventilation and minimal opportunity

for volatilization into room air. Before using either compound

in this method, the user should consult the latest material

safety data available.

16.10.13.3 Counting

16.10.13.3.1 Immediately after termination of the test, the

tubes are capped (or otherwise covered) and the contents mixed by

inversion. They can be stored at room temperature until the

eggs are examined for fertilization. Counts should be completed

within 48 hours and, 'if counts extend over two days, should be

made by replicate, i.e., count all replicate 1 tubes, then

replicate 2, etc.

16.10.13.3.2 At least 100 eggs from each test tube are examined

and scored for the prese~ce, or absence of an elevated

fertilization membrane. 'Newly fe~tilized eggs will almost always

have a completely elevated membrane around the egg (See Figures 5

and 6). Often a double membrane appears in sea urchin eggs, but

following storage, even of only several hours, the inner

(hyaline) membrane may disappear. Fertilized eggs may touch the

outer membrane, or the membrane(s) may partially collapse.

Because these phenomena only occur after preservation, eggs with

any elevation of the fertilization membr~ne are counted as

fertilized.

430
Normal Fertilized,

~gg

a. b. c.

d. , e. f. Unfertilized

g. h.

Figure 5. Examples of typical fertilized and unfertili:~ed sea urchin eggs

and a number of examples of atypical "fertilized" eggs (a through h). Normal

fertilized eggs have an outer fertilization membrane and,an inner hyaline

membrane. After preservation, the hyaline membrane sometimes disappears (a) i

in other cases the egg is displaced from the center and contacts the perimeter

either inside an enlarged hyaline envelope (b) or with no visible pyaline

membrane (c). In some instances there appears to be only a slight elevation,

of the outer membrane or only the hyaline membrane appears, fully (d),

partially (f), or only as a halo (g). In some batches of eggs the membrane(s)

appear to be fragile and some collapse (e). In rare cases sperm appear to

activate membrane elevation over only segments of the egg leading to a

blistered appearance (h). When eggs appearing as those irl examples f, g, and

h are common in a 'test, the results should be'examined closely to see if their

occurrence appears to be dose-related (indicating an effect on fertilization)"

not dose-related (indicating a problem with egg quality or preserv.ative), or

is common in the effluent egg control (indicating an effluent- produced false

fertilization). Eggs that are not mature are capable of being fertilized, but

should never be counted. These include obviously smaller (often denser) eggs,

normal sized eggs with a distinct, clear center, and very large eggs with

often irregular color and density.

431
• •

• •

• •

•

• • b. c. 8.

• • • • • • • • • • • • • • • • • •

. • . • • • • . • . . •

• • • . • • • • . • • • • • • • •

d. c. f.

Figure 6. Examples of typical fertilized and unfertilized sand

dollar eggs. Nearly all newly released eggs are characterized by

a surrounding sphere of small purple chromatophores embedded .

within the transparent gelatinous coat surrounding the egg. The

coat and the chromatophores may be lost or retained in the test

and subsequent handling. Typical fertilized eggs are represented

by (a) and (b). Some fertilized eggs (c) show only a wispy

remnant of the fertilization membrane. Eggs when spaw~ed usually

appear as in (d) and (e) or somewhere in between. The more

rounded "raisin" appearing egg in (d) is usually superior to the

"asteroid" appearing egg in (e) although the latter can provide

acceptable test results. However, the more irregularly shaped or

vacuolated the eggs appear, the poorer the control fertilization

is likely to be. The egg shown in (f), the "pitted olive," never

shows a fertilization membrane and should not be counted.

432
,

16.10.13.3.3 It, is convenient to concentrate the eggs prior to

counting. If the eggs are allowed to completel~r settle (ca 30

minutes after termination and mixing), most of 1:he overlying

solution can be removed with a pipet, leaving the' eggs

concentrated in a much smaller volume. 'I;'he eggs are then

resuspended by filling and emptying a 1 mL pipe1: about 5 times

from the remaining volume and finally transferring 1 mL of the

egg suspension into a 1 mL Sedgewick-Rafter counting chamber

(other volume counting chambers can be used) . .

16.10.13.3.4 Failure to completely resuspend the eggs can result

in biasing the counts towards higher percent fei:-ti;Lization Clue to

a tendency seen in rare batches of eggs in which unfertile eggs

tend to be adhesive.' This phenomenon may be further influenced

by the choice of preservative, the strength of t:he preservative,

and the period between preservation and countin9. However, other

sampling procedures may be used once d~monstrated not t,o bias

sampling and if no clumping of adhesive eggs is observed in' a

given test; for example, concentrated eggs may be picked ,up from

the test tube and deposited in a small drop on a microscope

slide, or eggs can be scored by examination with the test tubes

laying on their sides and viewed at low power OJ~ with an inverted

microscope ..

16.10.13.4 Endpoint

16.10.13.4.1 In a count of at least 100 eggs, record the number

of eggs with fertilization membranes and the nunilier of eggs

without fertilization membranes.

16.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

16.11.1 A summary of test conditions and test acceptabil~ty

criteria is listed in Table 7.

TABLE 7. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY'

CRITERIA FOR,. STRONGYLOCENTROTUS PURPCTRATUS AND

DENDRASTER EXCENTRICUS, FERTILIZATION TEST WITH

EFFLUENTS AND RECEIVING WATERS

l. Test type: Static non- renl8wal

2. Salinity: 34 ± 2%0

3. Temperature: 12 ,± 'l°C

4. Light quality: Ambient. labora'tory light

durinq test orl8oaration

433
5. Light intensity: 10-20 uE/m2 /s (Ambient

laboratory levels)

6. Test chamber size: 16 x 100 or ·16x 125 mm

7. Test solution volume: . 5 mL

8. Number of spawners: Pooled sperm from up to four

males and pooled eggs from up

to four females are u$ed per

test

9. No. egg and sperm cells About 1,120 eggs and not more

per chamber: than 3,360,000 sperm per test

tube I

10. No. replicate chambers 4

per concentration:

11. Dilution water: Uncontaminated l-/lm-filtered

natural seawater or

! hypersaline brine prepared i from natural seawater or

artificial sea salts I

I 12. Test concentrations: Effluents: Minimum of 5 and a

control

Receiving waters: 100% a

! control

I 13. Dilution factor: Effluents: ~0.5

Receiving waters: None or ~O.5

14. Test duration: 40 min (20 min plus 20 min)

15. Endpoint: Fertilization of eggs

16. Test acceptability ~70% egg fertilization in

criteria: controls; %MSD of <25%; and

appropriate sperm counts I

17. Sampling requirements: One sample collected at test , initiation, and preferably I used within 24 h of the time

I it is removed from the

sampling device (see Section

8, Effluent and Receiving

Water Sampling, and Sample

Preparation for Toxicity

! Tests)

18. Sample volume required: 1 L

434
16.12 ACCEPTABILITY OF TEST RESULTS

16.12.1 Test results are acceptable only if all the follow:Lng

requirements are met:

(1) Egg ,fertilization at the NOEC must be greater than 80%

of that in the controls.

(2) The minimum significant difference (%IvISD) is <25%

relative to the control.

(3) The sperm count for the final sperm stock must not

exceed 33,600,000/mL.

(4) If the sperm count for the final sperm stock is between

5,600,000 and 33,600,000/mL it must not exceed 2x of

the target density from the trial, or 'if no target

density was specified for the test (see 11.5.1), the

high sperm density controls (0.2 mL sperm stock) must

have at least 5% higher fertilization than tne low

sperm density controls (0.05 mL sperm stock) .

(5) Dilution water egg blanks and effluent egg blanks

should contain essentially no eggs with fertilization

membranes.

16.13 DATA ANALYSIS

16.13.1 GENERAL

16.13.1.1 Tabulate and summarize the data. Cal.culate the

proportion of fertilized eggs for each replicate. A sample set

of test data. is listed in Table 8.

16.13.1.2 The statistical tests described here must be used with

a knowledge of the assumptions upon which the tests are

contingent. The assistance of a,statistician is recommended for

analysts who are not proficient in stat~stics.

16.13.1.3 The endpoints of toxicity tests using' the sea urchin

and the sand dollar are based on the reduction in proportion of

eggs fertilized. The IC25 is calculated using the Linear

lnterpolation Method (see Section 9, Chrqnic Toxicity Test

Endpoints and Data Analysis). LOEC and NOEC values for fecundity

are obtained using a hypothesis testing approach such as

Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank Test

(Steel, 1959; Miller, 1981) (see Section 9). Separate ~nalyses

are performed for the estimation of th~ LOEC and NQEC endpoints

435
and for the estimation of the IC25. See the Appendices for

examples of the manual computations, and examples of data input

and program output.

TABLE 8. DATA FROM SEA URCHIN, STRONGYLOCENTROTUS PURPURATUS,

FERTILIZATION TEST

Effluent

Concentration No. of Eggs No. of Eggs Proportion

(\') Replicate Counted Fertilized F~rtilized

Control A 100 97 0.97

B 100 90 0.90

C '. 100 100 1. 00

0.05 A 100 100 1.00

B 100 100 1.00

C 100 98 0.98

O.J.O A 100 100 1. 00

B 100 97 0.97

C 100 99 0.99

0.J.5 A 100 98 0.98

B 100 96 0.96

C 100 97 0.97

0.20 A 10,0 94 0.94

B 100 88 0.88

C 100 97 0.97

0.40 A 100 43 0.43

B 100 63 0.63

C 100 46 0.46

0.60 A 100 2 0.02

B 100 1 0.01

C 100 9 0.09

0.80 A 100 0 0.00

B 100 0 0.00

C 100 0 0.00

436
16.13.2 EXAMPLE OF ANALYSIS OF SEA URCHIN, STRONGYLOCENTRpTUS.

PURPURATUS, AND SAND DOLLAR, DENDRASTEl? EXCENTRICUS,

FERTILIZATION DATA

16.13.2.1 Formal statistical analysis of the fertilization data

is outlined in Figure 7.

The response used in the analysis is the proportJon of fertilized

eggs in each test or control chamber. Separate analyses are

performed for the estimation of the NOEC and LOEC endpoints and

for the estimation of the IC25 endpoint. Concen1:rations at which

there are no eggs fertilized in any of the test chambers are

excluded from statistical analysis of the NOEC and LOEC, but

included in the estimation of the IC25.

16.13.2.2 For the case of equal numbers of replicates across all

concentrations and the control, the evaluation of the NOEC and

LOEC endpoints is made via a parametric test, Dul'mett' s

Procedure, or a nonparametric .test, Steel's Many'f-one Rank Test,

on the arc sine square root transformed data. illlderlying

assumptions of Dunnett's Procedure, normality an<i homogeneity of

variance, are formally tested. The test for normality is the

Shapiro-Wilk's Test, and Bartlett's Test is used to test for

homogeneity of variance. If either of these tes1~s fails, the

nonparametric test, Steel's Many-one Rank Test, is used to

determine the NOEC and LOEC endpoints.. If· the assumptions of

Dunnett's Procedure are met, the endpoints are estimated by the

parametric procedure. .

16.13.2.3 If unequal numbers of replicates occur among the

concentration levels tested, there are parametric and

nonparametric alternative analyses. The parametric analysis is a

t test with the Bonferroni adjustment (see Appendix D). The

wilcoxon Rank Sum Test with the Bonferroni adj us1:ment is the

nonparametric alternative.

16.13.2.4 Example of Analysi·s of Fecundity Data

16.13.2.4.1 This example uses toxicity data froIh a sea. urchin,

Strongylocentrotus purpuratus, fertilization tes1: performed with

effluent. The response of interest is the proportion of

fertilized eggs, thus each replicate must first .lJe transformed by

the arc sine square root transformation procedurl~ described in

Appendix B. The raw and transformed data, means I and variances of

the transformed observations at each effluent concentration and

control are listed in Table 9. The data are plo1:ted in Figure 8.

Because there is zero fertilization in all three I replicates for

the 0.80% effluent concentration, it was not included i.n the ' statistical analysis and is considered a qualita1:ive fecundity

effect. .

437
ENDPOINT ESTIMATE

IC25

HOMOGENEOUSVAR~CE

NO

YES

tTESTWITH

BONFERRONI

ADJUSTMENT

Figure 7. Flowchart for statistical analysis of sea urchin,

Strongylocentrotus pur,puratus, and sand dollar,

Dendraster excentricus, test.

438
nan v.wv -"

*

OA9 !!.8!! .

* IIIlMlAII!I'lDJEflIlIInUA1ION

ClDIIB:TIS11tEIIQNWIJoEl'OltlillCHc:otaJn'M1'Df

*

0.20

EFFLUENT CONCENTRATION (%).

1.0

0.9

0.8 Cl

w

N

- 0.7 -J

-

l- 0.6 a:::

w

u...

0.5 z

0

-

I- 0.4 CJJ a:::

0

Q..

0 0.3

a:::

Q..

0.2

0.1

0.0

0.00 0.05 9·10 ' - 0.15 - _-=--.._--- -~ -

Figure 8. Plot of proportion of fertilized sea urchin, Strongylocentrotus

purpuratus, eggs
~6.~3.2.5 Test for Normality

~6.~3.2.5.~ 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 10.

TABLE 9. SEA URCHIN, STRONGYLOCENTROTUS PURPURATUS,

FERTILIZATION DATA

Effluent Concentration (!l;)

Rep. Control 0.05 0.10 0.15 0.20 0.40 0.60

A 0.97 1.00 1.00 0.98 0.94 0.43 0.02

RAW B 0.90 1.00 0.97 0.9.6 0.88 0.63 0.01

C 1.00 0.98 0.99 0.97 0.97 0.46 0.09

ARC SINE A 1.397 1.521 1.521 1.429 1.323 0.7.15 0.142

SQUARE ROOT B 1.249 1.521 1.397 1.369 1.217 0.917 0.100

TRANSFORMED C 1.521 1.429 1.471 1.397 1.397 0.745 0.305

Mean ('11 ) 1.389 1.490 1.463 1.398 1.312 0.792 0.182

S~ 0.01854 0.00282 0.00389 6.00090 0.00819 0.01188 0.01173

i 1 2 3 4 5 6 7

TABLE 10. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S

EXAMPLE

Effluent 'Concentration (!l;)

Replicate Control 0.05 0.10 0.15 0.20 0.40· 0.60

A 0.008 0.031 0.058 0.031 0.011 -0.077 -0.040

B -0.140 0.031 -0.066 -0.029 -0.095 0.125 -0.082

C 0.132 -0.061 0.008 -0.001 0.085 -0.047 0.123

440
16.13.2.5.2 Calculate the denominator, D, of the statistic:

n

D = E (X - X) 2

i.l i

Where: Xi' = the ith centered observation

X = the overall mean of the centerE~d observations

n = the total number of centered observations

16.13.2.5.3 For this set of data, , n

X

21

_1_ (0.005)

21

0.000

D= 0.1159

16.13.2.5.4 Order the centered observations from smallest to

largest

xc!.) X(2) ... x(n)

where X(i) denot~s the ith ordered observation. The ordered

observations for this example are listed in Table 11.

16.13.2.5.5 From Table 4, Appendix B, for the nu~er of

observations, n, obtain the coefficients 'aI' a 2, ... ak where k is

n/2 if n is even and (n-1)/2 i~ n is odd. For the data in this

example, n = 21 and k = 10. The ai values are liBted in

Table 12.

16.13.2.5.6 Compute the test statistic, W, as follows:

k . 2

W = !-[La.(X(n-i+ll_x(il)]

D i.l

The differenc~s, x(n-i+l) - X(il, are listed in Table 12. For the

data in this example:

W = 1

0.1159

(0.3345)2 = 0.9654

16.13.2.5.7 The decision rule for this test is to compare W as

calculated in 2.6 to a critical value found in Table 6, Appendix

B. If the computed W is less than the critical value, .conclude

that the data are not normally distributed. For ·the data in this

441
TABLE 11. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S

EXAMPLE

i XCi) i XCiI

1 -0.140 12 0.008

2 -0.095 13 0.011

3 -0.082 14 0.031

4 -0.077 15 0.031

5 -0.066 16 0.031

6 -0.061 17 0.058

7 -0.047 18 0.085

8 -0.040 19 0.123

9 -0.029 20 0.125

10 -0.001 21 0.132

11 0.008

TABLE 22. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE

i ai x(n-i+l) - Xli)

2 0.4643 0.272 X (21 ) - X (1 )

2 0.3285 0.220 X I2O ) - X(2)

3 0.2578 0.205 X (19 ) - X (3 )

4 0.2119 0.162 X I1S ) - X(4)

5 0.1736 0.l.24 X I1 ?) - XIS)

6 0.l.399 0.092 X (16 ) - X (6 )

7 0.1092 0.078 X I1S ) - Xl?)

8 0.0804 0.071 X (14 ) - XIS)

9 0.0530 0.040 X (13 ) - X(9)

20 0.0263 0.009 X(12) - XC1O)

example, the critical value at a significance level of 0.01 and n = 21 observations is 0.873. Since W = 0.9654 is greater than the

critical value, conclude that the data are normally distributed.

16.13.2.6 Test for Homogeneity of Variance

16.13.2.6.1 The test used to examine whether the variation in

the proportion of fertilized eggs is the same across all effluent

442
concentrations including the control, is Bartlett'$ Test

(Snedecor and Cochran, 1980). The test statistic is as follows:

p p

[ ( V.) In 82 - V. In oS J

1.1 1·1

B = -~~---_..=....::._---

c

Where: Vi ~. degrees of freedom for each concentration and

control,

p = number of concentration levels including the control

n i = the number of replicates for concentration i.

In = loge

i = 1,2, ... , p where p is the number of: concentrations

including the control

p

( ~v.s;) B2 = ....,...::i_~l_~__

p

~V.

1.1

p p.

C .. 1+ (3 (p-1) r1(~1/V.- (~V.) -lJ

1.1 .1 1.1

16.13.2.6.2 For the data in this example (see Table 8), all

effluent concentrations including the co~trol have the same

number of replicates (ni = ) for all i). Thus, Vi = 2 for all i.

~6.~3.2.6.3 Bartlett's statistic is, therefore:

p.

B = [(14)ln(O.008279)-2Eln(S;)] /1.190S

1·1

=

=

=

[~4C-4.7940) - 2(-36.~047)']/1..~905

5.0934/~.1905

4.2784

16.~3.2.6.4 B is approximately distributed as chi-square with

p-1 degrees of freedom, when the variances are in fact the same.

Therefore, the approp+iate critical value for this test, at a

significance level of 0.01 with 6 degrees of freedom, is 16. 8:L~

Since B = 4.2784 is less than the critical value of 16: 8~, --

conclude that the variances are not different.

443
16.13.2.7 Dunnett's Procedure

16.13.2.7.1 To obtain an estimate of the pooled variance for the

Dunnett's Procedure, construct an ANOVA table as described in

Table 13.

TABLE 13. ANOVA TABLE

Source df Sum of Squares

(SS)

Mean Square (MS)

(SS/d~)

Between p - ]. SSB SB = SSB/ (p-].)

Within N - P SSW

2

Sw SSw/ (N-p)

Total N - J. SST

Where: p = number of concentration l~vels including the

control

N = total number of observations n 1 + n 2 ... + n p

ni = number of observations in concentration i

p

SSE. LT;/n1-G 2/N

1.1

Between Sum of Squares

Total Sum of Squares

SSW. SST-SSE Within Sum of Squares

the ~rand total of ali sample observations,

G" LT1 1.1

the total of the replicate measurements for

concentration i

G =

Ti =

Yij == the jth observation for concentration i

(represents the proportion of fertilized eggs for

concentration i in test chamber j)

444
16.13.2.7~2 For the data in this example:

N = 21

T1 = Yll + Y12 + Y13 4.167

.T2 Y21 + Y22 + Y23 = 4.471

T3 = Y31 + Y32 + Y33 4,389

T4 Y41 + Y42 + Y43 4.194

Ts = YS1 + YS2 + YS3 3.937

T6 = Y61 + Y62 + Y63 = 2.377

T7 Y71 + Y72 + Y73 0.547

G = T1 + T2 + T3 + T4 + Ts + T6 + T7 = 24.082

p

SSB = L T~/n ._G 2 /N . . . .

i=i t95~ 656) /3 - (24 .. 082) 2/21 4.269

SSW = SST -SSB

P n i

SST = LLy2._G2/N

~";ij3 2il 001 - (24 . 082 ) 2/21 = 4. 385

4.385 - 4.269 = 0:116

2

SB = SSB/(p-1) = 4.269/(7-1) = 0.7115

2

Sw = SSW/(N-p) = 0.116/(21-7) - 0.0083

16.13.2.7.3 Summarize these calculations in the ANOVA table

(Table 14) .

16.13.2.7.4 To perform the individual comparisons, calculate the

t statistic for each concentration, and control c~mbination as

follows:

! >

;

445
TABLE 14. ANOVA TABLE FOR DUNNETT'S PROCEDURE

EXAMPLE

Source df Sum· of Squares Mean Square (MS)

(SS) (SSjdf)

Between 6 4.269 o. 7115

Within J.4 0.116 0.0083

Total 20 4.385

Where: Yi == mean proportion fertilized 'eggs for concentration i

Y1 = mean proportion fertilized eggs for the contro.l

Sw = square root of the within mean square

n 1 = number of replicates for the control

ni = number of replicates for concentration i.

Since we are looking for a decreased response from the .control in

the proportion of fertilized eggs, the concentration mean is

subtracted from the control mean.

16.13.2.7.5 Table ,15 includes the calculated t values for each

concentration and control combination. In this example,

comparing the 0.05% concentration with the control the

calculation is as follows:

t = (1.389 - 1.490) = -1.358

2 0.0911"/(1/3)+(1/3)

16.13.2.7.6 Since the purpose of this test is to detect a

significant decrease in the proportion of fertilized eggs, a

one-sided test is appropriate. The critical value for this

one-sided test is found in Table 5, Appendix C. For an overall

alpha level of 0.05, 14 degrees of freedom for error and six

concentrations (excluding the control) the critical value is

2.53. The mean proportion of fertilized eggs for concentration i

is considered significantly less than the mean proportion of

fertilized eggs for the co~trol if t i is greater than the

446
critical value. Therefore, the b.40% and 0.60% concentrations

have a significantly lower mean proportion of fertilized eggs

than the control. Hence the NOEC is 0.20% effluent and the LOEC

is 0.40% effluent. .

TABLE 15. CALCULATED t VALUES

Effluent Concentratiqn (%).

0.05

0.10

0.15

0.20

0.40

0.60

i

2

3

4

5

6

7

-1. 358

-0.995

-0.121

1. 035

8.026

16.227

16.13.2.7.7 To quantify the sensitivity.of the test, the minimum

significant difference (M,SD) that can be statist:ically detected

may be calculated: .

MSD = d S w J(1/n 1 )+ (1/n)

Where: d =

Sw =

n

the critical value for Dunnett's Procedure

the square root of the within meOln.square

the common, number of replicates Ol.t each

concentration (this assumes equal replicati,on at

each concentration)

n l the number·of replicates in, the control.

16.13.2.7.8 In this example,

MSD = 2.53 (0.0911) V(1/3) + (1/3)

= 2. 53 ( 0 . 0911) (0. 816:5 )

= 0.188

16.13.2.7.9 TheMSD (0.188) is in transformed units. To.

determine the MSD in terms of proportion 'of ferti·lized eggs,

carry out the following. conversion.

1. Subtract the .MSD from the transformed cOIltrol mean.

1.389 - .0.188 = 1.201

447
2. Obtain the untransformed values for the control mean and

the difference calculated in step 1 of 13.2.7.9.

Sine (1. 389) J2 = 0.967

[ Sine (1. 201) J2 = 0.869

3. The untransformed MSD (MSDu ) is determined by sUbtracting

the untransformed values from step 2 in 14.2.7.9.

MSDu = 0.967 - 0.869- 0.098

16.13.2.7.10 Therefore, for this set of data, the minimum

difference in mean proportion of fertilized eggs between the

control and any effluent concentration that can be detected as

statistically significant is 0~098.

16.13.2.7.11 This represents a 10.2% decrease in the proportion

of fertilized eggs from the control.

16.13.2.8 Calculation of the rcp

16.13.2.8.1 The fertilization data in Table 7 are utilized in

this example. As can be seen from Figure 8, the observed means

are not monotonically non-increasing with respect to

concentration. Therefore, the means must be smoothed prior to

calculating the rc.

16.13.3.8.2 Starting with the observed control mean, Y1 = 0.957,

and the observed mean for the lowest effluent concentration, Y2

= 0.993, we see that Y1 is less than Y2 •

16.13.3.8.3 Calculate the smoothed means:

16.13.3.8.4 Since Y3 = 0.987 is larger' than M2 , average Y3 'with

the ~revious concentrations:

M1 = M2 = M3 = (M1 + M2 + ~3)/3 = 0.979.

16.13.3.8.5 Since M3 > Y4 = 0.970 > Ys =0.930 > Y6 = 0.507 > Y7 =

o. 040 > Ys = O. 0, set M4 = O. 970, Ms = O. 93 0, M6 = O. 5 07, M7 = 0.040, and Ms = 0.0. Table 16 contains the smoothed means and

Figure 10 gives a plot of the smoothed means and the interpolated

response curve.

16.13.2.8.6 An IC25 can be estimated using the Linear

Interpolation Method. A 25% reduction in mean proportion of

fertilized eggs, compared to the controls, ,would result in a mean

proportion of 0.734, where M1 (1-p/100) = 0.979(1-25/100).

Examining the means and their associated concentrations

448
(Table 16), the response, 0.734, is bracketed by Cs = 0.20%

effluent and C6 = 0.40% effluent.

16.13.2.8.7 Using the equation from Section 4.2 in Appendix L,

the estimate of the IC25 is calculated as follow's:

(C (j.l) -cj) ICp = cj + [M1 (1-p/100) -Mj ] --'-"---:---":"-

(M(j+l) -Mj )

IC25 = 0~20 + [0.979(1 - 25/100) - 0.930] (0.40 - 0.20)

,(0.507 - 0.930)

= 0.29%.

TABLE 16. SEA URCHIN, STRON'J!"LOCENTROTUSPURPURATUS,

MEAN PROPORTION OF FERTILIZED EGGS

Effluent Response Smo.othed

Cone. Means, Yi Mean~, Mi

(%) i (proportion) (proportion)

Control J. 0.957 0.979

0 .. 05 2 0.993 0.979

O.J.O 3 0.987 0.979

0.J.5 4 0.970 0.970

0.20 5 0.930 0.930

0.40 6 0.507 0.507

0.60 7 0.040 0.040

0.80 8 0.000 0.000

16.13.2.8.8 When the ICPIN program was used to analyze this set

of data, requesting 80 resamples, the estimate of: the IC25 was

0.2925%. The empirical 95.0% confidence interval for the true

mean was 0.2739% to 0.3241%. The computer program output for the

IC25 for this data set is shown in Figure 10.

16.14 PRECISION AND ACCURACY

16.14.1 PRECISION

16.14.1.1 Single-Laboratory Precision

449
J.6.J.4.J..J..J. Single-laboratory precision data for

Strongylocentrotus purpurtatus with the reference toxicant

copper, tested in natural seawater, are provided in Table 17.

The coefficient of variation based on the EC25 is 29%, and on

EC50 is 24%, showing acceptable precision. Single-laboratory

precision data for Dendraster excentricus with the reference

toxicant copper, tested in natural seawater, are provided in

Tables 18 and J.9. The coefficient of variation based on the

EC25, is 18% to 29% and EC50, is 21% to 33%, showing acceptable

precision.

16.14.1.2 Multi-laboratory Precision

J.6.14.J..2.1 Multi-laboratory precision data for

Strongylocentrotus purpuratus, with the reference toxicant

copper, tested in natural seawater, are provided in r~ble 20.

The coefficient of variation for the EC25 was 52%, based on data

from five laboratories.

16.J.4.2 ACCURACY

16.J.4.2.J. The accuracy of toxicity tests cannot be determined.

450
----~--.-

***-~­... - - - - - - .-:18_-""-",

1.0

0.8

0.8 0

I..LJ

N

- 0.7 -'

-

I- 0.6 0::

I..LJ

I..L.

0.5 z

0

IIi>o -

I- OA U1 0::

.... 0

a...

0 0.3 0::

a...

0.2

0.1

0.0

0.00 0.05 0.10 0.15 0.20

eFfI.lJEJ\ITOONCENTRAiION (;&)

0.40 0.60 0.80

Figure 9. plot of raw data, observed means, and smoothed means for the sea

urchin, -Strongylocentrotus pur,puratus, eggs.
Cone. ID 1 2 3 4 5 6 7 8

Cone. Tested o .05 .10 .15 .. 20 .40 .60 .80

Response 1 .97 1. 00 1. 00 .98 .94 .43 .02 0

Response 2 .90 1.00 .97 .96 .88 .63 .01 0

Response 3 1.00 .98 .99 .97 .97 .. 46 .09 0

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

*** Inhibition Concentration Percentage Estimate *** Toxicant/Effluent: Effluent

Test Start Date: Test Ending Date:

Test Species: Sea Urchin, Strongylocentrotus purpuratus

Test Duration: 40 minutes

DATA FILE: urchin.icp

OUTPUT FILE: urchin.i25

Cone. Number Concentration Response Std. Pooled

ID Replicates % Means Dev. Response Means

---------------------------~----------,--------------- ------------------

1 3 0.000 0.957 0.051 0.979

2 3 0.050 0.993 0.012 0.979

3 3 0.100 0.987 0.015 0.979

4 3 0.150 0.970 0.010 0.970

5 3 0.200 0.930 0.046 0.930

6 3 0.400 0.507 0.108 0.507

7 3 0.600 0.040 0.044 0.040

8 3 0.800 0.000 0.000 0.000

The Linear Interpolation Estimate: 0.2925 Entered P Value: 25

0.2917

Lower:

Lower:

0.22

Number of Resamplings: 80

The Bootstrap Estimates Mean:

Original Confidence Limits:

Expanded Confidence Limits:

Resampling time in Seconds:

Standard Deviation: 0.0141

0.2739 Upper: 0.3241

0.2533 Upper: . 0.3589

Random Seed: -25579058

Figure 10. ICPIN program output for the IC25.

452
TABLE 17. SINGLE LABORATORY PRECISION OF .THE SE1~ URCHIN,

STRONGYLOCENTROTUS PURPURATUS FERTILI~~ATION TEST

PERFORMED IN SEAWATER USING GAMETES'FROM ADULTS

MAINTAINED IN S·EAWATER AFTER BEING COIJLECTED FROM

NATURAL POPULATIONS WITH COPPER (CU JlG/L)' SULFATE.AS

THE REFERENCE TOXICANT I'

Test Number NOEC (p,g/L) EC25 (p,g/L) EC50 (fJ,g/L) : '. , . , '

1 6.9 9.7 14.3

2 23.0 26~2 ' ,

30.9

3 11.2 19.6 25.8

4 16.0 16.4 , 31,.1

5 15.3 17.8 24.6

,

'6 10.8 18.6 28.3

Mean 18.1 25.8

CV(%), "

29.0 24.0 ,

Tests 'performed by Sally Noack~ AScI, at EPA's Pacific Ecosystems

Branch of ERL-Narragansett, Newport, OR. ",

Copper concentrations were measured and within 10% of nominal;

nominal qoncentrations were 5, 8, 12, 17, 25, 35~ and 50 Jlg/L.

These tests used only three replicates per concentration.

453
TABLE 18. SINGLE LABORATORY PRECISION OF THE SAND DOLLAR,

DENDRASTER EXCENTRICUS FERTILIZATION TEST PERFORMED IN

SEAWATER USING GAMETES FROM ADULTS MAINTAINED IN

SEAWATER AFTER BEING COLLECTED FROM NATURAL POPULATIONS

WITH COPPER (CU ~G/L) SULFATE AS THE REFERENCE TOXICANT

Test Test NOEC (~g/L) EC25 (~g/L) EC50 (~g/L)

Date Number

7/11/94 1* 5.0 9.4 12.6

2** 5.0 14.6 17.5

3*** - 16.0 18.6

7/14/94 1* 12.0 16.7 20.9

2** <5.0 19.6 25.8 , " 3*** 17.0 23.0 30.5

7/17/94 1* 8.0 15.3 17.7

2** 5.0 13.5 16.4

3*** 12.0 13.4 17.0

7/19/94 1* 12.0 12.8 15.6 I 2** 17.0 18.6 22.1

3*** 12.0 13.3 16.0

i Mean 1 13.5 16.7

2 16.6 20.5

3 16.4 20.5

overall

SD 1 3.2 3.5 ; 2 3.0 4.3

3 4.6 6.7 , overall

CV(%) 1 24% 21%

2 18% 21%

3 28% 33%

overall

Tests performed at National Council of the Paper Industry for Air

and Stream Improvement, Inc. Anacortes, WA.

Copper concentrations were nominal; nominal concentrations were

5, 8, 12, 17, 25, 35, and 50 ~g/L.

* Tests conducted with nominal S:E ratio of 147:1

** Tests conducted with nominal S:E ratio of 166:1

*** Tests conducted with nominal S:E ratio of 224:1

454
TABLE 19. SINGLE LABORATORY PRECISION OF THE SAND DOLLAR,

DENDRASTER EXCENTRICUS FERTILIZATION TEST PERFORMED IN

SEAWATER USING GAMETES FROM ADULTS MAINTAINED IN

SEAWATER AFTER BEING COLLECTED FROM NATURAL POPULATIONS

WITH COPPER (CU ~G/L) SULFATE AS THE REFERENCE

TOXICANT.

Test Number NOEC (~g/L) EC25 (jlg/L) EC50 (jlg/L)

1 17.0 25.8 31.0

2 25.0 34.3 41.8

3 12.0 31.1 43.7

4 8.0 '- 14.2 19.8

5 25.0 27.2 30.5

Mean 26.5 33.4

CV (%) 29.0 29.0

Tests performed by Gary Chapman and Debra Denton at EPA's Pacific

Ecosystems Branch of ERL-Narragansett, Newport, OR.

Copper concentrations were nominalj nominal concentrations were

5, 8, 12, 17, 25, 35, and 50 ~g/L.

455
TABLE 20. MULTIPLE LABORATORY PRECISION OF THE SEA URCHIN,'

STONGYLOCENTROTUS PURPURATUS, FERTILIZATION TEST

PERFORMED WITH COPPER (CU ~G/L) SULFATE AS A REFERENCE

TOXICANT

Lab # of Tests Statistic EC25 (JLg/L)

A 3 Mean 7.8'

SD 3.0

CV(%} 38%

B 2 Mean 4.0

SD - CV (%) - '. C 6 Mean 18.0

SD 5.4

CV(%} 30%

D 2 Mean 14.9

NA - CV(%} -

E 6 Mean 19.3

SD 10.5

CV(%} 54%

# of Lab Statistic EC25

Means

~5 Mean 12.8

SD 6.6

CV(%} 52%

Tests performed as part of a methods evaluation effort organized

by the US EPA laboratory in Newport, Oregon; tests were' conducted

in 1991 by volunteer laboratories in California and Washington.

456
APPENDIX I .. PURPLE URCHIN AND SAND DOLLAR TEST: STEP-BY-STEP

SUMMARY

PREPARATION OF TEST SOLUTIONS

A. Determine test concentrations and appropriate dilution water

based on NPDES permit conditions and guidance from the

appropriate reg~latory agency. .,

B. Prepare effluent" test solutions by diluting' well mixed

unfiltered effluent using volumetric flasks and pipettes.

Use hypersaline brine where' necessary to ma,intain all test.

solutions at 34 ± 2%0. Include brine contro,ls in tests that

use brine.

t

C. Prepare a copper reference toxicant stock solution (2,000

mg/L) by adding 5.366 9 of copper chloride (CuC12 02H20) to 1

liter of reagent water. For each reference.toxicant test

prepare a copper sub-stock of 3 mg/L by diluting 1.5 mL of

stock to one liter with reagent water.

D. Prepare a control (0 jJ-g/L)' plus at least fiv'e consecutive

copper reference toxicant solutions (e.g., from the series

3.0, 4.4, 6.5, 9.5, 1.3.9, 20.4, and 30.0 J.Lg/L, by adding

0.1.0, 0.15, 0.22, 0.32, 0.46, 0.68, and. 1..00 mL of sub~stock

solution, respectively, to 1.00-L volumetric flasks and

filling to 1.00-mL with dilution water) .

E. Randomize numbers for test chambers and record the' chamber

numbers with their respective test concentrations on a

randomization data sheet. Store the data sheet safely untll

after the test samples have been analyzed .

.F. Sample effluent and reference toxic~nt solu1:ions for

physical/chemical analysis. Measure salini1:y, pH and'

dissolved oxygen from each test concentration.

G. Place test chambers in a water bath or environmental chamber

set to 12°C and allow temperature to equilibrate.

H. Measure the temperature in several temperature blanks during

the course of the test.

PREPARATION AND ANALYSIS OF TEST ORGANISMS

A. Obtain test organisms and hold or condition ,as necessary for

spawning.

B. On day of test, spawn organisms, examine gametes,_pool good

eggs, pool good sperm.

C. Determine egg and sperm densities and adjust as necessary.

457
D. Run trial sperm:egg fertilization tes't (optional).

E. Adjust sperm density for definitive test.

F. Inject sperm into test solutions.

G. 20 minutes later inject eggs into test solutions.

H. 20 minutes after egg addition, stop the test by the addition

of preservative.

I. Confirm sperm density in definitive test by hemacytometer

counts.

J. Count at least 100 eggs in each test tube.

K. Analyze the data.

L. Include standard reference toxiqant 'point estimate values in

the standard quality control charts.

458
APPENDIX II. USING THE NEUBAUER HEMACYTOMETER ,~?O ENUMERATE SEA c,(

URCHIN SPERM

.S

The Neubauer hemacytometer is a specialized microscope slide with

two counting grids and a coverslip.

TOP VIEW:

COVERSLIP

!I-t- SUPPORT

COUNTING GRIDS

(size exaggerated)

(see detail n~Xt page)

4~9
Together, the total area of each grid (1 mm2 ) and the ve~tical

distance between the grid and the coverslip (0.1 mm), provide

space for a specific microvolume of aqueous sample (0.1 mm3 ) •

SIDE VIEW:

Counting

Ala / Coverslip

-------:-:=rJ:----=,- ~~Well Loading

Notch

END VIEW THROUGH MID-CROSS SECTION:

Coverslip

Counting 1 Counting

Loading Areal +Area Loading

NOlch-C -- l' -.=:J-NOlch

Overflow Well

460
This volume of liquid and the cells suspended thereinr

• (e. g. ,

blood cells or sperm cells) represent lllO ,'OOOt:h ·of t.he liquid

volume and cell numbers of a full miLl.iliter (cm3 ) of the sampled

material. .

NEUBAUER

HEMACYTOMETEIR

GRID OF 400 SQUARES

If the full 400-squares of each grid are counted, this represents

the number of sperm in O.l mm3 • Multiplying this value times lO

yields the sperm per mm3 (and is the source of the hemacytometer

factor of 4,000 squares/mm3 ). If this product is multiplied by

1,000 mm3 /cm3 , the answer is the number of sperm in one

milliliter of the sample. If the counted sample represents a

dilution.of a more concentrated original sample, the above answer

is multiplied by the dilution factor to yield t4e' cell 'density in

the original sample. If the cells are sufficiently dense, it is

not necessary to count the entire 400-square field, and the final.

calculation takes into account the number of squares actually

counted:

cells/mL = (dilution) (4,OOO sguares/mm3 ) (l,OOO mm3 /cm3 ) (cell count)

(number of squares counted) ,

Thus, with a dilution of 4000 (O. 025 mL of semen: in 100' mL of

dilution water), 80 squares counted, and a count of 100, the

calculation becomes:

cells/mL = (4,OOO) (4,000) (1.000) (100)

80

= 20,000,000,000 cells/mL

461
There are several procedures that are necessary for counts. to be

consistent within and between laboratories. These include mixing

the sample, loading and emptying the hematocrit tube, cleaning

the hemacytometer and cover slip, and actual counting procedures.

Obviously, if the sample is not homogeneous, subsamples can vary

in sperm density: A few extra seconds in mixing can save a lot

of wasted minutes in subsequent counting procedures. A full

hematocrit tube empties more easily than one with just a little

liquid, so withdraw a full sample. This can be expedited by

tipping the sample vial.

Because the sperm are killed prior to sampling, they will slowly

settle. For this reason, the sample in the hematocrit tube

should be loaded onto the hemacytometer as rapidly as possible.

Two replicate samples are withdrawn in fresh hematocrit tubes and

loaded onto opposite sides of a hemacytometer.

Coverslip

Counting 1 Counting

Area Area Loading

r=::~~===--a::==Ci::r=:=5-2==;;-dt:.::l_Notch

Overflow Well

462
The loaded hemacytometer is left for 15 minutes to allow the

sperm to settle onto the counting field. If the coverslip is

moved after the samples are loaded, the hemacytometer should be

rinsed and refilled with fresh sample. After 15 minutes, the

hemacytometer is placed under a microscope and the counting grid

located at 100x. Once the grid is properly positioned, the

microscope is adjusted to 200x or 400x, and one of the corner

squares is positioned for counting (any one of the four corners

is appropriate). For consistency, use the same procedure each

time (Many prefer to start in the upper left corner of the

optical field, and this procedure will be used in 'the examples

given below) .

Examine the first large square in the selected corner. If no

sperm are visible, or if the sperm are so dense pr clumped to

preclude accurate counting; count a sample with a more'

appropriate dilution.

In making counts of sperm, it is necessary to adopt a consistent

method of scanning the smaller squares and counting sperm that

fall upon the lines separating the squares. Count the sperm in

the small squares by beginning in the upper left: hand corner

(square 1) and proceding'right to square 4, down to square 5,

left to square 8, etc. until all 16 squares are counted.

OJ 0

0 Jl

Jl

Q.

2 OJ 3 g. 1. 0 p 4

0 OJ

Q.

P p 0

p

0" 0 7 ",6 5" 0 " (p 0 if

" 0

" 9 if 10 11 0 12 a

0 a if g. " , " '" 160

15 " 14 013 0

463
Because sperm that appear Qll lines might be counted as being in ' either square, it is important to avoid double counting or noncounting. For this reason a convention is decided upon and used

consistently: paraphrasing the instructions received with one

(Hausser Scientific) counting chamber "to avoid counting (sperm)

twice, the best practice is to count all touching the top and

left, and none touching the lower and right, boundary lines."

Whatever convention is chosen, it must be adhered,to. The

example below shows a sperm count based upon a selected

convention of counting sperm that fallon the upper and left

lines, but not on the lower or right lines:

27

28

0=

Not

Counted

54

,,>2 0 13

1 03 p8 12 ' . 9 p14

4Q. 5 7,,> go d 60 10 IIp

Q. 25 in 180 15">

24 22 P16

P 021 ( D P

026 017 23 "> 19 0

029 032 33 36 37 4Jl() 039

300 034 0

cf 31 35 ~80 41 42d

52 0 d 46 0

cf 51 49 48 g. 44

47 043 ">

053 500 , 045

0 "" " '-'

In the above illustration, sperm falling on the lower and right

lines are not counted. The count begins at the upper left'as

illustrated in the preceding figure. A typical count sequence is

demonstrated by the numbers next to each sperm illustrated.

Sperm identified as numbers 1, 5, 13, 20, 27, 28, 33, 51 and 54

touch lines and are counted as being in the square below them or

to their right. 'The circled sperm arenQt counted as being in

this field of 16 small squares (but they would be included in any

counts of adjacent squares in which they would be on upper or

left hand lines) .

464
Once these counting conventions have been selected, it is

advisable to follow another strict protocol outlining the number

and sequence of large squares to be counted.: Because the sperm

may not be randomly distributed across the counting grid; it is

recommended to count an array of squares covering the entire

grid. The following procedure is recommended:

Count the number of sperm in the first large' sq,uare.

1. If the number is less than 10, count all 25 squares using

the same scanning pattern outlined above (left to right

through squares 1 to 5, down to square 6, ~eft through

square 10, down to 11, etc.). See pattern, no. 3.

2. If the number is between 10 and 19, count 9 large squares

using pattern no. 2.

3. If the number is 20 or greater, count 5 -large squares using

pattern no. 1.

1 2

3

4 5

Pattern no. 1

1 2

4 3

5

7 6

8 9

Pattern no. 2

I 1 2 3 4 5

10 9 8 7 6

11 12 13 14 15

20 19 18 17 16

21 22 23 24 25 , ,

Pattern no. 3 i

The final consideration in achieving good replicate counts is

keeping the hemacytometers andcoverslips clean~ They should be

rinsed in distilled water soon after use. Thecoverslips should

be stored in a good biocleanser such as hemasol. For an hour or

so prior to use, the hemacytometer slides should also be soaked

in the solution. Both slides and coverslips should then be

rinsed off with reagent water, blotted dry with'a lint-free'

tissue, and wiped with lens pape!.

" , ,

465
SECTION 17

GIANT KELP, Macrocystis pyrifera

GERMINATION AND GERM-TUBE GROWTH TEST METHOD

Adapted from a method developed by

Brian S. Anderson and John W.. Hunt

Institute of Marine Sciences, University of California

Santa Cruz, California

(in association with)

California Department of Fish and Game

Marine Pollution Studies Laboratory

34500 Coast Route 1, Monterey, CA 93940

TABLE OF CONTENTS

Appendix I

17.1

17.2

17.3

17.4

17.5

17.6

17.7

17.8

17.9

17.10

17.11

17.12

17.13

17.14

Scope and Application

Summary of Method

Interferences

Safety

Apparatus and Equipment

Reagents and Supplies

Effluents and Receiving Water Collection,

Preservation, and Storage

Calibration and Standardization

Quality Contro+

Test Procedures

Summary of Test Conditions and Test

Acceptability Criteria

Acceptability of Test Results

Data Analysis

Precision and Accuracy

Step-by Step Summary

466
SECTION 17

GIANT KELP I MACROCYSTIS PYRIFERA

GERMINATION AND GROWTH TEST

17.1 SCOPE AND ,APPLICATION

17.1.1 This method estimates the chronic toxicity of effluents

and receiving water to zoospores and embryonic ,gametophytes of

giant kelp, Macrocystis pyrifera .during a 48-h static non-renewal

exposure. The effects include the synergistic, antagonistic, and

additive effects of all the chemical, physical, and biological

components which adversely affect the physiological and

biochemical functions of the test organisms.

17.1.2 Detection limits of the toxicity of an effluent or

chemical substance are organism dependent.

17.1.3 Brief excursions in toxicity may not be detected using

24-h composite .samples. Also, because of the long sample

collection period involved in composite sampling' and be'cause the

test chambers are not sealed, highly volatile and highly

degradable toxicants in the source may not be detected in the

test.

17.1.4 This method is commonly used in one of .two forms: (1) a'

definitive test, consisting of minimum of 'five effluent

concentrations and a control, and (2) a receivin:g water test (s) ,

consisting of one or more receiving water concentrations and a

control.

17.1.5 This method should be restricted to use by, or under the

supervision of, professionals experienced in aquatic toxicity

testing. Specific experience with any toxicity test is usually

rieeded before acceptable results become routine.,

17.2 SUMMARY OF METHOD

17.2.1 This method provides step-by-step instru.ctipns for

performing a 48-h day static non-renewal ·toxicity test using

giant kelp to determine the toxicity of substances in marine and

467
estuarine waters. The test endpoints are germination of

gameophyte spores and length of embryonic gametophyte germination

tubes.

17.3 INTERFERENCES

17.3.1 Toxic substances may be introduced by contamin~nts in

dilution water, glassware, sample hardware, and testing equipment

(see Section 5, Facilities and Equipment, and Supplies) .

17.3.2 Improper effluent sampling and handling may adversely

affect test results (see Section 8, Effluent and Receiving Water

Sampling, and Sample Handling, and Sample Preparation for

Toxicity Tests) .

17.4 SAFETY

17.4.1 See Section 3, Health and Safety.

17.5 APPARATUS AND EQUIPMENT

17.5.1 Tanks, trays, or aquaria -- for holding and acclimating

giant kelp, e.g., standard salt water aquarium or Instant Ocean

Aquarium (capable of maintaining,seawater at 10-20°C), with

appropriate filtration and aeration system.

17.5.2 Air pump, air lines, and air stories -- for aerating water

containing broodstock or for supplying air to test solutions with

low dissolved oxygen.

17.5.3 Constant temperature chambers or water baths -- for

maintaining test solution temperature and keeping dilution water

supply, gametes, and embryo stock suspensions at test temperature

(15°C) prior to the test.

17.5.4 Water purification system -- Millipore Super-Q, Deionized

water (DI) or equivalent.

17.5.5 Refractometer

17.5.6 Hydrometer(s)

for determining sali~ity.

for calibrating.refractometer.

468
17.5.7 Thermometers, glass or electronic, laboratory grade

for measuring water temperatures.

17.5.8 Thermometer, National Bureau of Standards Certified (see

USEPA METHOD 170.1, USEPA, 1979) -- to calibrate laboratory

thermometers.

17.5.9 pH and DO meters -- for routine physical and chemical

measurements.

17.5.10 .standard or micro-Winkler apparatus - - ... for determining

DO (optional) and calibrating the DO meter.

17.5.11 Winkler bottles -- for dissolved oxygen determinations.·

17.5.12 Balance

0.0001 g.

Analytical, capable of accura.tely weighing to

17.5.13 Fume hood -- to protect the analyst from effluent or

formaldehyde fumes.

17.5.14 Glass stirring rods for mixing test solutions.

17.5.15 Graduated cylinders -- Class A, borosil:Lcate glass or

non-toxic plastic labware, 50-1000 mL for making test sol~tionsr

(Note: not to be used interchangeably for gametes or embryos and

test solutions).

17.5.16 Volumetric flasks - - Class A, borosilicc~te glass or non~

toxic plastic labware, 10-1000 mL for making test: solutions.

17.5.17 Pipets, automatic ~- adjustable, to cover a range of

delivery volumes from 0.010 to 1.000 mL.

17.5.18 Pipet bulbs and fillers -- PROPJPET@ or equivalent.

17.5.19 Wash bottles -- for reagent water, for t:opping off

graduated cylinders, for rinsing small glassware and instrument

electrodes and probes.

17.5.20 Wash bottles -- for dilution water.

469
~7.5.2~ 20-liter cubitainers or polycarbonate water cooler jugs

-- for making hypersaline brine.

17.5.22 Cubitainers, beakers, or similar chambers of non-toxic

composition for holding, mixing, and dispensing dilution water

and other general non-effluent, non-toxicant ,contact uses. These

should be clearly labeled and not used for other purposes.

7.5.23 Beakers, 250 borosilicate ,glas,s - - for mixing test

solutions.

17.5.24 Beakers, 1,000 mL borosilicate glass --' for holding

sporophyll blades.

~7.5.25 Inverted or compound microscope '-- for inspecting

zoopspores and embryonic gametophytes. '

~7.5.26 Hemacytometer (bright-line rbc)

zoospore density.

for measuring

~7.5.27 Counter, two unit, 0-999

zoopspores.

for recording counts of

~7.5.28 Light meter (irradiance'meter w/cosine corrected sensor)

-- for measuring light intensity.

17.5.29 Cool white fluorescent lights -- for providing light

during incubation of developing gametophytes.

~7.5.30 60 ~m NITEX® filter

~7.6 REAGENTS AND SUPPLIES

for filtering receiving water.

7.6 . Sample containers - - for sample shipment and storage ,(see

Section 8, Effluent and Receiving Water Sampling, arid Sample

Handling, and Sample Preparation for Toxicity Tests) .

~7.6.2 Data sheets (one set per test)' -- for data recording:

(Figures and 2) .

17.6.3 Tape, colored -- for labelling test chambers and

containers.

470
17.6.4 Markers, water-proof --for mark:tng containers, etc.

17.6.5 Parafilm -- to cover graduated cylinders and vessels.

17.6.6 Glo:ves, disposable - - for personal prot:ection from

contamination.

17.6.7 Pipets, serological 1-10 mL, graduat:ed.

17.6.8 Pipet tips

17.6.9 Coverslips

for automatic pipets.

for microscope slides.

17.6.10 Lens paper -- for clea:ning microscope optics.

17.6.11 Laboratory tissue wipes for cleaniIl~g and drying

electrodes, microscope slides, etc.

17.6.12 Disposablecountertop covering -- for protection of work

surfaces and minimizing spills and contamination.

17.6.13 pH buffers 4, 7, and 10 (or. as ~er instructions of

instrument manufacturer) --for standards and calibration check

(see USEPA Method 150.1, USEPA, 1979).

17.6.14 Membranes and filling solutions -- for dissolved oxygen

probe (see USEPA Method 36Q.l, USEPA, 1979), ori reagents for

modified Winkler analysis.

17.6.15 Laboratory quality assurance samples and standards --

for the above methods.

17.6.16 Test chambers -- 600 mL, five chambers per

concentration. The chambers should be borosilicate glass (for

effluents) or,nontoxic disposable. plastic labware (for reference

toxicants). To avoid contamination from the air and excessive

evaporation of test solutions during the .test, ~he chambers

should be covered during the test with safety glass plates or a

plastic sheet (6 mm thick) .

17.6.17 Glutaraldehyde -- for specimen preservation - optional;

(see Section 17.10.8.2).

471
17.6.18 Microscope slide (flat) -- for each test chamber to

serve as the substratum upon which the zoospores will settle.

17.6.19 Reference toxicant solutions (see Section 17.10.2.4 and

see Section 4, Quality Assurance) .

17.6.20 Reagent water -- defined as distilled or deionized water

that does not contain substances which are toxic to the test

organisms (see Section 5, Facilities, Equipment, and Supplies and

Section 7, Dilution Water).

17.6.21 Effluent and receiving water -- see Section 8, Effluent

and Surface Water Sampling, and Sample Handling, and Sample

preparation for Toxicity Tests.

17.6.22 Dilution water and hypersaline brine -- see Section 7,

Dilution Water and Section 17.6.24, Hypersaline Brines. The

dilution water should be uncontaminated l-~m-filtered natural

seawater. Hypersaline brine should be prepared from di.lution

water.

17.6.23 HYPERSALINE BRINES

17.6.23.1 Most industrial and sewage treatment effluents

entering marine and estuarine systems have little measurable

salinity. Exposure of larvae to these effluents will usually

require increasing the salinity of the test solutions.. It is

important to maintain an essentially constant salinity across all

treatments. In some applications it may be desirable to match

the test salinity with that of the receiving water (See Section

7.1). Two salt sources are available to.adjust salinities --

artificial sea salts and hypersaline brine (HSB) derived from

natural seawater. Use of artificial sea salts is necessary only

when high effluent concentrations preclude salinity adjustment by

HSB alone.

17.6.23.2 Hypersaline brine (HSB) can be made by concentrating

natural seawater by freezing or evaporation. HSB should be made

from high quality, filtered seawater, and can be added to the

effluent or to reagent water to increase salinity. HSB has

several desirable characteristics for use in effluent toxicity

testing. Brine deriyed from natural seawater contain~ the

necessary trace metals, biogenic colloids, and some of the

472
microbial components necessary for adequate growth, survival,

and/or reproduction of marine and estuarine organisms, and it can

be stored for prolonged periods without any apparent degradation.

However, even if the maximum salinity HSB (100~), is used as a

diluent, the maximum concentration of effluent (0%0) that can be

tested is 66% effluent at 34~ salinity (see Table 1).

TABLE 1. MAXIMUM EFFLUENT CONCENTRATION (%) THAT CAN BE TESTED

AT 34~ WITHOUT THE ADDITION OF DRY SALTS GIVEN THE

INDICATED EFFLUENT AND BRINE SALINITIES.

Effluent Brine Brine Brine Brine Brine

Salinity 60 70 80 90 , 100

%"0 %"0 ~o %"0

, 00

0 43.33 51.43 57.50 62.22 66.00

;

1 44.07 52.17 58.23 62.92 66.67 ,

2 44.83 52.94 58.97 63.64 67.35

!

3 , 45.61 53.73 59.74 64.37 I 68.04

I

4 46.43 54.55 60.5'3' 65.12 68.75

5 47.27 55.38 61. 33 65.88 , 69.47

10 52.00 60.00 65.71 70.00 I 73.33

15 57.78 65.45 70.77 74.67 77.6.5

20 65.00 72.00 76.67 80.00 82.50

25 74.29 80.00 83.64 86.15 88.00

17.6.23.3 High quality (and preferably high salinity) .seawater·

should be filtered to at least 10 Jlm before placing into the

freezer or the brine generator. Water should be. collected on an

incoming tide to minimize the possibility of cont:.amination.

17.6.23.4 Freeze Preparation of Brine

17.6.23.4.1 A convenient container .for making m?B by f.reezing is

one that has a bottom drain. One liter of brine can be made from

473
four liters of seawater. Brine may be collected by partially

freezing seawater at -10 to ~20°C until the remaining liquid has

reached the target salinity. Freeze for approximately six hours,

then separate the ice (composed mainly of fresh water) from the

remaining liquid (which has now become hypersaline) .

17.6.23.4.2 It is preferable to monitor the water until the

target salinity is achieved rather than allowing total freezing

followed by partial thawing. Brine salinity should never exceed

100~. It is advisable not to exceed about 70~ brine salinity

unless it is necessary to test effluent concentrations greater

than 50~.

17.6.23.4.3 After the required salinity is attained, the HSB

should be filtered through a 1 ~m filter and poured directly into

portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and the date the brine

was generated. Containers of HSB should.be stored in the dark at

4°C (even room temperature has been acceptable). HSB is usually

of acceptable quality even after several months in storage.

17.6.23.5 Heat Preparation of Brine

17.6.23.5.1 The ideal container for making brine using heatassisted evaporation of 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 to~ic

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 is to use. a thermostatically controlled heat

exchanger made from fiberglass. If aeration is needed, use only

oil-free air compressors to prevent contamination.

17.6.23.5.2 Before adding seawater to the brine generator,

thoroughly clean the generator, aeration supply tube, heater, and

any other materials that will be in direct contact with the

brine. A good quality biodegradable det~rgent should be used,

followed by several (at least three) thorough reagent water

rinses.

474
17.6.23.5.3 ' Seawat~r should be fil~ered to at ]~east 10 p.mbefore

being put into the brine generator. The temperSfture ot the

seawater is increased slowly t,o 40°C. The water, should, be

aerated to prevent temperature stratifi-cation 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%'0 and that the temperature does not exceed 40°C.

Add~tional seawater may be added to the brine· to obtain the

volume of brine required.

,

17.6.23.5.4 ,After the required salinity is attained, the HSB

should be filtered through a 1 p.m filter and poured directly into

portable containers (20-L cubitainers or polycarbonate water

cooler jugs are suitable). The brine storage containers should

be capped and labelled with the salinity and the1date the brine

was generated. Containers of HSB should be stored in the dark at

4°C (even room temperature has been acceptable) . ',HSB is usually

of acceptable quality even after several months :j.n storage."

17.6.23.6 Artificial Sea Salts

17.6.23.6.1 No data from giant kelp tests usingisea salts, or

artificial seawater (e.g., GP2) are available for eval~ation at

this time, and their use must be c~msidered provisional.

17.6.23.7 Dilution Water Preparation from Brine

17.6.23.7.1 Although salinity adjustment with brine is the

preferred method, the use of high salinity brines! and/or reagent

water has sometimes been associ'ated with discerni:ble adverse

effects on test organisms. For this reason, it is recommended

that only the minimum necessary volume of brine and reagent 'water

be used to offset the low salinity of the effluent, 'and that

brine controls be included in ,the test. The remaining dilui::.i<;:>n

water should be natural seawater. Salin~ty may be adjusted in

one of two ways. First, the' salinity of the highest effluent

test concentration may be adjusted to an acceptab~e salinity, and

then serially dilut~d. Alternatively, each efflu~nt

concentration can be prepared individually with a:ppropriate

volumes of effluent and brine.

17.6.23.7.2 When HSB and reagent water are use!:i, tho!oughly

mix together the reagent water and HSB before mixing in the

475
effluent. Divide the salinity of the HSB by the expected test

salinity to determine the proportion of reagent water to brine.

For example, if the salinity of the brine, is 100~ and the test

is to be conducted at 34~, 100~ divided by 34~ = 2.94 .. The

proportion of brine is 1 part, plus 1.94 parts reagent water: To'

make 1 L of dilution water at 34~ salinity from a HSB of 100~,

340 mL of brine and 660 mL of reagent water are required. Verify

the salinity of-the resulting mixture using a refractometer.

17.6.23.8 Test Solution Salinity Adjustment

17.6.23.8.1 Table 2 illustrates the preparation of test

solutions (up to 50% effluent) at 34~ by combining effluent,

HSB, and dilution water. Note: if the highest effluent

concentration does not exceed 50% effluent, it is convenient to

prepare brine so that the sum of the effluent salinity and brine

salinity equals 68~; the required brine volume is then 'always

equal to the effluent volume needed for each effluent

concentration as in the example in Table 2.

17.6.23.8.2 Check the pH of all test solutions and adjust to

within 0.2 units of dilution water pH by 'adding, dropwise, dilute

hydrochloric acid or sodium hydroxide (see Section 8.8.9,

Effluent and Receiving Water Sampling, Sample Handling,. and

Sample Preparation for Toxicity Tests) .

17.6.23.8.3 To calculate the amount, of brine to add to each

effluent dilution, determine the following quantities: salinity

of the brine (SB, in ~), the salinity of ·theeffluent (SE, in'

~), and volume of the effluent to be added (VE, in mL). Then

use the following formula to calculate the volume of brine (VB,

in mL) to be added:

VB = VE x (34 - SE)/(SB - 34)

17.6.23.8.4 This calculation assumes that dilution water

salinity is 34 ± 2~.

17.6.23.9 Preparing Test Solutions

17.6.23.9.1 Two hundred mL of test solution are needed for each

test chamber. To prepare test solutions at low effluent

concentrations «6%), effluents may be added directly to dilution

476
water. For, example, to prepare 1% effluent, add 10 mL of

effluent to a,l-liter volumet~ic flask using a volumetric pipet·

or calibrated automatic pipet. Fill the volumetric' flask to, the

1-Liter mark with dilution water, stopper it, and shake to mix.

Distribute equal volumes into the replicate test chambers.

17.6.23.9.2 To prepare a test solution at higher effluent

concentrations, hypersaline brine must u~ually be used., For

example, to prepare 40% effluent, add 400 mL of effluent to a 1-

liter volumetric flask. Then, assuming an efflu~nt salinity of

2~ and a brine salinity of 66~, add 400 'mL of brine (see

equation above and Table 2) and top off the flask with dilution I

water. Stopper the flask and shake well. Dist:ribute equal

volumes into the replicate test chambers.

17.6.23.10 Brine Controls

17.6.23.10.1 Use brine ,controls in all tests whE~re brine. is

u~ed. Brine ,controls contain the same volume of'brine as does

the highest effluent concentration using brine, plus the volume

of reagent water ,needed to reproduce the hyposalinity of the

effluent in the highest concentration" plus dilution water.

Calculate the amount of reagent water, to·add to brine controls by

rearranging ,the above equation, (See, 17.6.23.8.3) setting SE

0, and solving for VE.

VE = VB x (SB - 34.) / (34 - SE)

If effluent salinity is essentially O~, the, reagent water volume

needed in the brine control will equal t~e effluent volume at the

highest test concentration. However, as effluent. salinity and

effluent concentration increase, less reagent wa~er volume is

needed.

17.6.24 TEST 0RGANISMS

17.6.24.1 The test organisms for this method ~re the zoospores

of the giant kelp, Macrocystis pyrifera . . MacrocYistis is the

dominant canopy forming Laminarian alga in southe~n and central

California and forms extensive subtidal forests along t.he coast.

Giant kelp .forests support a rich diversity. of marine life and

provide habitat and food for hundreds of i~vertebrate and

vertebrate species (North, 1971; Foster and Schiel, 1985) . It

477
TABLE 2. EXAMPLES OF EFFLUENT DILUTION SHOWING VOLUMES OF

EFFLUENT (x~), BRINE, AND DILUTION WATER NEEDED FOR

ONE LITER OF EACH TEST SOLUTION.

FIRST STEP:

to achieve a

for dilution

Combine brine with reagent water or natural

brine of 68-x~ and, unless natural seawater

water, also a'brine-based dilution water of

seawater

is used

34~.

SERIAL DILUTION:

Step 1. Prepare the highest effluent concentration to be tested

by adding equal volumes of effluent and brine to the appropriate

volume of dilution water. An example using 40% is shown.

Effluent Cone. Effluent x§o Brine (68- . Dilution Water*

(t) x)§o 34§o

40 800 mL 800 mL 400 mL

Step 2. Use either serially prepared dilutions of the highest test

concentration or individual dilutions of 100% effluent.

Effluent Cone. (%) Effluent source Dilution Water* (34g-.,)

20 1000 mL of 40% lOOO mL

10 1000 mL of 20% 1000 mL

5 1000 mL of 10% 1000 mL

2.5 1000 mL of 5% 1000 mL

Control none 1000 mL

INDIVIDUAL PREPARATION

Effluent Cone. Effluent xg-., Brine (68-x) g-., Dilution Water*

(%) 34g-.,

40 400 mL 400 mL 200 mL

20 200 mL 200 mL 600 mL

10 100 mL , 100 mL 800 mL

5 50 mL 50 mL " 900 mL

2.5 25 mL 25 mL 950 mL

Control none none 1000 mL

*May be natural seawater or brine-reagent water equivalent.

478
is an appropriate tox;icity ,test species because: ,of its

availability, economic and ecological importance I history of

successful laboratory culture (North, 1976; Luning, 1980;

Kuwabara, 1981; Deysher arid Dean, 1984; Linfield, 1985), and

p~evious u~e in, toxicity, te,st.,ing (Smith and Harrison, 197~.i ~ames

et al., ,1987; Ande!son and,Hunt, 1988; Hunt et al., 1989;

Anderson et al., 1990'). Other Laminarian alga spe,cies have

proven to be useful for laboratory toxicity tes'ting (Chung and

Brinkhuis, 1986; Thompson and Burrows, 1984; Hopkin and Kain,

1978; see Thursby et al., 1993 ~or review). ' ,

17.6.24.2 Like all kelps, MacrocystiE? has a life cycle that

alternates between a microscopic gametophyte st<;ige and a

macroscopic sporophyte stage.,' It is the sporophyte stage that'

forms, kelp forests.,." These p:Lants pro<:iuce, repro(~uctive blades

'(sporophylls) at their base. The sporophylls dE~velop patches

(sari) iri which bi'flagellate, haploid zoospores are produced.

The zoospores are released into the water column where they swim

~nd eventually s,~tt:l:~ a,nto. t:q.e., botto~ ang g_~rminate. _ Th~ " , .

~aioe~ious ,spor~'s' de-Y,elop into eit~e~ rnalear fenlale' gametophytef/

The male gametophytes produce .flagellated gametE~s which may

fertilize eggsproduGeo.by the femalegametophytes. ~ertilized;

eggs develop into sporophytes within,12~ 15 days, completing'the

lifecycle.

17.6.24.3 The method described here focuses on 'germination of'

the zoospore,s an<:i tb:e' 'lnitial growth of the devei~ping

gametophytes. It involves the controlled release of ,zoospores" "

from the sporophyll bladesl followed by the introducti0I?- .of a '

spore' suspension: of known 'density into the' testcont'ainers·. The

,zoosporesswimthrough'the test' solution 'and eventually settle

onto glass mi'crQscopeslides. The settled spores germinate by

-extruding the cytoplasm of, the spore through the germ-t,ube into

the first gamet ophytic cell. ',This stage is often referred' to ~s:

the '''dumbell'' stage. The two endpoints measured after 48 hours

are germ,ination ,SUCCE;:!SS and growth of, t,he ,embryonic gametophytes"

. (germ-tube length) .

,17.6.24.4, Species Identification

17.6.24.4.1 Altho~gh there is som'e debate over ,t.he "taxonomy' of>'

the genus Macrocystis, Abbott and Hollenberg (19'i76) consider only

two species in California: M. pyrifera, and M. Lategrifolia. The

479
two are distinguished from each other based on habitat and the

morphology of their holdfasts. Macrocystis pyrifera occurs

sUbtidally while M. integrifolia occurs 'in the low intertidal

and shallow subtidal zones.' Macrocystis pyrifera has a conical

holdfast while M. integrifolia has a more flattened, c~eeping

holdfast. Consult Abbott and Hollenberg (1976) for a more

detailed taxonomic discussion of the two species.

~7.6.24.5 Obtaining Zoospores

17.6.24.5.1 Macrocystis zoospores are obtained from the

reproductive blades (sporophylls) of the adult plant., The

sporophylls are located near the base of the plant just above its

conical holdfast. Sporophylls must be collected subtidally and

should be collected from at least five different plants in any

one location to give a good genetic representation of the

population. The sporophylls should be c~llected from areas free

of point and non-point source pollution to minimize the

possibility of genetic or physiological adaptation to pollutahts.

In situations where a thermocline is present at the collection

site, the sporophylls should be collected from below the

thermocline to ensure adequate spore release. Sporophylls are

identified in the field by the presence of darkened patches

called sori. The zoospores develop within the sori. In addition,

the sporophylls are distinguished from vegetative blades by their

thinner width, basal location on the adult plant, and,general

lack of pneumatocysts (air bladders) . Collection of a,lgae is

regulated by California law. Collectors must obtain a scientific

collector's permit from the California Department of Fish and

Game and observe any regulations regarding collection and

transport ,of kelp. For further information regarding sporophyll

collection, contact the Marine Pollution ,Studies Laboratory,

34500 Coast Route 1, Granite Canyon, Monterey CA, 93940, (408)

624-0947.

17.6.24.6 Broodstock Culture and Handling

17.6.24.6.1 After collection, the sporophylls should be, kept

damp and not exposed to direct sunlight. , Avoid immersing the

blades in seawater, however, to prevent premature spore release.

The sporophylls should be rinsed thoroughly in 0.2 ~m filtered

seawater to remove diatoms and other epipqytic organisms. The

individual blades can be gently rubbed between fingers under

480
running filtered seawater or brushed with a soft bristled brush.

The blades are stored between moist paper towels (lasagna style

so that the sporophylls do not overlap' each other, and each layer

of sporophylls are separated by a layer of paper towels) at

approximately 9-12°C until needed. The zoosporel3 must be

released within 24 hours of collection to insure their viability.

Preliminary data indicate that prolonged storage times may affect

test results (Bottomley et al., 1991); however as long as

germination rates meet control acceptability criteria this should

not affect test results. Sporophylls should be kept shaded to

prevent damage to the spores. For holding or transport times

longer than approximately six hours, the sporophylls should. be

placed in an ice chest with blue ice. The blue ice should be

wrapped in newspaper (10 layers) for insulation,! then plastic to

prevent leaking.

17.7 EFFLUENT AND RECEIVING WATER COLLECTION, P:RESERVATION,AND

STORAGE

17.7.1 See Section 8, Effluent and Receiving Waiter Sampling,

Sample Handling, and Sample Preparation 'for Toxil;::ity Tests.

17.8 CALIBRATION AND STANDARDIZATION

17.8.1 See Section 4, Quality Assurance.

17.9 QUALITY CONTROL

17.9.1 See Section 4, Quality Assurance.

17.10 TEST PROCEDURES

17.10.1 TEST DESIGN

17.10.1.1 The test consists of at least five effluent

concentrations plus a dilution water control ..T~sts that use

brine to adjust salinity must also contain five replicates of a

brine control.

17.10.1.2 Effluent concentrations are expresse~ as percent

effluent.

17.10.2 TEST SOLUTIONS

. .481
17.10.2.1 Receiving waters

17.10.2.1.1 The sampling point is determined by the objectives

of the test. At estuarine and marine sites, samples are,usually

collected at mid-depth. Receiving water toxicity is' determined

with samples used directly as collected or with samples passed

through a 60 ~m NITEX® filter and compared without dilution,

against a control. Using five replicate chambers per test, each

containing 200 mL, analysis would require approximately 1 L of

sample per test.

17.10.2.2 Effluents

17.10.2.2.1 The selection of the effluent test concentrations

should be based on the objectives of the study. A dilution

factor of at least 0.5 is commonly used. A dilution factor of

0.5 provides hypothesis test discrimination of ± 100%, and

testing of a 16 fold range'of' concentrations. Hypothesis test

discrimination shows little improvement as dilution-facto'rs are

increased beyond 0.5 and declines rapidly'if smaller dilution

factors are used. USEPA reoommends that one of the five'effluent

treatments must be a concentration of effluent mixed with

dilution water whioh corresponds to the permittee's ins'tream

waste conoentration (IWC). At least two of the effluent

treatments must be of lesser effluent concentration than the IWe,

with one being at least one-half the concentration of the Iwe.

If 100~ EBB is used as a diluent, the maximum concentration of

effluent that can be tested will be 66% at 34~ salinity. . .

17.10.2.2.2 If the effluent is known or suspected to be highly

toxic, a lower range of effluent concentrations should be used

(such as 25%, 12.5%, 6.25%, 3.12% and 1.56%).

17.10.2.2.3 The volume in eaoh test chamber is 200 mL.

17.10.2.2.4 Effluent dilutions should be prepared for all

replicates in each treatment in one container to minimize

variability among the ~eplicates. Dispense into the appropriate

effluent test chambers.

17.10.2.3 Dilution Water

17.10.2.3.1 Dilution water should be uncontaminated l-~m482
filtered natural seawater or hypersaline brine prepared from,

uncontaminated natural seawater plus reagent water (see Section

7, Dilution Water). Natural seawater may be, un<::ontaminated

receiving water., This water is used in all dilution steps anc;i as

the control water.

17.10.2.4 Reference Toxicant Test

17.10.2.4.1 Reference toxicant tests should be conduc~ed as

described in Quality Assurance (see Section 4.7).

17.10.2.4.2 The preferred reference toxicant fqr giant :j{elp is

copper chloride (CuCI 2 02H20). Reference toxicant~ tests provide

an indication of the sensitivity of the test orsranisms and the

suitability of the testing laboratory (see Section 4 Quality

Assurance). Another toxicant may be specified by the app~opriate

regulatory agency. Prepare a 10,000 ~g/L coppei' stock 'solution

by adding 0.0268 g of copper chloride (CuCI 202H20) to one liter

of reagent water in a polyethylene volumetric flask.

Alternatively, certified standard solutions can be ordered from

commercial companies.

17.10.2.4.3 Reference toxicant solutions should be' , five , ,

replicates each of 0 (control), 5.6, lO, l8, 32, lOO, and lao

~g/L total copper. Prepare one liter of each concentration by

adding 0, 0.56, 1.0, 1.8, 3.2, 5.6, 10.0, and 18i.o mL of· stock

solution, ,respectively, to' one-liter volumetric £.lasks' and fill

with dilution water. Start with control solutions and progress

to the highest concentration to minimize 'contamination.

, .

17.10.2.4.4 If the ,effluent and reference toxicant tests are to.

be run concurrently, then the tests must use zoospores from the

same release. The tests must be handled in the i~ame way and test

solutions'delivered to the test chambers at the same time.

'Reference toxicant tests must be conducted at 34 ± 2%0.

17.10.3 RELEASE OF ZOOSPORES FOR THE TEST

17.10.3.1 Zoospores are released by slightly del3icca.ting the

sporophyll blades, and then placing them in filtered seawater.

To desiccate the sporophylls, blot the blades with paper towels

and expose them to air for 1 hour.

483
~7.~0.3.2 The number of sporophyll blades needed depends upon

their maturity; usually 25-30 blades (~ 100 grams wet weight) are

sufficient. After 1 hour the blades should be rinsed again

thoroughly using 0.2 ~m-filtered seawater, then placed in a one

L glass or plastic beaker filled with 0.2 ~m filtered seawater at

~5-16DC. The release water should never exceed 18DC.

~7.~0.3.3 After one hour, a sufficient number of zoospores

should be present to conduct the test. The presence of zoospores

is indicated by a slight cloudiness in the water. To verify

whether zoospores are present, periodically sample the solution

and observe the sample microscopically (100x).

17.~0.3.4 To insure that the zoospores are viable and have not

begun to germinate before they are exposed to the toxicant, the

zoospore release process should not be longer·thantwo .hours. If

it takes longer than two hours to get an adequate density of

zoospores (~7,500 zoospores/mL of test solution), repeat the

release process with a new batch of sporophylls.

~7.~0.3.5 After the zoospores are released, remove the

sporophylls and let the spore mixture settle for 30 minutes.

After 30 minutes, decant 250 mLs from the top of the spore

solution into a separate clean glass beaker. Sample the spore

solution and determine the spore density using a bright-line

hemacytometer (100x). Spores may be counted directly, or to

obtain a more accurate count, fix a sample of spores by mixing

nine milliliters of spore solution with ~-mL of 37% buffered

formalin (or acetic acid) in a test tube. S~ake the sample well

before placing it on the hemacytometer.

~7.10.3.6 After counting, the density is multiplied by 1.111 to

correct for the dilution caused by adding 1 mL of formalin to the

sample. Use at least five replicate counts. After the density

is determined, calculate the volume of zoospores necessary to

give approximately 7,500 spores/mL of test solution. Toprevent

over-dilution of the test solution, this,volume should not exceed

~% of the test solution volume. If this . volume exceeds. 1% of the

test solution volume, it should be noted in the results.

~7.~0.3.7 Test solutions must be prepared while the zoospores

are releasing from the sporophylls. Test solutions must be

mixed, sampled, and temperature equilibrated in time to receive

484
the swimming· zoospores as soon as they are count~ed. Zoospore

release and counting should be done in a room separate from that

used for.toxicant preparation, and care should be takeD; to.avoid

contaminating the zoospores prior to testing.

17.10.4 START OF THE TEST

17.10.4.1 Prior to Beginning the Test

17.10.4.1.1 The test·should begin as soon as po:ssible,

preferably within 24 h of sample collection. The maximum holding

time following retrieval of the.sample from the ,sampling device

should not exceed 36h for off-site toxicity tests unless

permission is granted by the permitting authority. In no case

should the sample be used in a test more ,than 72 h after sample

collection (see Section 8 Effluent and Receiving Water Sampling,

Sample Handling, and Sample Preparation for Toxicity, Test) .

17.10.4.1.2 Just prior to test initiation (appr,oximately 1 h),

the temperature of a sufficient quantity of the sample to make

the test solutions should be adjusted to .the tes't temperature (15

± laC) and maintained at that temperature during the addition o~

dilution water..

17.10.4.1.3 Increase the temperature of the water bath, room, ·or

incubator to the required test temperature (15 ± laC).

17.10.4.1.4 Randomize the placement of test chambers in the

temperature-controlled water bath, room, or incubator at the

beginning of the test, using a position chart. l~ssign numbers

for the position of each test chamber using a random numbers or

similar process (see Appendix A, for an example of

randomization). Maintain the chambers in this configuration

throughout the test, using a position chart. Record these'

numbers on a separate data sheet together with the concentration

and replicate numbers to which they correspond. Identify this

sheet with the date, test organism, test ,number, laboratory, and

investigator's name, and safely store it away unt,il after the

gametophyte spores have been examined at the end of the test.

•

17.10.4.1~5 Note:

invalidate the test

afterwards. Make a

Loss of the randomization sheet would

by making it impossible to an.alyze the data

copy of the randomization sheiet and store

485
separately. Take care to follow the numbering system exactly

while filling chambers with the test solutions.

~7.~0.4.~.6 Arrange the test chambers randomly in the water bath

or controlled temperature room. Once chambers have been labeled

randomly and filled with test solutions, they can be arranged in

numerical order for convenience, since this will also ensure

random placement of treatments.

~7.10.4.2 Estimation of Zoospore Density

~7.~0.4.2.1 After determining the zoospore density a~d

calculating the volume yielding 7,500 zoospores/mL test solution,

add this volume to each test chamber (this is the start time of

the test). Observe a sample of zoospores microscopically to

verify that they are swimming before adding them to the test

chambers.

~7.~0.4.2.2 Incubate the developing gametophytes for 48 hours in

the test chambers at 15°C under 50 ~E/m2/s. The zoospores

germinate and develop to the Ildumbell ll gametophyte stage during

the exposure period .

. ~7.10.5 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE

7.10.5.1 The lights used in this method are:;- cool whit·e

fluorescent lights adjusted to give .50 ~E/m2/s at the top of each

test chamber. Each test chamber must receive the same quanta of

light (50 ± 10 ~E/m2/s). Areas of increased light can be

eliminated by taping the outside of the light diffuser or

wrapping the fluorescent bulbs with aluminum foil.

~7.~0.5.2 The water temperattire in the test chambers should be

maintained at 15 ± 1°C. If a water bath is used to maintain the

test temperature, the. water depth surrounding the test cups

should be as deep as possible without floating the chambers.

~5.10.5.3 The test salinity should be in the range of 34 ± 2~.

The salinity should vary by no more than ±2%'o among the '"chambers

on a given day. If effluent and receiving water tests ·are

conducted concurrently, the salinities of these tests should be

similar.

486
15.10.5.4 Rooms or incubators with high volume yentilation

should be used with caution because the volatili2:ation of the

test solutions and evaporation of dilutiqn water may cause wide

fluctuations in salinity. Covering the test charr~ers with clean

polyethylene plastic may, help prevent volatilization and

evaporation of the test solutions.

17.10.6 DISSOLVED OXYGEN (DO) CONCENTRATION

17.10.6.1 Aeration may affect the toxicity of ef,fluent and

should be used only as a last resort to maintain a satisfactory

DO. The DO concentration should be measured on new solutions at

the start of the test (Day 0). The DO should not fall below 4.0

mg/L (see Section 8, Effluent and Receiving Water; Sampling,

Sample Handling, and Sample Preparation for Toxicity Tests). If

it is necessary to aerate,' all treatments and the control should

be aerated. The aeration ~ate should not exceed that necessary

to maintain a minimum acceptable DO and under no circumstances

should it exceed 100 bubbles/minute, using a pipet with a 1-2 mm

orifice, such as a 1 mL KIMAX® serological pipet No. 37.033,' or

equivalent.

17.10.7 OBSERVATIONS DURING THE TEST

17.10.7.1 Routine Chemical and Physical .Observations

17.10.7'.1.1 DO is measured at the beginning of t;.l~e exposure

period in one test chamber at each tes't concentration and in the

control.

I.

i

17.10.7.1.2 Temperature, pH, and salinity are measured at the

beginning of the exposure period in one ~est chamber at each '

concentration and in the control. Temperature should also be

monitored continuously or observed and recorded daily for at

least two locations in the environmental control system or the

samples. Temperature should be measured in a sufficient number

of test chambers at the end of the test to determine temperature '

variation in the environmental chamber.

17.10.7.1.3 Record all the measurements 'on the data sheet.

17.10.8 TERMINATION OF THE TEST

487'
17.10.8.1 Ending the Test

17.10.8.1.1 Record the time the test is terminated.

17.10.8.1.2 Temperature, pH, dissolved oxygen, and salinity are

measured at the end of the exposure period in one test chamber at

each concentration and in the control.

17.10.8.2 Sample preservation

17.10.8.2.1 In some cases it may be convenient to preserve the

kelp cultures for later analysis. Preliminary work by Anderson

and Hunt (Marine Pollution Studies Laboratory unpublished data)

indicates that cultures can be preserved in 0.1% glutaraldehyde

(final concentration) and that preservation has no significant

effect on germination or germ-tube growth. Other researchers

have used higher glutaraldehyde concentrations and found adequate

preservation with no effect on spore germination or gametophyte

growth (K. Goodwin, Calif. Inst. of Tech.", unpublished data).

17.10.8.2.2 Because data on the effects of preservation are

preliminary, it is recommended that anyone interested in

preserving kelp cultures for later analysis first demonstrate

that preservation does not affect test results. This can be

accomplished by comparing germination and germ-tube growth in

preserved vs non-preserved kelp cultures: We also recommend that

if it is necessary to preserve kelp cultures for later analysis,

a complete test should be preserved so that if any repl.icates are

read preserved, all of the replicates should be read preserved.

In the case where concurrent reference toxicant and complex

effluent tests are conducted, it may be convenient to fix one

test in glutaraldehyde and read the other test immediately.

17.10.8.2.3 When fixing kelp cultures, it is important to

minimize disturbance to the gametophytes. Make sure that the

culture slides are fixed and stored horizontally. We have used

disposable petri dishes for preservation chambers; these allow

individual replicate slides to be labelled and preserved

separately to avoid mixing replicates. Note: Glutaraldehyde is

toxic. If you intend to use this materi~l as a preservative,

study the material data safety sheets from the supplier and

488
follow strict safety precautions. Make sure test chambers and

solutions contaminated with this material are d.:i..spose~ of

properly.

17.10.8.3 Counting

17.10.8.3.1 After 48 hours, the test is terminated. Because it

takes a considerable amount of time to read theitest, reading can

begin after 45 hours and must be completed with1n six hours.

Remove the slide without decanting the test solution. The test

sl~de can be lifted from the bottom of the test chamber with a

separate clean microscope slide. Blot thebottpm on a paper

towel and place an lS-mm square cover slip on the slide. Blot

the excess water around the edge of the cover slip to eliminate

the flow of water under the cover slip,

17.10.8.4 Endpoints

17.10.8.4.1 The endpoints measured for the 48 hour Macrocystis

method are percent germination success and germination tube

length. Germination is considered successful if a germ-tube is

present on the settled zoospore. Germination is considered to be

unsuccessful if no germination tube is visible. To differentiate

between a germinated and non-germinated zoospore, observe the

settled zoospores at 400x magnification and detE~rmine whether

they are circular (non:-germinated)or haye a protuberance that

extends at least one spore diameter (about 3.0 jj:m) from the edge

of the spore (germinated). Spores with a germination tubes less

than one spore diameter are considered non-germi1nated ..

17.10.8.4.2 The first 100 spores encountered while moving across

the microscope slide are counted for each replicate of each

treatment. Note: Sewage effluents may contain certain objects,

such as ciliates, which look similar to rion-germ;inated kelp

spores. It is important to ensure that only kelp spores are

counted for this endpoint. Kelp spores are green-brown in color,

spherical, and lack mobility. Also, components of the cytoplasm

of kelp spores appear to fluoresce a light green: color when the

spore is slightly out of focus. If a particular object cannot be

identified, it should not be counted.

17.10.8.4.3 The growth endpoint is the measurement of the total

length of the germination tube from the edge of the original

489
spore membrane. Only germinated spores with straight germination

tubes and within the same focal plane are measured; if a spore is

not completely in focus from tip to tip it should not be

measured. The spores to be measured are randomly selected by

moving the microscope stage to a new field of view without

looking through the ocular lens.

17.10.8.4.4 Measure the germination-tube length of th~ spore

whose spore case center is nearest the micrometer in each field;

the spores case can be distinguished from the growing tip because

it is usually clear (empty) at 48 hours,' and it is more circular

than the growing tip. If more than one spore case is touching

the micrometer, both (or all) germinated spores are measured. A

total of 10 spores for each replicate of each treatment are

measured. It is easier> to measure germ-tube length with a

micrometer having a 10 mm linear scale (0.1 mm subdivisions);

measure lengths to the nearest, micron (typically to the nearest

half micrometer unit; see Section 10200E, Standard Methods 17th

edition, for micrometer/microscope calibration ,procedures). In

situations where germination is significqntly inhibited it may be

difficult to find germinated spores for germ-tube growth

measurement using the random search technique.

17.10.8.4.5 To expedite reading, the slide can be scanned to

find germinated spores if germination is 30% or less. In this

situation the first 10 spores encountered are measured for germtube length.

17.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA

..'

17.11.1 A summary of test conditions and test acceptability

criteria is listed in Table 3.

17.12 ACCEPTABILITY OF TEST RESULTS

17.12.1 For tests to be considered acceptable, the following

requirements must be met:

(1) Mean control germination must be at least 70% {n the

controls.

(2) Mean germination-tube length in~he .controls must be at

least 10 ~m in the controls.

490
(3) The germination-tube growth NOEC must be bE~low 35 p.g/liter

in the reference toxicant test.

(4) The, minim:um significant difference (%MSD) is <20% relative

to the control for both germination and germ-tube l~ngth in

the reference toxicant test. "

TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY

CRITERIA FOR GIANT KEL~" MACROCYSTIS :PYRIFERA, , ' I"

GERMINATION AND GERM-TUBE LENGTH TEST WITH EFFLUENTS " '

AND RECEIVING WATERS

~. Test type: Static non-renewal

2. Salinity: 34 ± 2%0,

\

3 . Temperature: 15 ± 1°C :

"

4. Light quality: , Ambient' laboratory illumination

5. Light intensity: 50 ± ~O ,J.E/m2 /s

6. Photoperiod: ~6 h light, 8 h da·rkness

7. Test chamber size: 600 mL'

8. Test solution volume: 200'mL/replicate

9. Spore de:q.sity per test 7500/mL of test solution

chamber:

replicate chambers i~O. No. 5

:' per concentration:

~~. Dilution water: Uncontaminated ~-p.m-filt~red natural

seawater or hypersaline brine

prepared from natural seawater

12. Test concentrations: Effluents: Minimum of 5 and a

control I

!

Receiving I '

waters: :100% receiving

water and a control

~3. Dilut.ion factor: Effluents: ~0.5

Receiving waters: None or ~0.5

~4. Test duration: 48 h

491
15. Endpoints: Germination and germ-tube length

16. Test acceptability ~70% germination in the controls; I

criteria: ~10 /lm germ-tube length in the

controls and the NOEC must be below

35 /lg/L in the re·ference toxicant I

test; must achieve a %MSD of <20 for

both germination and germ-tube

length in the reference toxicant. I

17. Sampling requirements: One sample collected at test

initiation, and preferably used

within 24 h of the time it is

removed from the sampling device

(see Section 8, Effluent and

Receiving Water Sampling, Sample

Handling, and Sample Preparation for I

Toxicity Tests)

I

18. Sample volume 2 L per test

required:

17.13 DATA ANALYSIS

17.13.1 GENERAL

17.13.1.1 Tabulate and summarize the data. Table 4 presents a

sample set of germination and growth data.

17.13.1.2 The endpoints of the giant kelp 48-hour chronic test,

are based on the adverse effects on germination and growth. The

IC25 endpoints are calculated using point estimation techniques

(see Section 9, Chronic Toxicity Test Endpoints and Data

Analysis). LOEC and NOEC values for germination and growth are

obtained using a hypothesis testing approach such as Dunnett's

Procedure (Dunnett, 1955) or Steel's Many~one Rank Test (Steel,

1959; Miller, 1981) (see Section 9). Separate analyses are

performed for the estimation of the LOEC.and NOEC endpoints and

for the estimation of the IC25 endpoints. Concentrations at

which there is no germination in any of the test chambers are

excluded from the statistical analysis of the NOEC and ·LOEC for

germination and growth, but included in the estimation of the

492
IC25. See the Appendices for examples of the manual

computations, and examples of data input an4 prqgram outpu~.

17.13.1.3 The statistical tests described here must be used with

a knowledge of ,the assumptions upon which the tE~sts are.

contingen't. .The assi,stance of a statistician ii:l recommended for

analysts who are not proficient in statistics.

17.13.2 EXAMPLE OF ANALYSIS OF GIANT KELP, MACROCYSTIS PYRIFERA,

GERMINATION DATA

17.13.2.1 Formal statistical analysis of the germination data is

outlined in Figure 1. The response used in the analysis' is the

proportion of germinated spores in each test or control chamber.

Separate analyses are performed for the estimation of the NOEC

and LOEC endpoints and for the estimation of the' IC25 endpoint.

Concentrations at which there is no germ~nation in any of the

test chambers are excluded from statistical analysis of the NOEC

and LOEC, but included in the estimation of the ICendpoints.

17.13.2.2 For the case of equal numbers of replicates across all

concentrations and the control, the evaluation of the NOEC and

LOEC endpoints is made via a parametric test, Du~nett's

Procedure, or a nonparametric test, Stee~'s Many-one Rank Test,

on the arc sine squ~re root transformed data. rnpderlying

assumptions of Dunnett's Procedure, normality and homogeneity of

variance, are formally· tested. The test for normality .is the

Shapiro-Wilk's Test, and Eartlett's Test is used to test for

homogeneity of variance. If either of these test:.s fails, the

nonparametric test, Steel's Many-one Rank Test, :Ls used to

determine the NOEC and LOEC endpoints. If the, a~;sumptions of

Dunnett's Procedure are met, the endpoints are estimated by the

parametric procedure.

17.13.2.3 If unequal numbers of replicates occur among the

concentration levels tested, there are parametric and

nonparametric alternative analyses. The parametric analysis. is a

t test with the Bonferroni adjustment (see Appendix D). The

Wilcoxon Rank Sum Test with the Bonferroni adjust.ment is the

nonparametric alternative.

493
TABLE 4. DATA FROM GIANT KELP, MACTOCYSTIS PYRIFERA GERMINATION AND

GROWTH TEST

Copper Cone. Replicate Number Number proportion Mean

(/Lg/L) Chamber Counted Germinated Germinated Length

Control 2 200 89 0.89 29.58

2 200 88 0.88 28.75

3 200 85 0.85 29.14

4 100 89 0.89 16.50

5 100 91 0.92 17.93

5.6 1. 1.00 82 0.82 1.8.26

2 200 55 0.55 1.6.25

3 100 84 0.84 16.39

4 100 96 0.96 1.8.70

5 100 85 0.85 15.62

20.0 1 1.00 90 0.90 1.3.31.

2 1.00 90 0.90 1.8.92

'3 1.00 70 0.70 15.62

4 1.00 83 0.83 14.30

5 1.00 87 0.87 15.29

2B.0 1 100 88 0.B8 18.59

2 100 52 0.52 12.88

3 1.00 83 0.83 16.28

4 100 54 0.54 15.38

5 100 49 0.49 19.75

32.0 1 100 71 0.71 12.54

2 100 82 0.82 10.67

3 100 86 0.86 1.5.95

4 100 81. 0.81 12.54

5 100 82- 0.82 11.. 66

56.0 1 100 84 0.84 11.44

2 100 68 0.68 11..88

3 1.00 62 0.62 11.88

4 100 80 0.80 11.00

5 100 83 0.83 11.. 55

100.0 1 1.00 66 0.66 7.92

2 100 72 0.72 7.59

3 100 63 0.63 8.25

4 100 72 0.72 9.13

5 100 71 0.71. 8.80

280.0 1 100 37 0.37 6.49

2 100 69 0.69 7.25

3 100 0 0.00

4 1.00 32 0.32 7.63

5 100 48 0.48 8.13

494
Figure 1 ..Flowchart for statistical analysis of giant kelp,

Macrocystis pyrifera, germination datsl.

495
17.13.2.4 Example of Ana~ysis of Germination Data

17.12.2.4.1 This example used toxicity data from a giant kelp,

Macrocystis pyrifera, germination and growth test performed with

copper. The response of interest is the proportion of germinated

spores, thus each replicate must be transformed by the arc sine

square root transformation procedure described in Appendix B.

The raw and transformed data, means and variances of the

transformed observations at each concentration including the

control are listed in Table 5. A plot of the survival data is

provided in Figure 2.

17.13.2.5 Test for Normality

17.13.2.5.1 The first step of the test for normality is to

center the observations by subtracting the mean of all

observations within a concentration from each observation in that

concentration. The centered observations are listed in Table 6.

17.13.2.5.2 .Calculate the denominator, D, of the test statistic:

Where: Xi the ith centered observation

X the overall mean of the centered observations

n the total number of centered observations.

17.13.2.5.3 For this set of data, n 40

X _1_(-0.002) 0.,000

40 .

D 0.9281

17.13.2.5.4 Order the centered observations from smallest to

largest:

496
TABLE 5. GIANT KELP, MACTOCYSTIS PYRIFERA GERMIN1~TICN DATA

COPPER ARC SINE

CONCENTRA'rION REPLICATE RAW SQUARE ROOT MEAN

(f.lg/L ) CHAMBER DATA TRANSFORMED • ! Y S?1

Control 1 0.89 1.233 l' ' 1.224 0.00114

2 0.88 1.217

3 0.85 1.J.73

4 0.89 1.233

5 0.91 1.266'

5.6 1 0.82 1.133 2i 1.134 0.03670

2 0.55 0.835

3 0.84 1".159

4 0.96 1.369

5 0.85 1.173 I

10.0 1 0.90 1.249 3: 1.167 0.Oi152

2 0.90 1.249

3 0.70 0.991

4 0.83 1.146

5 0.87 1.202

18.0 1 0.88 1.217 4 0.954 0.04423

2 0.52 0.805

3 0.83 1.146

4 0.54 0.825

5 0.49 0.775

32.0 1 0.71 1.002 5, 1.115 0.00466

2 ,0.82 1.133

3 0.86 1.187-

4 0.81 1.120

5 0.82 1.133

56.0 1 0.84 1.159 6' 1. 058. 0.01272

2 0.68 0.970

3 0.62 0.907

4 0.80 1.107

5 0.83 1.146

100.0 1 0.66 0.948 7 0.979 0.00191

2 0.72 1.013-

3 0.63 0.917

4 0.72 1.013

5 0.71 1. 002

180.0 1 0.37 0.654 8 0.610 0.11914

2 0.69 0.980

3 0.00 0.050

4 0.32 0.601

5 0.48 0.765

497
1.01 ***--- * --- CCIICrI,.......WWflCallOl~

0.9 * *

*

* z· 0.8

0

- 0.7i I- * ""- ./ '1' :* ---...:z * c::

0

0..

0 0.6 c::

0.5

* 0..

z \ * 0

- If:- I- 0.4

1.0 «

OJ z

- 0.3 1 * :::;;;

c::

w

<..')

0;2

0.1

.a.0

0.0 5.6 10.0 18.0 32.0 66.0 100.0 180.0

COPPER CONCENTRATION (ugIL)

Figure 2. Plot of germination proportions of the giant kelp,

Macrocystis pyrifera, at each treatment level.
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WJ:LK'S EXAMPLE

Copper Concentration (ggh)

Rep Control 5.6 10.0 18.0 32.0 56.Cl 1.00.0 180.0

1 0.009 -0.001 0.082 0.263 -0.113 o.10J. -0.031 0.044

2 -0.007 -0.299 0.082 -0.149 0.018 -0.08€1 0.034 0.370

3 -0.051 0.025 -0.176 0.192 0.072 -0.151 -0.062 -0.560

4 0.009 0.235 -0.021 -0.129 0.005 0.049 0.034 -0.009

5 0.042 0.039 0.035 -0.179 0.018 0.088 0.023 0.155

TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WI.LK IS EXAMPLE

i X(i) i X(i)

1 -0.560 21 0.018

2 -0.299 22 0.023

3 -0·179 23 0.025

4 -0.176 24 0.034

5 -0.151 25 0.034

6 -0.149 26 0.035

7 -0.129 27 0.039

8 -0.113 28 0.042

9 -0.088 29 0.044

10 -0.062 30 0.049

1.1. -0.051. 3"1. 0.072

1.2 -0.031. 32 0.082

1.3 -0.021. 33 0.082

1.4 -0.009 34 0.088

1.5 -0.007 35 0.J.01

1.6 -0.001. 36 0.1.55

17 0.005 37 0.192

1.8 0.009 38 0.235

1.9 0.009 39 0.263

20 0.018 4'0 0.370

Where X(i) is the ith ordered observation. These ordered

observations are listed in Table 7.

499
17.13.2.5.5 From Table 4, Appendix B, for the number of

observations, n, obtain the coefficients a l , a 2, .... , ak where k

is n/2 if n is even and (n-1)/2 if n is odd. For the ~ata in

this example, n = 40 and k = 20. The ai values are listed in

Table 8.

17.13.2.5.6 Compute the test statistic, W, as follows:

k 2

w,. ~[Eai(Xln-i.1)_xli)]

D i-1

The differences Xln-i +l ) - Xli) are listed in Table 8.

in this example:

W = 1 (0.9230)2 = 0.918

0.9281

For this data

TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE

i ai x(n-i+1) - Xli)

1 0.3964. 0.930 X(40) X l1}

2 0.2737 0.562 x (39 ) - X (2 )

3 0.2368 0.414 X(38) - Xl3}

4 0.2098 0.368 X(37) - Xl4 }

5 0.1878 0.306 X(36) - XIS}

6 0.1691 0.250 XI3S) - X(6)

7 0.1526 0.217 X(34) - X (7)

8 0.1376 0.195 X(33) - X(8)

9 0.1237 0.170 X(32) -' X(9)

10 0.1108 0.134 XI31} - X(10)

11 0.0986 0.100 X(30) - X(].].)

12 0.0870 0.075 X(29) - X(12)

13 0.0759 0.063 X(28} - X(13)

14 0.0651 0.048 X(27) - X(1 4)

15 0.0546 0.042 X(26) - X(1S)

16 0.0444 0.035 X(2S) - X(16)

17 0.0343 0.029 X(24) - X(17)

18 0.0244 0.016 X( 2 3) - X(18)

19 0.0146 0.014 Xl22} - X(19)

20 0.0049 0.000 X(21 ) - X(20)

500
TABLE 9. ASSIGNING RANKS TO THE CONTROL AND,S. () p.g/L

CONCENTRATION LEVEL FOR STEE~'S MANY-ONE RANK TEST

Rank

1

2

3

4.5

4.5

6

7.5

.7.5

9

10

Transformed

Proportion

Germinated

0.835

1.133

1.159

1.173

1.173

1.217

1.233

1.233 .

1.266

1.369

TABLE 10. TABLE OF RANKSl

eoncentration

5.6 P.g/L

5.6 IJ-g/L

5.6 IJ-g/L

5.6 IJ-g/L

C<:;mtrol

Control

Control

. Control

Control

5.6 IJ-g/L

Concentration (p.g/L)

Rep. Control

1 1.233(7.5,6.5,~.5,8.5,8.5,8.5,8.S)

2 1.217(6,5,6.5,7,7,7,7)

. 3 1.173 (4.5',3,5,5,6,6,6)

4 1.233(7.5,6.5,8.5,8.5,8.5,8.5,8.5)

5 1.266(9,10,10,10,10,10,10)

5.6

1.133 (2) "

0.835(1)

1.159(3)

1. 369 (10)

1.173 (4.5)

10.0

1.249(8.5)

1.249(8.5)

0 ..991 (1)

1.146(2)

1.202(4)

--------- -.- --- -------- --------------- -------'- --------- - -.- - -------- ------- Concentration (p.g/L) (Continue~d)

Rep. 18.0 32.0 56.0 100~O 180.0

1 1.217(6.5) 1.002(1) 1.159(5) 0.948(2) 0.654(3)

2 0.805(2) 1. 133 (3.5) 0.970(2) 1.013(4.5) 0.980(5)

3 1.146 (4) 1.187 (6) 0.907(1) 0.917 (1) 0.050(1)

4 0.825(3) 1.120(2) 1.107(3) 1.013 (4.5) 0.601(2)

5 0.775(1) 1.133(3.5) 1.146(4) 1.002{3) 0.765(4)

lControl ranks are given in the order of the concentration with 'which

they were, ranked.

501
'17.13.2.5.7 The decision rule for· this test is to compare W as

calculated in Subsection 5.6 with the critical value found ·in

Table 6, Appendix B. If the computed W is less than the critical

value, conclude that the data are not normally distributed .. For

this set of data, the critical value at a significance level of

0.01 and n = 40 observations is 0.9·19. Since W = 0.918 is less

than the critical value, conclude that the data are noe normally

distributed.

17.13.2.5.8 Since the data do not meet the assumption of

normality, Steel's Many-one Rank Test will be used to analyze the

germination data.

17.13.2.6 Steel's Many-one Rank Test

17.13.2.6.1 For each control and concentration combination,

combine the data and arrange the observ~tions in order of size

from smallest to largest. ~ssign the ranks (1, 2, ... , 10) to

the ordered observations with a rank of 1 assigned to the

smallest observation, rank of 2 assigned to the next larger

observation, etc. If ties occur when ranking, assign the average

rank to each tied observation.

17.13.2.6.2 An example of assigning ranks to the combined data

for the control and 5.6 ~g/L copper concentration is given in

Table 9. This ranking procedure is repeqted for each

control/concentration combination. The complete set of rankings

is summarized in Table 10. The ranks are then summed for each

concentration level, as shown in Table 11.

17.13.2.6.3 For this example, determine if the survival in any

of the concentrations is significantly lower than the survival in

the control. If this occurs, th~ rank sum at that concentration

would be significantly lower than the rank sum of the control.

Thus compare the rank sums for the survival at each of the

various concentration levels with some "minimum" or critical rank

sum, at or below which the survival would be considered

significantly lower than the control. At a significance level of

0.05, the minimum rank sum in a test with seven concentrations

(excluding the control) and five replicates is 16 (See Table 5,

Appendix E) .

502
~7.13.2.6.4 Since the rank sum for the 32.0 ~g/L concentration

is equal to the critical value and the rank sums for the 56.0,

100.0 and 180. 0 ~g/L concent:r:ations are less than the cri~tical

value, the germination propor'tions in those concentrations are

considered significantly less than that in the control. Hence,

the NOEC and the LOEC are considered to 'be 18.0 ~g/L and.32.0

~g/L, respectively.

TABLE 11.

Concentration

5.6

10.0

18.0

32.0

59. 0

100.0

180.0

17.13.2.7 Calculation of the ICp

RANK SUMS

'Rank Sum

20.5

124.0

16.5

16.0

15.0

15.0

15.0

17.13.2.7.1 The germination data from Table 4 'and Figure 2 are

utilized in this example. As can be seen from;the figure, the

observed means are not monotonically non-increasing with respect

to concentration. Theref,ore, the means must be smoothed prior to

calculating 'the IC.

17.13.2.7.2 Starting with the observed control mean, Yl = 0.884

is less than the observed mean for the lowest effluent

concentration, Y2 = 0.804, so set M1 = 0.884.

17.13.2.7.3 Comparing Y2 to Y3 = 0.840, we see' that Y2 is less

than Y3 •

17.13.2.7.4 Calculate the smoothed means:

503
17.13.2.7.5 Since M3 is larger than Y4 = 0.652, set M4 = 0.652.

Since Ys = 0.804 is larger than M4 , these means must be smoothed.

17.13.2.7.6 Calculate the smoothed means:

17.13.2.7.7 Since Y6 = 0.754 is larger than Ms , average Y6 with

the two previous concentrations:

17.13.2.7.8 Since M6 > Y7 = 0.688 > Ya = 0.372, set M7 ::: 0.688

and Me = 0.372. Table 12 contains the smoothed means and

Figure 3 gives a plot of the smoothed means and the interpolated

response curve.

17.13.2.7.9 An IC25 can be estimated using the Linear

Interpolation Method. A 25% reduction in germination, compared

to the controls, would result in a mean germination of 0.663,

where M1 (1-p/100) = 0.884(1-25/100). Examining the smoothed

means and their associated concentrations (Table 12), the

response, 0.663, is bracketed by C7 = 100.0 ~g/L and Ca = 180.0

J.Lg/L.

17.13.2.7.10 Using the equation in Section 4.2 from Appendix L,

the estimate of the IC25 is calculated as follows:

IC25 = 100.0 + [0.884(1 - 25/100)

= 106.3 ~g/L.

0.688] (180.0 - 100.0)

(0.372 - 0.688)

17.13.2.7.11 When the ICPIN program was used to analyze this set

of data, requesting 80 resamples, the estimate of the IC25 was

106.3291 J1.g/L. The empirical 95.0% confidence interval for the

true mean was 94.6667 ~g/L to 117.0588 ~g/L. The computer

program output for the IC25 for this data set is shown in Figure 4.

504
1.0 J * * * IIIMUL~"'~ * --- ClClMB:11ne~....W108

* - - - - - - c:aMCI'ITtEMXJnB)""~

o.e1Ii; *

*

* 2 0.8

b

-

t- 0.7

* 'r'

* -~ * 0:::

0

a.. * 0 0.8 0::: * a..

-·2 0.5 \ * 0

-

t- 0.4

111 «

2 0 - 0.3 1 * 111 ::E

0:::

w

t:l

0.2

0.1

0.0

0.0 5.8 10.0 18.0 32.0 58.0 100.0 180.0

COPPER CONCENT'RATION (ugIL)

-~- -- ~- ----- - - -- - ---~

F~gure 3. Plot of raw data, observed means, and smoothed means for the

giant kelp, Macrocystis pyrifera, germination data from

Tables 4 and 13.
Cone. ID 1. 2 3 4 5 6 7 8

Cone. Tested o 5.6 1.0 1.8 32 56 100 180

Response 1. .89 .82 .90 .88 .71 .84 .66 .37

Response 2 .88 .55 .90 .52 .82 .68 .72 .69

Response 3 .85 .84 .70 .83 .86 .62 .63 0

Response 4 .89 .96 .83 .54 .81 .80 .72 .32

Response 5 .91. .85 .87 .49 .82 .83 .71 .48

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

*** Inhibition Concentration Percentage Estimate *** Toxicant/Effluent: Copper

Test Start Date: Test Ending Date:

Test Species: Giant Kelp, Macrocystis pyrifera

Test Duration: 48 hours

DATA FILE: kelpgerm.icp

OUTPUT FILE: kelpgerm.i25

Cone.

ID

Number

Replicates

Concentration

ug/L

Response

.Means

Std.

Dev .

Pooled

Response Means

1. 5 0.000 0.884 0.022 0.884

2 5 5.600 0.804 0.i52 0.822

3 5 10.000 0.840 0.083 0.822

4 5 18.000 0.652 0.187 0.737

5 5 32.000 0.804 0.056 0·.737

6 5 56.000 0.754 0.098 0.737

7 5 1.00.000 0.688 0.041 0.688

8 5 1.80.000 0.372 0.252 0.372

The Linear Interpolation Estimate: 1.06.3291 Entered P Value: 25

105.8680

Lower:

Lower:

0.28

Number of Resamplings: 80

The Bootstrap Estimates Mean:

Original Confidence Limits:

Expanded Confidence Limits:

Resampling time in Seconds:

Standard Deviation: 5.6981

94.6667 Upper: 117.0588'

88.8354 Upper: 122.4237

Random Seed: 390692880

Figure 4. ICPIN program output for the IC25.

506
17.13.3 EXAMPLE OF ANALYSIS OF GIANT KELP, MACROCYSTIS PYRIFERA,

GROWTH DATA

17.13.3.1 Formal statis.tica.l analysis of the growth data is

outlined in Figure 5. The response used in the I~tatistical

analysis is mean germ-tube length per replicate. An IC25 can be

calculated for the growth data via a point estilnation techniqu~

(see Section 9, Chronic Toxicity Test Endpoints· and Data

Analysis). Hypothesis testing can be used to obtain the NOEC and

LOEC for growth.

17.13.3.2 The, statistical analysis using hypothesis tests

consists of a parametric test, Dunnett's Procedure, and a

nonparametric test, Steel's Many-one Rank Test. The underlying

assumptions of the Dunnett IS Procedure.f normalit:y and homogeneity

of variance, are formally tested. The test for normal~ty is the

Shapiro-Wilk's Test and Bartlett's Test is used 'to test for

homogeneity of variance. If either of these tesltsfai;Ls,the

nonparametric test, Steel1s ,Many-pneRank Test,is used to

determine the NOEC and LOEC endpoint~. If the cfssumptions of

Dunnett's Procedure are met, the endpoints are determined by the

parametric test.

17 .l-3. 3.3 Additionally, if. unequal numbers of rep1icat'es occur

among the concentration levels tested, there are parametric and

nonparametric alternative analyses. The parametric analysis is a

t test with the Boriferroni adjustment. The Wilcoxon Rank Sum

Test with the Bonferroni adjustment is t~e nonp~rametric

alternative. For detailed information on the Bonferroni

adjustment, see Appendix D.

17.13.3.4 The data, mean and variance of the observations at

each· concentration including the control for this example are

listed in Table 13. A plot of the data is proviped in Figure 6.

17.13.3.5 Test for Normality

17.13.3.5.1 The first step of the test for norm~lity ~s to

center the observations by subtracting the mean 9f all

observations within a concentration from each ob~ervation in that

concentration. The centered observations are lil3ted in Table 14.

507
Figure 5. Flowchart for statis~ical analysis of giant kelp,

Mac~ocystis pyrifera, growth data.

508
TABLE 13. GIANT KELP, MACROCYSTIS PYRIFERA, GROWTH DATA

Copper Concentration </lg/L)

Rep Control 5.60 10.0 18.0 32.0 56.0 100.0 180.0

1 19.58 18.26 13.31 18.59 12.54 11.44 7.92 6.49

2 18.75 16.25 18.92 12.88 10.67 11. 88 7.59 7.25

3 19.14 16.39 15.62 16.28 15.95 11.88 8.25

4 16.50 18.70 14.30 15.38 12.54 11.00 9.13 7.63

5 17.93 15.62 15.29 19.75 11.66 11.55 8.80 8.13

Mean <'4) 18.38 17.04 15.49 16.58 12.67 11.55 8.34 '7.38

8i 1.473 1.827 4.498 7.327 3.953 0.133 0.396 , 0.478

i 1 2 3 4 5 6 7 8

TABLE 14. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

Copper Concentration (ugh)

Rep Control 5.6 10.0 18.0 32.0 56.0 100.0 180.0

1 1.20 1.22 -2.18 2.01 -0.13 -0.11 -0.42 -0.89

2 0.37 -0.79 3.43 -3.70 -2.00 0.33 -0.75 -0.13

3 0.76 -0.65 0.13 -0.30 3.28 0.33 -0.09

4 -1. 88 1.66 -1.19 -1. 20 -0.13 -0.55 0.79 0.25

5 -0.45 -1.42 -0.20 3.17 -1. 01 0.00 0.46 0.75

17.13.3.5.2 Calculate the denominator, D, of the statistic:

n - 2 D " L.J (X .-X)

i.l

Where: Xi = the ith centered observation

X = the overall mean of the centered observations

509
n = the total number of ,centered observations

17.13.3.5.3 For this set of data, n = 39

x = __1__ (-0.03) = 0.000

39

D = 79.8591

17.13.3.5.4 Order the centered observations from smallest to

largest

where Xli) denotes the ith ordered observation. The ordered

observations for this example are listed in Table 15.

TABLE 15. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

i Xli) i Xli)

1 -3.70 21 -0.11

2 -2.18 22 -0.09

3 -2.00 23 0.00

4 -1.88 24 0.13

5 -1.42 25 0.25

6 -1.20 2"6 0.33

7 -1.19 27 0.33

8 -1.01 28 0.37

9 -0.89 29 0.46

10 -0.79 30 0.75

11 -0.75 31 0.76

12 -0.65 32 0.79

13 -0.55 33 1.20

14 -0.45 34 1.22

15 -0.42 35 1.66

16 -0.30 36 2.01

17 -0.20 37 3.17

J.8 -O.B 38 3.28

J.9 -0.13 39 3.43

20 -0.J.3

510
* * * 1IIll'IXML1I!ft.IatlE.......natfJllml ----c:cIIIIiICN'ntEMliMWIE,.94::HCll:JNC:IIRM1II

*

58.0 100.0 180.0

*

32.0

*

18.0

*

*

5.8 10.0

20

18

18

:c 14 le>

z

w 12- --'

w

al 10 =:J

lI U'1 8 ::E J-1 a:: J-1 -w

e> e

4

2

0

0.0

-- .. ---- .------ - - COPPERCONCENTRATlON(i.ijiIL)

Figure 6. Plot of mean growth data for the giant kelp,

Maaroaystis pyrifera, tests.
17.13.3.5.5 From Table 4, Appendix B, for the number of

observations, n, obtain the coefficients aI' a 2 , ••• a k where k is

n/2 if n is even and (n-1)/2 if n is odd. For the data in this

example, n = 39 and k = 19. The ai values are listed in

Table 16.

17.13.3.5.6 Compute the test statistic, W, as follows:

k 2

W s [ Ea. (X (n-i.l) _ X (i) ) ]

D i-I

The differences xln-i+ll - Xli) are listed in Table 16.

of data:

For this set

W = 1 (8.7403)2

79.8591

0.957

TABLE 16. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE

i ai X ln-i ...1) - Xli)

1 0.3989 7.13 x(39) - X(l)

2 0.2755 5.46 XI3S) - X(2)

3 0.2380 5.17 X( 37 ) - X( 3)

4 0.2104 3.89 Xl36l - X(4)

5 0.1880 3.08 XI 3S ) - XIS)

6 0.1689 2.42 X(34) - Xl61

7 0.1520 2.39 X(33) - X( 7)

8 0.1366 1.80 X(32 ) - XIS)

9 0.1225 1.65 X(31} - X(9)

10 0.1092 1.54 X(30) - X(lO)

11 0.0967 1.21 X( 29 ) - X( 11 )

12 0.0848 1. 02 X(2S) X(12)

13 0.0733 0.88 X(27 ) - X( 13 )

14 0.0622 0.78 X(26) - XI H )

15 0.0515 '0.67 XI 2S ) - XI 1S )

16 0.0409" 0.43 X(24 ) - Xl16l

17 0.0305 0.20 X (23) - X( 17 )

18 0.0203 0.04 X(22 ) - XI 1S )

19 0.0101 0.02 Xl~l) - X(19 )

512
17.13.3.5.7 The decision rule for this test is 'to compare Was

calculated in Subsection 5.6 to a critical value found in

Table 6, Appendix B. If the computed W is less than the critical

value, conclude that the data are not normally distributed. For

this set of data, . the critical value at a significance level,of

0.01 and n = 39 observations is 0.917. Since W= 0.957 is

greater than the critical value, conclude that the data are

normally distributed.

17.13.3.6 Test for Homogeneity of Variance

17.13.3.6.1 The test used to examine whether the variation in

mean weight of the mysids is the same across all concentration

levels including the control, is Bartlettts Test (Snedecor and

Cochran, 1980). The test statistiq is as follows:

p p

[(EVi ) In "52 - LVi In Sf]

B = __i_.l i._l _

c

Where: Vi . . .

degrees of freedom for each copper concentration

and control, Vi = (ni - 1)

p = number of concentration levels including the

control

i 1, 2, ... , p where p is the number of

concentrations including the control

n i the number of replicates for concentration i.

p

(LVisf)

"52 = _i_ol _

p

LVi

i=l

p p

C = 1+[3(p-I)r1 [Ll/v.-(LV.)-1)

i.l i-l

513
17.13.3.6.2 For the data in this example (See Table 13), all

concentrations including the control have five replica~es except

the 180 ~g/L concentration which has four replicates (ni = 5 for

i = 1 - 7 i n s = 4). Thus, Vi :::: 4 for i :::: 1 - 7 and Va ::::·3.

17.13.3.6.3 Bartlett's statistic is therefore:

p

Ba [(31)ln(2.5761) - EViln("s;)] /1.0977

i.l

::: [31(0.9463) - [4In(1.4729) +

= [29.3353 - 9.4481] / 1.0977

"" 18.12

+ 3In(0~4780)] / 1.0977

17.13.3.6.4 B is approximat~ly distributed as chi-.squa're with p

- 1 degrees of freedom, when the variances are i~ fact the same.

Therefore, the appropriate critical value for 'this test, at a

significance level of 0.01 with seven degrees ~f freedom, is

18.48. Since B = 18.12 .is less ,than the critical value, conclude

that the variances are not different.

17.13.3.7 t Test with Bonferroni r~ Adjustt;nent

17,.13.3.7.1 To obtain an est~mate ,of the pooled variance for the

t test with Bonferroni's adjustment, construct an ANOVA table as

described in Table 17.

TABLE l7. ANOVA TABLE

Source

Between

Within

Total

df

p - l

N - P

N - l

Sum of Squares

(SS)

SSB

ssw

SST

Mean Square(MS)

(SS/df)

SB SSB/ (p-l)

2

Sw SSW/(N-p)

Where: p = numbe~ of concentration levels including the

control

514
,N = total number of observations n1 + n2 ••• + np

ni = number of observations in concentration i

. SSB

SST

Bet;:ween Sum of Squares

Total Surnof Squares

SSW = SST -SSB

G =

Within Sum of Squ~~es

the grand total of all sample observations,

p

G = ET.

1.1

Ti - the total of the replicate measurements for

concentration i

Yij - the' jth observation for concentration i

(represents the mean length ,of th~ germ-tubes £or

concentration i in test chamber ~)'

17.13.3.7.2 For the data in this example:

N = 39

T1 = Yll + Y12 + Y13 + 'Y14 + 'Y15 = 91.90

Ti = Y21. + Y 22 + Y 23 + Y 24 + Y~!; = 85.22

T3 = Y 31 + Y 32 + Y 33 + Y 34 + Y 3S' = 77.~4

T4 = Y41 + Y42 + Y 43 + Y 44 + Y4S = 82.88

Ts = Y S1 + Y S2 + Y S3 + Y S4 + YS5 = 63.36

T6 = Y61 + Y6~ + Y63 + Y64 + Y6~'.. = 57.75

T7 - Y71 + Y n + Y 73 + Y74 + .Yn:. = 41.69'

Ta = Y S1 + YS2 + Y S3 + 'YS4 = 2·9,.50

G = T1 + T2 + T3 + T4 + Ts + T6 + :T7 + Ts = 529.74

p

SSB ET~/n ._G 2 /N

1.1

515
= 7749.905 - (529.74)2 = 554.406

39

p nJ.

SST: E"£Y;j-G 2 /N

i.lj.l

= 7829.764 - (529,74)2

39

634.265

SSW : SST-SSB = 634.~65 - 554.406 = 79.859

S~ SSB/(p-1) = 554.406/(8-1) = 79.201

sa = SSW/(N-p) 79.859/(39-8) 2.576

17.13.3.7.3 Summarize these calculations in the' ANOVA table

(Table 1.8).

TABLE 18. ANOVA TABLE FOR THE t TEST WITH BONFERRONI'S

ADJUSTMENT EXAMPLE

Source df Sum of Squares Mean Square (MS)

(SS) (SS/df)

Between 7 554.406 79.201

Within 31 79.859 2.576

Total 38 634.265

17.13.3.7.4 To perform the individual comparisons, calculate the

t statistic for each concentration" and control combination as

follows:

t. :

516
Where: Yi = mean length for concentration i

Y1 = mean length for the control

Sw = square root of the within mean square

n 1 = number of replicates for the control

n i = number of replicates for concentraltion i

17.13.3.7.5 Table 19 includes the ca~culated t:values for each

concentration and control combination. In this ,example,

comparing the 5.6 J.lg/L concentration with ,the control, the

calculation'is as follows:

(18.38 -17.04)

t 2 = '-:[--:1-.-=6-::05~J;:::( ;:1I=;:,5;::)=+::;(;:::1::;:/:;:5=)-:-]

= 1.320.

TABLE 19. CALCULATED t VALUES

Concentration (/J.g/L) i t:;,

5.6 2, :1. 320

10.0 3 ,2.847

18.0 4 ,1.773

32.0 5 5.625

56.0 6 6.728

J.OO . 0' 7 9.891

280.0 8 J.0.836

17.13.3.7.6 Since the purpose of this test is to dete~t a

significant reduction in mean length, a one-sided test is .

appropriate. The critical value for this one-sided test is found

in Table 5, Appendix D. For an overall alpha lev:el of 0.05, 31

degrees of freedom for error and seven concentrations (excluding

the control) the approximate critical value is 2.597. The mean

weight for concentration "i" is considered significantly less

than the mean weight for the control if t i is greater than the

critical value. Therefore, the 10 0 IJ-g/L, 32 IJ-g/L, 56.'0 IJ-g/L,

517
..

100.0 ~g/L, 180.0 ~g/L concentrations have significantly lower

mean length than the control. Because the 10.0 ~g/L

concentration shows signigicantly lower mean length than the

control while the higher 18.0~g/L concentration does not, these

test results are considered to have an anomalous dose-response

relationship and it is recommended that the test be repeated. If

an NOEC and LOEC must be determined for this test, the lowest

concentration with significant growth impairment versus the

control is considered to the LOEC for growth. Thus, for this

test, the NOEC and LOEC would be 5.6 ~g/L and 10.0 ~g/L,

respectively.

17.13.3.8 Calculation of the ICp

17.13.3.8.1 The growth data from Table 13 and Figure 3 are

utilized in this example. As can be seen in the figure, the

observed means are not monotonically non-increasing with respect

to concentration. Therefore, the means must be smoothed prior to

calculating the IC

17.13.3.8.2 Starting with the observed control mean, Y1 = 18.38

is greater than the observed mean for the lowest copper

concentration, Y2 = 17.044, so set M1 = 18.38. Likewise, Y2 is

greater than the observed mean for the next copper concentration,

Y3 = 15.488, so set M2 = 17.044.

17.13.3.8.3 Comparing Y3 to Y4 = 16.576, we see that Y3 is less

than Y4 •

17.13.3.8.4 Calculate the smoothed means: '

J.7.J.3.3.8.5 Since M4 > Ys = 12.672 > Y6 = 11.550 > Y7 = 8.338 > Ya

= 7.375, set Ms = 12.672, M6 = 11.550, M7 = 8.338 and Ma ,= 7.375.

Table 20 contains the smoothed means and Figure 7 gives a plot of

the smoothed response curve. .

518
TABLE 20. GIANT KELP, MACROCYSTIS PYRIFERA, MEAN

GERM-TUBE LENGTHS AFTER SMOOTHING

Copper

Cone.

(j.Lg/L)

Control

5.6

10.0

18.0

32.0

56.0

100.0

180.0

i

1

2

3

4

5

6

7

8

Response

Means

Yi (mm)

18.380

17.044

15.488

16.576

12.672

11. 550

8.338

7.375

Smoclthed

Means

Mi (mm)

18.380

17.044

16. (132

16. Ci32

12.672

11. S50

8.3,38

7.3.75

17.13.3.8.7 Using the equation in Section 4.2 from Appendix L,

the estimate of the IC25 is calculated as follows:

IC25 = 18.0 + [18.380(1 - 25/100) - 16.032]

= 27.36 J1g/L.

(32 . 0 - 18. 0 )

!(12. 672' -16.032)

17.13.3.8.6 An IC25 can be estimated using the Linear

Interpolation Method. A 25~ reduction in length,: compared to the

controls, would result in a mean length of 13.785 mm, where M1 (1-

p/100) = 18.380(1-25/100). Examining the smoothed means and

their associated concentrations (Table 20), the response, 13.785

mm, is bracketed by C4 = 18.0 J1g/L and Cs = 32.0 J1g/L.

519
2O"h. * * * * _ ......-,_........

* * ---~--'...-- I 18 ,"'--- "" ------ CI:OICfI_~"__

18

:I: 14 lt:>

Z

w 12 ....J

w

CD 10 ::>

IU1 I 8 l\) :::;:

0 0::-

w 1 * t:> 8

"

2

0 ,

0.0 5.8 10.0 18.0 32.0 58.0 100.0 - 180.0

COPPER CONCENTRATION (ugIL)

Figure 7. Plot of raw data, observed means, and smoothed means for the giant

Kelp, Macrocystis pyrifera, growth data from Tables 13 and 20.
17.13.3.8.8 When the ICPIN program was used to analyze t1).is set

of data, requesting 80 resamples, the estimate ,of the TC25 was

27.3625 f.l.g/L. The empirical 95.0% confidence interval for the

true mean was 20.8734 f.l.g/Lto 42.3270 f.l.g/L. The computer program

output for the IC25 for this data set is shown in Figure 8.

17.14 PRECISION AND ACCURACY

17.14.1 PRECISION

.17.14.1.1 Single-Laboratory Precision

17.14.1.1.1 Single-laboratory precision data for the giant kelp

48-hour test method with the reference toxicants copper chloride

and sodium azide with natural seawater are provided in Tables 21-

22. The coefficient of variation (CV) of the germinatio.n EC50s

using copper was 38.8%; the CV of the germ-tube length IC40s

using copper was 32.9% (Table 21). The coefficient of variation

(CV) of the germination EC50s using azide was 3.6.7%; the CV of

the germ-tube length IC25s using azide was 30.8%, the CV of the

germ-tube length IC50s using azide was 28.4% (Table 22) .

17.14.1.2 Multi-laboratory Precision

17.14.1.2.1 Multi-laboratory precision data for the kelp 48.-:-hour

test method with the reference toxicant copper ,bhloride are

provided in Table 23. The coefficient of varia'tion of the IC50s

for the germ-tube length endpoint ranged.betweep 8.4% and 55.5%

using copper chloride. The coefficierit of variation of the IC50s

for the germination endpoint ranged between >1.1% and 67.6% using

copper chloride.

17.14.2 ACCURACY

17.14.2.1 The accuracy of toxicity tests cannot be determined.

521
Cone. ID 1 2 3 4 5 .. 6 7 8

Cone. Tested o 5.6 10 18 32 56 100 180

Response

Response

Response

Response

Response

1 19.5818.2613.3118.5912.5411.44 7.92 6.49

2 18.7516.2518.9212.8810.6711.88 7.59 7;25

3 19.1416.3915.6216.2815.9511.88 8.25

4 16.5018.7014.3015.3812.5411.00 9.13 7.63

5 17.9315.6215.2919.7511.6611.55 8.80

*** Inhibition concentration Percentage Estimate *** Toxicant/Effluent: Copper

Test Start Date: Test Ending Date:

Test Species: Giant kelp, Macrocystis pyrifera

Test Duration: 48 hours

DATA FILE: kelpgrow.icp

OUTPUT FILE: kelpgrow.i25

Cone.

ID

Number

Replicates

Concentration

ug/L

Resp~nse

Means

Std.

Dev.

Pooled

Response Means

1 5 0.000 18.380 1.214 18.380

2 5 5.600 17.044 1.352 17.044

3 5 10.000 15.488 2.121 16.032

4 5 18.000 16.576 2.707 16.032

5 5 32.000 12.672 1.988 12.672

6 5 56.000 11.550 0.365 11.550

7 5 100.000 8.338 0.629 8.338

8 4 180.000 7.375 0.691 ".375

The Linear Interpolation Estimate: 27.3625 Entered P Value: 25

27.5292

Lower:

Lower:

0.28

Number of Resamplings: 80

The Bootstrap Estimates Mean:

Original Confidence Limits:

Expanded Confidence Limits:

Resampling time in Seconds:

Standard Deviation: 4.7812

20.8734 Upper: 42.3270

17.6289 Upper: 49.8093

Random Seed: -35158431

Figure 8. ICPIN program output for the IC25.

522
TABLE 21. SINGLE LABORATORY PRECISION OF THE GIANT KELP,

MACROCYSTIS PYRIFERA GERMINATION AND GERM-,TUBE LENGTH

TEST WITH COPPER (CO ~G/L) CHLORIDE AS THE REFERENCE

TOXICANT

Test Number Germ-Tube Length I, Germination \

NOEC IC40 NOEC EC50

,1 <5.6 122.7 10.0 67.5

2 10.0 43.1 18.0 73.5

3 18.0 70.7 18.0, ' 124.3

4 5.6 88.0 56.0' 101.6

5 32.0 124.7 56.0, 122.9

Mean 89.8 1 90.7

CV 38.8% 32.9%,

Data from Anderson et al., 1994

TABLE 22.' S,INGLE LABORATORY PRECISION OF THE GI1lliT KELP,

MACROCYSTIS PYRIFERA GERMINATION AND GERM-TUBE LENGTH

TEST WITH SODIUM AZIDE (MG/L) .AS THE REFERENCE

TOXICANT

Test Date Germ-Tube Length 1 Germination

NOEC IC25 IC50 NOEC EC50'

2/11/92 18.0 39.5 133.7 18.0 52.3

2/18/92 18.0 34.1 96.5 32.0' '

72.6"

6/29/92 32.0 57.5 142.2, ' 32.0 132.1

7/07/92 10.0 33.1 92.5 :LO.O 79;2

7/15/92 18.0 42.8 138.9 28.0 117.8

7/16/92 5.6 25.0 68.4 10.0 48.3

7/22/92 10.0 30.2 80.6 J,8 . 0 62.4

10/09/92 5.6 25.1 80.0 '5.6 60.3

7/02/92 10.0 24.8 80.1 18.0 84.0

Mean, 34.7 101.4 " 78.8

CV 30.8% 28.4% 36.7%

Data from Hunt et al., 1991

523
TABLE 23. MULTI-LABORATORY PRECISION OF THE GIANT KELP,

MACROCYSTIS PYRIFERA GERMINATION AND GERM-TUBE LENGTH

TEST PERFORMED WITH COPPER CHLORIDE (~G/L) AS THE

REFERENCE TOXICANT

Lab Germ-tube length Germination CV CV

NOEC IC40 NOEC EC50 Germ-tube Germi'nat

ion

,

March J.990 3. 5.6 122.7 10.0 46.9 8.4% >1.1%

2 32.0 117. 8 . 32.0 46.2

3 18.0 104.1 32.0 *

May 1990 1 10.0 43.1 18".0 112.0 39.9% 59.3%

2 <5.6 99.1 32.0 164.2

3 18.0 68.7 18.0 67.9

I

May 1990 1 18.0 70.7 18.0 112.0 45.3% nc

I

I 2 18.0 91.3 56.0 64.5

3 32.0 134.2 32.0 158.0

i December 1 5.6 88.0 56.0 77.7 45.3%" nc

J.990 2 5.6 45.3 18".0 *

September 1 32.0 124.7 56.0 127.4 55.5%" 7.4%"

J.990 2 18.0 54.4 56.0 114.8

September 1 <10.·0 89.3** 56.0 115.5 44.5% 67.6%

1989 2 <10.0 171. 8** 56.0 327.7

November 1 32.0 >180.0 <10.0 >180.0 nc nc

J.989 2 10.0 >180.0 18.0 >180.0

I: May 1988 1 <56.0 232.0*** <56.0 211. 0 nc 50.0%

2 <56.0 * 56.0 100.7

* No ECSO calculated because response was less than SO%".

** Only concentration means available, therefore no IC40 values were

calculated.

nc Not calculated (Insufficient numbers to calculate the coefficient of

variation) . .

*** IC50 value, not IC40

Data from Hunt et al., 1991

524
APPENDIX I. MACROCYSTIS TEST: STEP-BY-STEP SUMMARY

PREPARATION OF TEST SOLUTIONS

appropriate dilution water

and guidance from the

I

A. Determine test concentrations and

based on NPDES permit conditions

appropriate regulatory agency.

B.Prepare effluent test solutions by diluting well mixed

unfiltered effluent using volumetric flasks and pipettes.

Use hypersaline brine where necessary to maintain all test

solutions at 34 ± 2%0. Include brine contr<;>ls in tests that

use brine.

C. Prepare a copper reference toxicant stock solution (10,000

~g/L) by adding 0.0268 of copper chloride (CuC1202H20) to 1

liter of reagent water.

D. Prepare copper reference toxicant solution of 0 (control)

5.6, 10, 18, 32, 100 and 180 ~g/L by adding 0, 0.56, 1.0

1.8, 3.2, 10.0 and 18.0 mL of stock solution, respectively,

to a 1-L volumetric flask and fill'ing to 1:::L with dilution

water. . I

E. Sample ~ffluentandreferencetoxicant ~olutions for

physical/chemical analysis. Measuresalini:ty, pH ~nd

dissolved oxygen from each test concentrati:on.

F. Randomize numbers for test chambers arid record the chamber

numbers with their respective test concentrations on a

randomization data sheet. Store the· data sheet safely until

after the test samples have been analyzed. I '

G. Place test chambers in a water bath or environmental chamber

set to 15°C and allow temperature to equilibrate.

H. Measure the temperature daily" in one random replicate (or

separate 'chamber) of each test conce~tration. Monitor the

temperature of the water bath or environmental chamber

continuously.

I. At the end of the test, measure salinity, pH, and dissolved

oxygen concentration from each test concentration.

525
PREPARATION AND ANALYSIS OF TEST ORGANISMS

A. Collect sporophylls and rinse in 0.2. ~m filtered seawater.

Store at 9-12°C for no more than 24 hours before zoospore

release.

B. Blot sporophylls and leave exposed to air for one hour.

C. Place 25-30 sporophylls one liter of 0.2 ~m filtered

seawater for no more than two hours. The presence of

zoospores is indicated by a slight cloudiness in the water.

D. Take a sample of the zoospore solution from the top 5

centimeters of the beaker and determine the spore density

using a hemacytometer. Determine the volume of water

necessary to give 7,500 spores/mL of test solution. This

volume should not exceed one percent of the test solution

volume.

E. Verify that the zoospores are swimming, then pipet the

volume of water necessary to give 7,500 spores/mL into each

of the test chambers. Take zoospores from the top 5

centimeters 'of the release beaker so that only swimming

zoospores are used.'

F. At 48 ± 3 hours, count the number of germinated and nongerminated spores of the first 10'0 spores' encountered in

each replicate of each concentration. Measure the'length of

10 randomly selected germination tubes (or preserve with

0.1% glutaraldehyde for later examination) .

G. Analyze the data.

H. Include standard reference toxicant point estimate values in

the standard quality control charts.

526
Data Sheet for Kelp Toxicity Test

Test Start Date: Start Time:

Test End Date: End Time:

Reference Toxicant:

Kelp SpeCies :

Collection!Arrival Date:

Kelp Source:

Sample Source: Microscope Model:

Sample Type: Solid Ehitriate Pore Water Water Effluent RefTox Micrometer COl1lversion Factor:

Test Number Numoer

Cont. Station of Spores. of Spores . Length Measurements (in ocular micrometer undts) Notes

# Code Germ. Not Germ. Ll L'.l Lj L4 L:5 Lo L7 US VJ LlU

1

2

3

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527
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541
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Quality Criteria for the Protection of Aquatic Life and Its

Uses. Federal Register, Vol. 45, No. 231, Friday, November

28, 1980.

USEPA. 1980b. Proposed good laboratory practice guidelines for

toxicity testing. Paragraph 163.60-6. Fed. Reg.

45:26377-26382, April 18, 1980.

USEPA. 1980c. Physical, chemical, persistence, and ecological

effects testing; good laboratory practice standards

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USEPA. 1981. In situ acute/chronic toxicological monitoring of.

industrial effluents for the NPDES biomonitoring program

using fish and amphibian embryo/larval stages as test '

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Enforcement and Permits, U. S .. Environmental Protection

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USEPA. 1982. Methods for organic chemical analysis of municipal

and industrial wastewater. Environmental Monitoring and

support Laboratory, U. S. Environmental Protection Agency,

Cincinnati, OR 45268. EPA/600/4-82/057.

USEPA. 1983. Guidelines and format for EMSL-Cincinnati methods.

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U. S. Environmental Protection Agency, Cincinnati,' OR 45268.

EPA/600/8-83/020.

USEPA. 1984. Effluent and ambient toxicity testing and instream

community response on the Ottawa Ri~er, Lima, Ohio. Mount,

D.I., N.A. Thomas, T.J. Norberg, M. T. Barbour, T.R. Roush,

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Environmental Protection Agency, Duluth, MN 55804.,

EPA/600/3-84/080.

542
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effluents to fre~hwater and marine organisms. Third

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Monitoring and Support Laboratory, U. S. Env~ironmental

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USEPA. 1985c. Validity of effluent and ambient toxicity tests

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Ohio. Mount, D.l., and T.J. Norberg-King (eds.).

Environmental Research Laboratory, U. S. Environmental

Protection Agency, Duluth, MN 55804. EPA/600/3-85/044.

USEPA. 1985d. Validity of effluent and ambient 'toxici'ty te'sting

for predicting biological impact on Five Mile Creek,

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Norberg-King (eds.). Environmental Research Laboratory, U.

S. Environmental Protection Agency, .Duluth, loiN 55804. '

EPA/600/8~85/015.,

USEPA. '1985e. Validity of' effluent and ambient toxicity tests

for predicting biological impact, Ohio River, near Wheeling,

West Virginia. Mount, D.l., A.'E~ Bteen, and T.J.

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S. Environmental Protection Agency, Duluth, ~~ 55804.

EPA/600/3-85/071.

USEPA.1986a. Validity of effluent and ambient toxicity tests

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Norberg-King (eds.). Environmental Research Laboratory,

U. S. Environmental Protection Agency, Duluth, MN 55804.

EPA/600/8~86/001.

USEPA. 1986b. Validity of effluent and ambient toxicity tests

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, Environmental Research Laboratory, U. S. Environmental

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USEPA. 1986c. Valldity of effluent and ambient tqxicity tests

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543
Environmental Research Laboratory, U. S. Environmental

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USEPA. 1987b. Guidance manual for conducting complex effluent

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USEPA. 1987c. Guidance manual for rapid chronic toxicity tests

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544
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USEPA. '1987f. Guidance manual for conducting sexual.

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of effluents and receiving waters to marine and estuarine

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USEPA. 1988b. NPDES compliance inspection manual. Office of

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USEPA. 1988c. Methods for aquatic toxicity identification

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D. I. Nount and L. Anderson-Carnahan. Environm!ental Research

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545
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USEPA. 1989b. Toxicity reduction evaluation protocol for

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Risk Reduction Engineering Laboratory, U. S. Environmental

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USEPA. 1989c. Generalized methodology for conducting industrial

toxicity reduction evaluations (TREs). J:A. Fava, D.

Lindsay, W.H. Clement, R. Clark, G.M. DeGraeve.tJ..D. Cooney,

S. Hansen, W. Rue, S. Moore, and P. Lankford (eds.). Risk

Reduction Engineering Laboratory, U. S. Environmental

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USEPA. 1989d. Methods for' aquqtic toxicity identification

evaluations: Phase fl toxicity identification procedures.

D.l. Mount" and L. Anderson-Carnahan. Environmental

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Duluth, MN 55804. EPA-600/3-88/035.

USEPA. 198ge. Methods for aquatic toxicity identification

evaluations: Phase III, toxicity confirmation procedures.

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USEPA. 1990a. Macroinvertebrate field and laboratory methods

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Klemm, D.J., P.A. Lewis, F. Fulk, and J.M. Lazorchak.

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USEPA. 1990b. Supplemental methods and status reports for

short-term saltwater toxicity tests. G. Morrison and G.

Chapman. ERL Contrib. No. 1199. Environmental Research

Laboratory, U. S. Environmental Protection Agency,

Narragansett, RI 02882. 127 pp.

546
USEPA. 1991a. Technical support document for walter qualitybased toxic controls. Office of Water Enforcement and

Permits and Office of Water Regulations and 'Standards, U. S.

Environmental Protection Agency, Washington, 'DC 20460.

EPA/505/2-90/001.

USEPA. 1991b. Methods for aquatic toxicity iden;tification

evaluations: Phase I, toxicity characterization procedures.

2nd ed., T. Norberg-King, D.L Mount, E. Dur,han, G. Ankley,

L. Burkhard, J. Amato, M. Ludasewycz, M. Schubauer-Berigan,

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Laboratory, U. S. Environmental Protection Agency, Duluth,

MN 55804. EPA/600/6-91/003.

USEPA. 1991c. Manual for the evaluation of laboratories

performing aquatic toxicity tests. Klemm, D'.J., L.B.

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USEPA. 1993a. Methods for measuring the acute toxicity of

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Environmental Monitoring Systems Laboratory, U. S.

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USEPA. 1993b. Short-term methods for estimating the chronic

toxicity of effluents' and surface waters to f~reshwater

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USEPA. 1993c. Fish field and laboratory methods .for evaluating

the biological integrity of surface watrs. Klemm, D.J.,

547
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USEPA. 1994. Short-term methods for estimating the chronic

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JWPCF
APPENDICES

Page

A.

B.

C.

D.

E.

Independence, Randomization, and Outliers

1. Statistical Independence

2. Randomization .

3 Outliers. . . . . . . . . . . . . .

Validating Normality and Homogeneity of Variance

Assumptions . . . . . . . . . . . . . . . . .

1. Introduction .

2. Tests for Normal Distribution of Data

3. Test for Homogeneity of Variance

4. Transformations of the Data

Dunnett's Procedure

1. Manual Calculations

2. Computer Calculations

t test with the Bonferroni Adjustment

Steel's Many-one Rank Test

566

566

566

... 572

575

575

575

582

584

587

587

595

.602

.609

F. Wilcoxon Rank Sum Test with the Bonferroni Adjustment 615

G. Single Concentration Toxicity Test - Comparison of

Control with 100% Effluent or Receiving Water or

Comparison of Dilution and Controls ... 622

H.

I.

J.

K.

Probit Analysis

Spearman-Karber Method . . . .

Trimmed Spearman-Karber Method

Graphical Method

564

627

. ,631

638

643
L. Linear Interpolation Method . ,. .648

l. General Procedure . .' · 648

2. Data Summary and Plots · 648

3. Monotonicity .648

4. Linear Interpolation Method .649

5. Confidence Intervals .650

6. Manual Calculations .651

7. Computer Calculations .655

Cited References . . : .. • · 659

565
APPENDIX A

INDEPENDENCE, RANDOMIZATION, AND OUTLIERS

1. STATISTICAL INDEPENDENCE

1.1 Dunnett's Procedure and the t test with Bonferroni's

adjustment are parametric procedures based on the assumptions

that (1) the observations within treatments are independent and

normally distributed, and (2) that the variance of the

observations is homogeneous across all toxicant concentrations

and the control. Of the three possible departures from the

assumptions, non-normality, heterogeneity of variance, and lack'

of independence, those caused by lack of independence are the

most difficult to resolve (see Scheffe, 1959). For toxicity

data, statistical independence means that given knowledge of the

true mean for a given concentration or control, knowledge of the

error in anyone actual observation would provide no information

about the error in any other observation. Lack of independence

is difficult to assess and difficult to test for statis·ticallY.

It may also have serious effects on the true alpha or beta level.

Therefore, it is of utmost importance to be aware of the need for

statistical independence between observations and to be

constantly vigilant in avoiding any patterned experimental

procedure that might compromise independence. One of the best

ways to help insure independence is to follow proper

randomization procedures throughout the test.

2 • RANDOMIZATION

2.1 Randomization of the distribution of test organisms among

test chambers, and the arrangement of treatments and replicate

chambers is an important part of conducting a valid test. The

purpose of randomization is to avoid situations where 'test

organisms are placed serially into test chambers, or where all

replicates for a test concentration are located adjacent to one

another, which could introduce bias into the test results.

2.2 An example of randomization of the distribution of test

organisms among test chambers, ,and an example of randomization of

arrangement of treatments and replicate chambers are d~scribed

using the topsmelt, Atherinops affinis, Survival and Growth test.

For the purpose of the example, the test design is as follows:

566
Five effluent concentrations ,are tested in addition to the

control. The effluent concentrations are as fbllows: 6.25%,

12.5%, 25.0%, 50.0%, and 100,~0%. There are five replicate

chambers per treatment. Each replicate chamber contains five

larvae.

2.3 RANDOMIZATION,OF FISH TO REPLICATE CHAMBERS EXAMPLE

2.3.1 Consider first the random assignment of the fish to the

replicate chambers. The first step is to label each of the

replicate chambers with the control or effluent concentration and

the replicate number. The next step is to assign each replicate

chamber three double-digit numbe~s. Anexqmple of this

assignment, is provided in Tab,leA.1. Note that the double digits

00 and 91 through 99 were not used,

2.3.2 The random numbers used to carry out the random assignment

of fish to replicate chambers are provided in Table A.2.

The third step is to choose a starting position in Table A.2, and

read the first double digit number. The first number read

identifies the replicate chamber for the first fish taken from

the tank. For the example, the first entry in row 2 was chosen

as the starting 'position. The first number in this row is 37.

According to Table A.l, this number corresponds to, replicate

chamber 2 of the 6.25% effluent concentration.· Thus, the first

fish taken from the ,tank is to be placed in replicate chamber 2

of the 6.25% effluent concentration~

I

2.3.3 The next step is to read the double digit number to the

right of the first one. The second number identifies the

replicate chamber for the second fish taken from the tank.

Continuing the example, the second number read in row 2 of Table

A.2 is 54. According to Table A;l" this number corresponds to

replicate chamber 4 of, the 50 . O,%efflueht concentration. Thus,

the second fish taken from the tank is to be placed in replicate

chamber 4 of the 50.0% effluent concentration.,

,

2.3.4 Contibue in this fashion until all the :fish have been

randomly assigned to a replicate chamber. In order to fill each

replicate chamber with ten fish, the assigned numbers will be

used more than once. ,If a number is read from the table that was

not assigned to a replicate chamber, then ignore it and continue

to the next number. If a replicate chamber becomes filled and a

567
number is read from the table that corresponds to it, then ignore

that value and continue to the next number. The first ten random

summarized in Table A.3.2.3.5 Three double-digit numbers were

assigned to each replicate chamber (instead of one or two doubledigit numbers) in order to make efficient use of the random

number table (Table A.2). To illustrate, consider the assignment

of only one double-digit number to each replicate chamber: the

first column of assigned numbers in Table A.1. Whenever the

numbers 00 and 31 through 99 are read from Table A.2, they will

be disregarded and the next number will be read.

TABLE A.1. RANDOM ASSIGNMENT OF FISH' TO REPLICATE CHAMBERS EXAMPLE

ASSIGNED NUMBERS FOR EACH REPLICATE CHAMBER

Assigned Numbers Replicate Chamber

01, 31, 61 Control, replicate chamber 1

02, 32, 62 Control, replicate chamber 2

03, 33, 63 Control, replicate chamber 3

04, 34, 64 Control, replicate chamber 4

OS, 35, 65 Control, replicate chamber 5

06, 36, 66 6.25% effluent, replicate chamber 1

07, 37, 67 6.25% effluent, replicate chamber 2

08, 38, 68 6.25% effluent, replicate chamber 3

09, 39, 69 6.25% effluent, replicate chamber 4

10, 40, 70 6.25% effluent, replicate chamber 5

11, 41, 71 12.5% effluent, replicate chamber 1

12, 42, 72 12.5% effluent, replicate chamber 2

13, 43, 73 12.5% effluent, replicate chamber ,3

14, 44, 74 J.2.5% effluent,- replicate chamber 4

15, 45, 75 12.5% effluent, replicate chamber 5

16, 46, 76 25.0% effluent, replicate chamber J.

17, 47, 77 25.0% effluent, replicate chamber 2

18, 48, 78 25.0% effluen,t, replicate chamber 3

19, 49, 79 25.0% effl~~" replicate chamber 4

20, 50, 80 25.0% effr't)le~" replicate chamber 5

2J., 51, 81 50.0% effluent, replicate chamber 1

22, 52, 82 50.0% effluent, replicate chamber 2

23, 53, 83 50.0% effluent, replicate chamber 3

24, 54, 84 50.0% effluent, replicate chamber 4

25, 55, 85 50.0% effluent, replicate chamber 5

26, 56, 86 100.0% effluent, replicate chamber 1

27, 57, 87 100.0% effluent, replicate chamber 2

28, 58, 88 100.0% effluent, replicate chamber 3

29, 59, 89 100.0% effluent, replicate chamber 4

30, 60, 90 100.0% effluent, replicate chamber 5

568
TABLE A.2. TABLE OF RANDOM NUMBERS (Dixon and Massey, 1983)

10 09 73 25 33 76 52 01 35 86 34 67 35 43 76 80 95 90 91 17 39 29 27 49 45

37 54 20 48 05 64 89 47 42 96 24 80 52 40 37 20 63 61 04 02 00 82 29 .16 65

08 42 26 89 53 19 64 50 93 03 23 20 90 25 60 15 95 33 47 64 35 08 03 36 06

99 01 90 25 29 09 37 67 07 15 38 31 13 11 65 88 67 67 43 97 04 43 62 76 59

12 80 79 99 70 80 15 73 61 47 64 03 23 66 53 98 95 11 68 77 12 27 17 68 33

66 06 5747 17 34 07 27 68 50 36 69 73 61 70 65 81 33 98 85 11 19 92 91 70

31 06 01 08 05 45 57 18 24 06 35 30 34 26 14 86 79 90 74 39 23 40 30 97 32

85 26 97 76 02 02 05 16 56 92 68 66 5748 18 73 05 38 52 47 18 62 38 85 79

63 57 33 21 35 05 32 54 70 48 90 55 35 75 48 28 46 82 87 09 83 49 12 56 24

73 79 64 57 53 03 52 96 47 78 35 80 83 42 82 60 93 52 03 44 35 27 38 84 35

98 52 01 77 67 14 90 56 86 07 22 10 94 05 58 60 97 09 34 33 50 50 07 39 98

11 80 50 54 31 39 80 82 77 32 50 72 56 82 48 29 40 52 42 01 52 77 56 78 51

83 45 29 96 34 06 28 89 80 83 13 74 67 00 78 18 47 54 06 10 68 71 17 78 17

88 68 54 02 00 86 50 75 84 01 36 76 66 79 51 90 36 47 64 93 29 60 91 10 62

99 59 46 73 48 87 51 76 49 69 91 82 60 89 28 93 78 56 13 68 23 47 83 41 13

65 48 11 76 74 17 46 85 09 50 58 04 77 69 74 73 03 95 71 86 40 21 81 65 44

80 12 43 56 35 17 72 70 80 15 45 31 82 23 74 21 11 57 82 53 14 38 55 37 63

74 35 09 98 17 77 40 27 72 14 43 23 60 02 10 45 52 16 42 37 96 28 60 26 55

69 91 62 68 03 66 25 22 91 48 36 93 68 72 03 76 62 11 39 90 94 40 05 64 18

09 89 32 05 05 14 22 56 85 14 46 42 75 6788 96 29 7788 22 54 38 21 45 98

91 49 91 45 23 68 47 92 76 86 46 16 28 35 54 94 75 08 99 23 37 08 92 00 48

80 33 69 45 98 2694 036858 70 29 73 41 35 53 14 03 33 40 42 05 08 23 41

44 10 48 19 49 85 15 74 79 54 32 97 92 65 75 57 60 04 08 81 22 22 20 64 13

12 55 07 37 42 11 10 00 20 40 12 86 07 46 97 96 64 48 94 39 28 70 72 58 15

63 60 64 93 29 16 50 53 44 84 40 21 95 25 63 43 65 17 70 82 07 20 73 17 90

61 19 69 04 46 2645 74 77 74 51 92 43 37 29 65 39 45 95 93 42 58 26 05 27

15 47 44 52 66 95 27 07 99 53 59 36 78 38 48 82 39 61 01 18 33 21 15 94 66

94 55 72 85 73 67 89 75 43 87 54 62 24 44 31 91 19 04 25 92 92 92 74 59 73

42 48 11 62 13 97 34 40 87 21 1686 84 87 67 03 07 11 20 59 25 70 14 66 70

23 52 37 83 17 73 20 88 98 37 68 93 59 14 16 26 25 22 96 63 05 52 28 25 62

04 49 35 24 94 75 24 63 38 24 45 86 25 10 25 61 96 27 93 35 65 33 71 24 72

00 54 99 76 54 64 05 18 81 59 96 11 96 38 96 54 69 28 2391 23 28 72 95 29

35 96 31 53 07 26 89 80 93 45 33 35 13 54 62 77 97 45 00 24 90 10 33 93 33

59 80 80 83 91 45 42 72 68 42 83 60 94 97 00 13 02 12 48 92 78 56 52 01 06

46 05 88 52 36 01 39 09 22 86 77 28 14 40 77 93 91 08 36 47 70 61 74 29 41

32 1790 05 97 87 37 92 52 41 05 56 70 70 07 86 74 31 71 57 85 39 41 18 38

69 23 46 14 06 20 11 74 52 04 15 95 66 00 00 18 74 39 24 23 97 11 89 '63 38

19 56 54 14 30 01 75 87 53 79 40 41 92 15 85 66 67 43 68 06 84 96 28 52 07

45 15 51 49 38 19 47 60 72 46 43 66 79 45 43 59 04 79 00 33 20 82 66 95 41

94 86 43 19 94 36 16 81 08 51 34 88 88 15 53 01 54 03 54 56 05 01 45 11 76

98 08 62 48 26 45 24 02 84 04 44 99 90 88 96 39 09 4734 07 35 44 13 18 80

33 18 51 62 32 41 94 15 09 49 89 43 54 85 81 88 69 54 19 94 3754 87 30 43

80 95 10 04 06 96 38 27 07 74 20 15 12 33 87 25 01 62 52 98 94 62 46 11 71

79 75 24 91 40 71 96 12 82 96 69 86 10 25 91 74 85 22 05 39 00 38 75 95 79

18 63 33 25 37 98 14 50 65 71 31 01 02 46 74 05 45 56 14 27 77 93 89 1936

74 02 94 39 02 77 55 73 22 70 97 79 01 71 19 52 52 75 80 21 80 81 45 17 48

54 17 84 56 11 80 99 33 71 43 05 33 51 29 69 56 12 71 92 55 36 04 09 03 24

11 66 44 98 83 52 07 98 48 27 59 38 17 15 39 09 97 33 34 40 88 46 12 .33 56

48 32 47 79 28 31 24 96 47 10 02 29 53 68 70 32 30 75 75 46 15 02 00 99 94

69 07 49 41 38 8763 79 19 76 35 58 40 44 01 10 51 82 16 15 I 01 84 87 69 38

569
2.4 RANDOMIZATION OF REPLICATE CHAMBERS TO POSITIONS EXAMPLE

2.4.1 Next consider the random assignment of the 30 replicate

chambers to positions within the water ba~h (or equivalent) .

Assume that the replicate chambers are to be positioned in a five

row by six column rectangular array. The first step is to label

the positions in the water bath. Table A.4 provides an example

layout. assignments of fish to replicate chambers for the 'example

are

TABLE A.3. EXAMPLE OF RANDOM ASSIGNMENT OF FIRST TEN FISH TO REPLICATE

CHAMBERS

Fish Assignment

First fish taken from tank 6.25% effluent, replicate chamber 2

Second fish taken from tank 50.0% effluent, replicate chamber 4

Third fish taken from tank 25.0% effluent, replicate chamber 5

Fourth fish taken from tank 25.0% effluent, replicate chamber 3

Fifth fish taken from tank Control', replicate chamber 5

Sixth fish taken from tank Control, replicate 'chamber 4

Seventh fish taken from tank 100.0% effluent, replicate chamber 4

Eighth fish taken from tank 25.0% effluent, replicate chamber 2

Ninth fish taken from tank 12.5% effluent, replicate chamber 2

Tenth fish taken from tank 50.0% effluent, replicate chamber 4

TABLE A.4. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS: EXAMPLE

LABELLING THE POSITIONS WITHIN THE WATER BATH

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

19 20, 21 22 23 24

25 26 27 28 29 30

570
2.4.2 The second step is to

three double-digit numbers.

provided in Table A.5. Note

through 99 were not used.

assign each of the 30 positions

An example of this assignment is

that the double digits 00 and 91

2.4.3 The random numbers used to carry out the random assignment

of replicate chamb~rs to positions are provided in Table A.2.

The third step is to choose a starting positior~ in Table A. 2, and

read the first double-digit number. The first number. read

identifies the position for the first replicate chamber of the

control For the example, the first entry in :r'ow 10 of Table A.2

was chosen as the starting position. The first. number in this

row was 73. According to Table A.5, this number corresponds to

position 13. Thus, the first replicate chamber for the control

will be placed in position 13.

2.4.4 The next step is to read the double-digit number to the

right of the first one. The second number identifies the

position for the sec~nd replicate chamber of the control.

Continuing the example, the second number read in row 10 of Table

A.2 is 79. According to Table A.5, this number corresponds to

position 19. Thus, the second replicate 'chamber for the control

will be placed in position 19.

2.4.5 Continue in this fashion until all the replicate chambers

have been assigned to a position. The first five numbers read

will identify the positions for the control replicate chambers,

the second five numbers read will identify the ]positions for the

lowest effluent concentration replicate chamberis, and so on. If

a number is read from the table that was not assigned to a

position, then ignore that val1..le and continue t(J the next number.

If a number is repeated in Table A.2, then ignore the repeats and

continue to the next number. The complete randomization of

replicate chambers to positions for the example is displayed in

Table A.6.

2.4.6 Three double-digit numbers were assigned to each position

(instead of one or two) in order to make effici~nt use of the

random number table (Table A.2). To illustrate, consider the

assignment of only ,one double-digit number to each position: the

first column of assigned numbers in Table A.5. Whenever the

numbers 00 and 31 through 99 are read from Table A.2, they will

be disregarded and the next number will be read.

571
3. OUTL~ERS

3.1 An outlier is an inconsistent or question~ble data point

that appears unrepresentative of the general trend .exhibited by

the majority of the data. Outliers may be detecteq by tabulation

of the data, plotting, and by an analysis of the residuals. An

explanation should be sought for any questionable data points.

Without an explanation, data points should be discarded only with

extreme caution. If there is no explanation, the analysis should

be performed both with and without the outlier, and the results

of both analyses should be reported.

3.2 Gentleman-Wilkls A statistic gives a test for the condition

that the extreme observation may be considered an outlier. For a

discussion of this, and other techniques for evaluating outliers,

see Draper and John (1981).

572
TABLE A.5. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS:

EXAMPLE ASSIGNED NUMBERS FOR EACH POSITION

Assigned Numbers Position

01. 31. 61 1

02. 32. 62 2

03. 33. 63 3

04. 34. 64 4

05.35. 65 5

06. 36. 66 6

07. 37. 67 7

08. 38. 68 8

09. 39. 69 9

10. 40. 70 10

11. 41. 71 11

12. 42. 72 12

13. 43. 73 13

14. 44, 74 14

15. 45. 75 15

16. 46.,76 16

17. 47. 77 I 17

18. 48. 78 18

19, 49. 79 19

20. 50. 80 20

21. 51. 81 21

22. 52. 82 I

22

23. 53. 83 23

24. 54. 84 24

25. 55.' 85 25

26. 56. 86 : 26

27. 57, 87 I 27

28. 58. 88 i 28

29. 59. 89 29

30. 60. 90 I30

573
TABLE A.S. EXAMPLE OF RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO

POSITIONS:

ASSIGNMENT OF ALL 30 POSITIONS

25. o!I; 50.0% 6.25%- Control 6.25% 100.0~

25. o!I; 12.5% 50.0%- 25.0% 50.0% 12.5%

Control 12.5% 100.0% 100.0%- 6.25% 6.25%

Control 12.5% 100.0% 6.25% Control 25.0%

100.0% 25.0% Control 50.0% 50.0% 12.5%

574
APPENDIX B

(

VALIDATING NORMALITY AND HOMOGENEITY OF VARIANCE ASSUMPTIONS

1. INTRODUCTION

1.1 Dunnett's Procedure and the t test with Bonferroni's

adjustment are parametric procedures based on the assumptions

that the observations within treatments are independent and

normally distrib~ted, and that the variance of the observations

is homogeneous across all toxicant concentrations and the

control. These assumptions should be checked prior to using

these tests, to determine if they have been met.. Tests for

validating the assumptions are provided in the following

discussion. If the tests fail (if the data do not meet the

assumptions), a nonparametric procedure such as Steel's Many-one

Rank Test may be more appropriate. However, the decision on

whether to use parametric or nonparametric tests may be a

judgement call, and a statistician should be consulted 'in

selecting the analysis.

2. TEST FOR NORMAL DISTRIBUTION OF DATA

2.1 SHAPIRO-WILK'S TEST

2.1.1 One formal test for normality is the Shapiro-WiLk's Test

(Conover, 1980). The test statistic is obtained by dividing the

square of an appropriate linear combination of the sample order

statistics by the usual sYmmetric estimate of variance. The

calculated W must be greater than zero and less than or equal to

one. This test is recommended for a sample size of 50 or less.

If the sample size is greater than 50, the Kolmogorov "D"

statistic (Stephens, 1974) is recommended. An example of the

Shapiro-Wilk's test is provided below.

2.2 The example uses growth data from the Mysid Larval Survival

and Growth Test. The same data are used later in the discussions

of the homogeneity of variance determination in: Section 3 of this

appendix and Dunnett's Procedure in Appendix C. The data, the

mean and variance of the observations at each concentration,

including the control, are listed in Table B.1 ..

575
TABLE B.1. MYSID, HOLMESIMYSIS COSTATA, GROWTH DATA

Concentration (%)

Replicate Control 1.80 3.20 5.60

1 0.048 0.055 0.057 0.041

2 0.058 0.048 0.050 0.040

3 0.047 0.042 0.046 0.041

4 0.058 0.041 0.043 0.043

5 0.051 0.052 0.045 0.040

Mean (Yi) 0.052 0.048 0.048 0.041

Sf 0.0000283 0.0000373 0.0000307 0.0000015

i 1 2 3 4

•

2.3 The first step of the test for nbrmality is to center the

observations by subtracting the mean of all observations within a

concentration from each observation in that concentration. The

centered observations are listed in Table B.2.

TABLE B. 2. CENTERED OBSERVATIONS FOR SHAPIRO-WILK" S EXAMPLE

'Concentration (%)

Replicate Control 1.80 3.20 5.60

1 -0.004 0.007 0.009 0.000

2 0.006 0.000 0.002 -0.001

3 -0.005 -0.006 -0.002 0.000

4 0.006 -0.007 -0.005 0.002

5 -0.001 0.004 -0.003 -0.001

2.4 Calculate the denominator, D, of the statistic:

n -2

D: E (X - X)

i.l i

Where: Xi = the ith centered observation

576
X the overall mean of the centereid observations

n = the total number of centered observations

2.4.1 For this set of data, n = 20

x = _1_ (0.001)

20

D = 0.000393

=0.000 I

,

2.5 Order the centered observations from smallest to largest

where Xli) denotes the ith ordered observation. 'rhe ordered

observations for this example are listed in Table B.3.

TABLE B.3. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE

i Xli) i :x: (il

1 -0.007 11 0.000

2 -0.006 12 0.000

3 -0.005 13 Cl.OOO

4 -0.005 14 0.002

5 -0.004 15 0.002

6 -0.003 16 0.004

7 -0.002 17 0.006

8 -0.001 18 0.006

9 -0.001 19 0.007

10 -0.001 20 0.009

2.6 From Table B.4,

the coefficients all

(n-1)/2 if n is odd.

= 10. The ai values

for the number of observations, n, obtain

a 2 , ••• a k where k is n/2 if n is even and

For the data in this example, n = 20 and k

are listed in Table B.S.

577
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (Conover, 1980) , '

;\n

Number of Observations

2 3 4 5 6 7 8 9 10

1 0.7071 0.7071 0.6872 0.6646 0.6431 0.6233 0.6052 0.5888 0.5739 '

2 0.0000 0.1667 0.2413 0.2806 0.3031 0.3164 0.3244 0.3291

3 0.0000 0.0875 0.1401 0.1743 0.1976 0.2141

4 0.0000 0.0561 0.0947 0.1224

5 0.0000 0.0399

;\ Number of.Dbservations

11 12 13 14 15 16 17 18 19 20

1 0.5601 0.5475 0.5359 0.5251 0.5150 0.5056 0.4968 0.4886 0.4808 0.4734

2 0.3315 0.3325 0.3325 0.3318 0.3306 0.3290 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 O~ 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 0.0$93 0.0725 0.0837 0.0932 0.1013

8 0.0000 0.0196 0.0359 0.Ol;96 0.0612 0.0711

9 0.0000 0.0163 0.0303 0.0422

10 0.0000 0.0140

;~n Number of'Observations

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

578
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (CONTINUED)

i\ Number of Observations

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.(1515 0.0546

16 0.0000 0.0068 0~0131 0.0187 0.0239, 0.0287 0.0331 0.0372 0.(1409 0.0444

17 0.0000 0.0062 0.0119 0.0172 0.0220 0.0264 0.()305 0.0343

18 0.0000 0.0057 0.0110 0.0158 0.()203 0.0244

19 0.0000 0.0053 0.C1101 0.0146

20 O.C<OOO 0.0049

j\ Number of Observations

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 0.1t~92 0.1691

7 0.1531 0.1535 0.1539 0.1542 0.1545 Q,.1548 0.1550 0.155l 0.1!553 0.1554

8 0.1384 0.1392 0.1398 0.1405 0.1410 0.1415 0.1420 . 0.1423 0.11~27 0.1430

9 0.1249 0.1259 0.1269 0.1278 0.1286 0.1293 0.1300 0.1306 0.1:512 0.1317

10 0.1123 0.1136 0.1149 0.1160 0.1170 0.1180 0.1189 0.1197 0."~05 0.1212

11 0.1004 0.1020 0.1035 0.1049 0.1062 0.1073 0.1085 0.1095 0."105 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 0.01\32 0.0846

15 0.0575 0.0602 0.0628 0.0651 0.0673 0.0694 0.0713 0.0731 0.0i'48 0.0764

16 0.0476 0.0506 0.0534 0.0560 0.0584 0.0607 0.0628 0.0648 0.0£167 0.0685

17 0.0379 0.0411 0.0442 0.0471 0.0497 0.0522 0.0546 0.0568 0.0~i88 0.0608

18 0.0283 0.0318 0.0352 0.0383 0.0412 0.0439 0.0465 0.0489 0.0~j11 0.0532 ,r

19 0.0188 0.0227 0.0263 0.0296 0.0328 0.0357 0.0385 . 0.0411 0.0~,36 0.0459

20 0.0094 0.0136 0.0175 0:0211 0.0245 0.0277 0.0307 0.0335 0.m;61 0.0386

21 0.0000 0.0045 0.0087 0.0126 0.0163 0.0197 0.0229 0.0259 0.02!88 0.0314

22 0.0000 0.0042 0.0081 0.0118 0.0153 0.0185 0.02:15 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

579
2.7 Compute the test statistic, W; as follows:

, k 2

W : 2. [La (X (n-1+l1 -x (!l ) ]

D !.1 !

The differences XCn- i +1 ) - XCi) are listed in Table B. 5. For this

set of data:

w = 1 (0.0194)2

0.000393

0.958

TABLE B.5. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE

i ai x(n-i+l) - Xli)

1 0.4734 0.016 X(20) - X (1 )

2 0.3211 0.013 X(19) - x C2l

3 0.2565 0.011 X(lS) - X(3)

4 0.2085 0.011 X I1?) - X(4)

5 0.1686 0.008 X C1.6 ) - xes)

6 0.1334 0.005 X(lS) - X(6l

7 0.1013 0.004 X (:l.4) - x(?)'

B 0.0711 0.001 X (13 ) XIS)

9 0.0422 0.001 X (:l.2) - X (9 )

10 0.0140 0.001 X(ll) - X(lO)

2.8 The decision rule for this test is to ,compare the computed W

to the critical value found in Table B.6. If the computed W is

less than the critical value, conclude that the data are not

normally distributed. For this set of qata, the critical value

at a significance level of 0.01 andn = 20 observations is 0.868.

Since W = 0.958 is greater than the crit~cal value, conclude that

the data are normally distributed.

2.9 In general, if the data fail the test for normality, a

transformation such as to log'values may normalize the data.

After transforming the data, repeat the Shapiro Wilk's Test for

normality.

580
TABLE B.6. QUANTILES OF THE SHAPIRO WILK'S TEST STATISTIC (Conover. 1980)

n 0.01 0.02 0.05 ,0.10 0.50 0.90 0.95 0.98 0.99

./

3 0.753 0.756 0.767 0.789 0.959 0.998 0.999 1.000 1.000

4 0.687 0.707 0.748 0.792 0.935 0.987 0.992 0.996 0.997

5 0.686 0.715 0.762 0.806 0.927 0.979 0.986 0.991 0.993

6 0.713 0.743 0.788 0.826 0.927 0.974 0.981 0.986 0.989

7 0.730 0.760 0.803 0.838 0.928 0.972 0.979 0.985 0.988

8 0.749 0.778 0.818 0.851 0.932 0.972 0.978 0.984 0.987

9 0.764 0.791 0.829 0.859 0.935 0.972 0.978 '0.984 0.986

10 0.781 0.806 0.842 0.869 0.938 0.972 0.978 0.983 0.986

11 0.792 0.817 0.850 0.876 0.940 0.973 0.979 0.984 0.986

12 0.805 0.828 0.859 0.883 0.943 0.973 0.979 0.984 0.986

13 0.814 0.837 0.866 0.889 0.945 0.974 0.979 0.984 0.986

14 0.825 0.846 0.874 0.895 0.947 0.975 0.980 0.984 0.986

15 0.835 . 0.855 0.881 O~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 0.9'90

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 p.989 0.9.91

41 0.920 0.929 0.94.1 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 10.990 0.991

48 0.929 0.937 0.947 0.954 0.974 0.985 0.988 la.990 0.991

49 0.929 0.937 0.947 0.955 0.974 0.98!: 0.988 1:1.990 0.991

50 0.930 0.938 0.947 0.955 0.974 0.985 0.988 1:1.990 0.991

'\

581
3 • TEST FOR HOMOGENEITY OF VARIANCE

3.1 For Dunnett's Procedure and the t test with Bonferroni's

adjustment, the variances of the data obtained from each toxicant

concentration and the control are assumed to be equal.

Bartlett's Test is a formal test of this assumption. In using

this test, it is assumed that the data are normally distributed.

3.2 The data used in this example are growth data from a Mysid

Survival and Growth Test, and are the same data used in Appendix

C. These data are listed in Table B.7, together with the

calculated variance for the control and each toxicant

concentration.

TABLE B.7. MYSID, HOLMESIMYSIS COSTATA, GROWTH DATA ,

Concentration (% )

Replicate Control 1.80 3.20 5.60

0.048 0.055 0.057 0.04~

2 0.058 0.048 0.050 0.040

3 0.047 0.042 0.046 0.04~

4 0.058 0.04~ 0.043 0.043

5 0.05~ 0.052 0.045 0.040

Mean (Yi ) 0.052 0.048 0.048 0.041

Sf 0.0000283 0.0000373 0.0000307 0.0000015

i 2 3 4

3.3 The test statistic for Bartlett's Test (Snedecor and

Cochran, 1980) is as follows:

B =

p p

[(Evi ) In Ef - EVi In S:l i~ i~

c

Where: Vi = degrees of freedom for each effluent concentration

and control, (Vi = ni - 1)

p = number of levels of toxicant concentration

including the control

582
ln = loge

i = 1, 2, ... , p where p is the number of

concentrations including the control

n i =the number of replicates for concentration i.

p

(LViS;)

82 = i·l

p

LV1

1-1

p P

c=1+ [3 (p-1) r 1 [L.1/V _ (LV )-1] i· i

1-1 i·l

3.4 Since B is approximately distributed as chi-square with p -

1 degrees of freedom when the variances are equal, the

appropriate critical value is obtained from a table of the

chi-square distribution for p - 1 degrees of freedom and a

significance level of 0.01. If B is less than the critical value

then the variances are assumed to be equal.

3.5 For the data in this example, all concentrations including

the control have the same number of replicates (ni - 5 for all

i). Thus, Vi = 4 for all i. For this data, p = ~h, 8 2 = 0.0000245, and C = 1.104. Bartlett's statistic is therefore:

P ,

B = [(16) 1n (0.0000245) -4L1n (S;) ] /1.104 i·1

= [16(-10.617) 4(-44.470)]/1.104

= [-169.872 - (-177.880)] /ll104 '

7.254

3.6 Since B is approximately distributed as chi-square with p -

1 degrees of freedom'when the variances are equal, the

appropriate critical value for the test is 9.21 for a

significance level of 0.01. Since B = 7.254 is less than 9.21,

conclude that the variances are not different.

\

583
4 • TRANSFORMATIONS OF THE DATA

4.1 When the assumptions of normality and/or homogeneity of

variance are not met, transformations of the data may remedy the

problem, so that the data can be analyzed by parametric

procedures, rather than nonparametric technique such as Steel's

Many-one Rank Test or Wilcoxon's Rank Sum Test. Examples of

transformations include log, square root, arc sine square root,

and reciprocals. After the data have been transformed, the

Shapiro-Wilkls and Bartlett's tests should be performed on the

transformed observations to determine whether the assumptions of

normality and/or homogeneity of variance are met.

4.2 ARC SINE SQUARE ROOT TRANSFORMATION (USEPA, 1993).

4.2.1 For data consisting of proportions from a binomial

(response/no responsej live/dead) response variable, the variance

within the ith treatment is proportional to Pi (1 - Pi), where Pi

is the expected proportion for the treatment. This clearly

violates the homogeneity of variance assumption required by

parametric procedures such as Dunnett's Procedure or the t test

with Bonferroni1s adjustment, since the existence of a treatment

effect implies different values of Pi for different treatments,

i. Also, when the observed proportions are based on small

samples, or when Pi is close to zero or one, the normality

assumption may be invalid. The arc sine square root (arc sine

) transformation is commonly used for such data to

stabilize the variance and satisfy the normality requirement.

4.2.2 Arc sine transformation consists of determining the angle

(in radians) represented by a sine value.. In the case of arc

sine square root transformation of mortality data, the organism

response proportion (proportion dead or affected; proportion

surviving) is taken as the sine value, the square root of the

sine value is determined, and the angle (in radians) for the

square root of the sine value is determined. Whenever the

response proportion is 0 or l,a special modification of the arc

sine square root transformation must be ~sed (Bartlett, 1937).

An explanation of the arc sine square root transformation and the

modification is provided below.

4.2.3 Calculate the response proportion (RP) at each effluent

concentration, in this case proportion surviving where:

584
RP = (number of surviving or unaffected organif~ms)/ (number

exposed) . I

. Example: If 12 of 20 animals in a given treatment .replicate

survive:

RP = 12/20

= 0.60

4.2.4 Transform each RP to its arc sine square root, as follows:

4.2.4.1 For RPs greater than zero or less than one:

Angle (radians) = JRP

Example: If RP = 0.60:

Angle arc sine

arc sine 0.7746

= 0.8861 radians

4.2.4.2 Modification of the arc sine square root when RP = O.

Angle (in radians) = arc sine VI/4N

Where: N = Number of animals/treatment replicate

Example: If 20 animals are used:

Angle = arc sine VI/SO

= arc sine 0.1118

= 0.1120 radians

585
4.2.4.3 Modification of the arc'sine square root when RP = 0

Angle = 1.5708 radians - (radians for RP = 0)

Example: Using above value:

Angle = 1.5708 - 0.1120

= 1.4588 , radians

586
.APPEND:IX C

DUNNETT'S PROCEDURE

L "l .. _'. '.,'

1 . MANUAL CALCULAT:IONS

1.1 Dunnett's Procedure (Dunnett, 1955; Dunnett, 1964) is used

to compare each concentration mean with the cont:rol mean to

decide if any of the concent~ations differ from the control.

This test has an overall error rate of alpha,which accounts for

the multiple comparisons with the control. It is based on the

assumptions that the observations are independent and normally

distributed and that the variance of the observations is

homogeneous across all concentrations and control. (See Appendix

B for a discussion on validating the assumptions). Dunnett's

Procedure uses a pooled estimate of the varianCE!, which is equal

to the error value calculated in an analysis of variance.

Dunnett's Procedure can only be used when the same number of

replicate test vessels have been used at each concentration and

the control. When this condition is not met, the t test with

Bonferroni's adjustment is used (see Appendix D) .

1.2 The data used in this example are growth da.ta from a Mysid

Survival and Growth Test, and are the same data used in Appendix

B. These data are listed in Table C.1.

TABLE C.1. MYSID, HOLMESIMYSIS COSTATA, GROWTH DATA

Conc.entratioll (%)

Replicate Control 1.80 3.20 5.60

1 0.048 0.055 0.057 0.041

2 0.058 0.048 0.050 0.040·

3 0.047 0.042 0.046 0.041

4 0.058 0.041 0.043 0.043

5 0.051 0.052 0.045 0.040

Mean (Yd 0.052 0.048 0.048 0.041

Total (Td 0.262 0.238 0.241 0.205

i 1 2 3 4

587
1.3 One way to obtain an estimate of the pooled variance is to

construct an ANOVA table including all sums of squares, as

described in Table C.2:

TABLE C. 2 • 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)

2

Sa SSB/ (p-1)

2

Sw SSW/ (N-p)

Where: p = number of effluent concentrations including

the control:

N = the total sample size; 1

ni = the number of replicates for concentration "i"

SST=L Yl/-G2/N

J.j

SSB=LTJ. 2/nJ.-G2 /N 1

SSW=SST-SSB

Total Sum of Squares

Between Sum of Squares

Within Sum·of Squares

G = the grand total of all sample

p

observations,· G=£.JT1

the total of the replicate measurements for

concentration i

588
N = the total sample size;

ni the number of ~eplicates for concentration i

Yij = the jth observation for concentration i

1.4 For the data in this example:

n1 = n 2 = n 3 = n4 = n5 5

N = 20

T1 = Yll + Y12 + Y13 + Y14 + Y15

T2 = Y21 + Y22 + Y23 + Y24 + 'Y25 =

T3 = Y31 + Y32 + Y33 + Y34 + Y35

T4 = Y41 + Y42 + Y43 + Y44 + Y45 ==

G = T1 + T2 + T3 + T4 = 0.946

p

SSB = LT:lni -G2/N

i-I

_1_(0.225) - (0.946)2.

5 20

p n i

SST = L'L,Yi>G2/N

, i-lj-l

= 0.0455 - (0.946)2

20

0.262

0.238

0.24]:

0.20=;

0.000254

0.000754

SSW = SST-SSB = 0.000754 - 0.000254 = 0.000500

S~ SSB/(p-1) = 0.000254/(4-1) = 0.0000847

S~ SSW/(N-p) = 0.000500/(20-4) =0.0000313

1.5 Summarize these data in the ANOVA table, as shown in Table

C.3:

589
TABLE C.3. COMPLETED ANOVA TABLE FOR D~TTIS PROCEDURE EXAMPLE

Source df Sum of Squares Mean Square (MS)

(S8) (SS/df)

Between 3 0.000254 0.0000847

Within J.6 0.000500 0.00003J.3

Total J.9 0.000754

1.6 To perform the individual comparisons, calculate the t

statistic for each concentration and control combination, as

follows:

t '=

i -s-.,-;-"'V;=(=l=/n=l=)=+==(=l/=n=i=)

Where: Y1 = mean for the control

Yi = mean for each concentratioon i

Sw = square root of the within mean square

n1 = number of replicates in the control

ni = number of replicates for concentration i.

1.7 Table C.4 includes the calculated t values for each

concentration and control combination.

590
TABLE C. 4 • CALCULATED t VALUES

Concentration (ppb)

1.80

3.20

5.60

i t i

2 1.131

,

3 1.131

4 3.111

1.8 Since the purpose of the test is only to detect a decrease

in growth from the control, a one-sided test is appropriate. The

critical value for theone-sided comparison is read from the

table of Dunnett's "t" values (Table C.5; this table assumes an

equal number of replicates in all treatment concentrations and

the control). For this set of data, with an overall alpha,level

of 0.05, 16 degrees of freedom and three concentrations excluding

the control, the critical value is 2.23. " The mean weight for

concentration "i" is considered significantly less than the mean

weight for the control if t i is greater than the critical value.

Comparing each of the calculated values in Table C.4 with the

critical value, a significant decrease in growth from the control

is detected in the 5.60% concentration. Therefore, the" NOEC and

the LOEC for growth,are 3.20% and 5.60%, respectively.

591
TABLE C.S. DUNNETTIS "TII VALUES (Miller, 1981)

U1

U>

I. , -- --- - ..._..

(ODe-tailed) d"J<

a ••05 a.O.l

I

I 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

I

I

5 2.02 2.44 2.68 2.85 2.98 3.08 3.16 3.24 3.30 3.37 3.90 4.21 4.43 4.60 4.73 4.85 4.94 5.03

6 1.94 2.34 2.56 2.71 2.83 2.92 3.00 3.07 3.12 3.14 3.61 3.88 4.07 4.21 4.33 4.0 4.51 4.59

7 1.89 2.27 2.48 2.62 2.73 2.82 2.89 2.95 3.01 3.00 3.42 3.56 3.83 3.96 4.07 4.15 4.23 4.30 I

8 1.86 2.22 2.42 2.55 2.66 2.74 2.81 2.87 2.92 2.90 3.29 3.51 3.67 3.79 3.88 3.96 4.03 4.09

I

9 1.83 2.18 2.37 2.50 2.60 2.68 2.75 2.81 2.86 2.82 3.19 3.40 3.55 3.66 3.75 3.82 3.89 3.94

10 1.81 2.15 2.34 2.47 2.56 2.64 2.70 2.76 2.81 2.76 3.11 3.31 3.45 3.56 3.64 3.71 3.78 3.83 i

11 1.80 2.13 2.31 2.44 2.53 2.60 2.67 2.72 2.77 2.72 3.06 3.25" 3.38 3.48 3.56 3.63 3.69 3.74

12 1.78 2.11 2.29 2.41 2.50 2.58 2.64 2.69 2.74 2.68 3.01 3.19 3.32 3.42 3.50 3.56 3.62 3.67

13 1.77 2.09 2.27 2.39 2.48 2.55 _2.61 "2.66 2.71 2.65 2.97 3.15 3.27 3.37 3.44 3.91 3.56 3.61

14 1.76 2.08 2.25 2.37 2.46 2.53 2.59 2.64 2.69 2.62 2.94 3.11 3.23 3.32 3.40 3.46 3.51 3.56

15 1.75 2.07 2.24 2.36 2.44 2.51 2.57 2.62 2.67 2.60 2.91 3.08 3.20 3.29 3.36 3.42 3.47 3.52

16 1. 75 2.06 2.23 2.34 2.43 2.50 2.56 2.61 2.65 2.58 2.88 3.05 3.17 3.26 3.33 3.39 3.44 3.48

17 1. 74 2.05 2.22 2.33 2.42 2.49 2.54 2.59 2.64 2.57 2.86 3.03 3.14 3.23 3.30 3.36 3.41 3.45

18 1.73 2.04 2.21 2.32 2.41 2.48 2.53 2.58 2.62 2.55 2.84 3.01 3.12 3.21 3.27 3.33 3.38 3.42

19 1. 73 2.03 2.20 2.31 2.40 2.47 2.52 2.57 2.61 2.54 2.83 2.99 3.10 3.18 3.25 3.31 3.36 3.40

20 1.72 2.03 2.19 2.30 2.39 2.46 2.51 2.56 2.60 2.53 2.81 2.97 3.08 3.17 3.23 3.29 3.34 3.38

24 1.71 2.01 2.17 2.28 2.36 2.43 2.48 2.53 2.57 2.49 2.77 2.92 3.03 3.11 3.17 3.22" 3.27 3.31 . 30 1. 70 1.99 2.15 2.25 2.~3 2.40 2.45 2.50 2.54 2.46 2.72 2.87 2.97 3.05 3.11- 3.16 3.21 3.24

40 1.68 1.97 2.13 2.23 2.31 2.37 2.42 2.47 2.51 2.42 2.68 2.82 2.92 2.99 3.05 3.10 3.14 3.18

60 1.67 1.95 2.10 2.21 2.28 2.35 2.39 2.44 2.48 2.39 2.64 2.78 2.87 2.94 3.00 3.04 3.08 3.12

120 1.66 1.93 2.08 2.18 2.26 2.32 2.3'7 2.41 2.45 2.36 2.60 2.73 2.82 2.90 2.94 2.99 3.03 3.06
1.9 To quantify the sensitivity of the test, the minimum

significant differ~nce :(MSD) m~y be' calculated,; The formula is

as ,follows:

MSD=d S"V (lln1 ) + (lin),

Where:d critical value ,for the Dunnett's Procedure

Sw = the square root of the within mean square

n the number of replicates at each concentration"

assuming an equal number of replicates at all

treatment concentrations

n 1 number of replicates in the control

For example:

MSD = 2.23(O.00559)V,(l/S)+(1/5)

2.23 (0.00559) (0.632)

.:' '

= 0.00788

1.10 Therefore, for this set of data, the minitnum difference

between the control'mean and a concentration mean that can be

detected as statistically significant is .0.007813' mg. This

represents a 15.2% reduction in mean weight from the control.

1.11 If the data have not been transformed:, the MSD (and the'

percent decrease from the control 'mean that it represents) can be

reported as is.

1.11.1 In the case where the data have been transformed, 'the,MsD

would be in transformed units. In this case carry out the

following conversion to determine the MSD in unitraI;1sformed units.

1.11.2 Subtract the MSD from the transformed cohtrol mean. Call

this difference D. 'Next, obtain untransformed values for the'

control, mean and the difference, D. Finally, c~~~pute the

untransformed MSD as follows:

I·

59~
MSDu = controlu - Du

Where: MSDu = the minimum significant difference for

untransformed data

Controlu = the untransformed control mean

Du = the untransformed difference

1.11.3 Calculate the percent reduction from the control that

MSDu represents as:

Percent Reduction

Controlu

X 100

1.11.3.1 An example of a conversion of the MSD to untransformed

units, when the arc sine square root transformation was used on

the data, follows.

Step 1.

Step 2.

Subtract the MSD from the transformed control mean.

As an example, assume the data in Table C.1 were

transformed by the arc sine square root

transformation. Thus:

0.052 - 0.00788 = 0.04412

Obtain untransformed values for the control mean

(0.052) and the difference (0.04412) obtained in

Step 1, above.

I

[ Sine (0 . 052) ] 2

[ Sine (0.04412)]2

0.00270

0.00195

Step 3. The untransformed MSD (MSDu ) is determined by

subtracting the untransformed values obtained in

Step 2.

MSDu = 0.00270 - 0.00195 = 0.00075·

In this case, the MSD would represent a 1.4% decrease in survival

from the control [( 0.00075/0.052) (100)] .

594
2 . COMPUTER CALCULATIONS

2.1 This computer program incorporates two analyses: an

analysis of variance (ANOVA)" and a multiple comparison of

treatment means with the control me~n '(Dunnett's Procedure). The

ANOVA is used to obtain the err~r value: Dunnett's Procedure

indicates which toxicant concentration means (if any) are

statistically different ,from the control mean at the 5% level of

significance. The program also provides 'the minimum difference

between the control and treatment means that could be detected as

statistically significant, and tests the validity of the

homogeneity of variance assumption by Bartlett's Test. The

multiple comparison is performed based on procedures described by

Dunnett (1955). I

2.2 The source code for the Dunnett's program is structured into

a series of subroutines, controlled by a driver routine. Each

subroutine has a specific function in the Dunne,tt' s Pro.cedure,

such as data input, transforming the data, testing for equality

of variances, computing p values, and calculating the one-way

analysis of variance. . .

2.3 The program compares ,up to seven toxicant concentrations

against the control, and can accommodate up to 50 replicates per

concentration.

2.4 If the number of replicates at each toxicant concentration

and control are not equal, a t test with the Bonferroni

adjustment is performed instead of Dunnett's Procedure (see

Appendix D) .

2.5 The program was written in IBM-PC FORTRAN by Computer

Sciences Corporation, 26 W. Martin Luther King Drive, Cincinnati,

Oll 45268. A compiled version of the program can be obtained from

EMSL-Cincinnati by sending a diskette with a written request.

2.6 DATA INPUT AND OUTPUT

2.6.1 The mysidgrowth data from Table C.1 are used to

illustrate the data input and output for this program..

2.6.2 Data ~nput

595
2.6.2.1 When the program is entered, the user is asked to select

the type of data to be analyzed:

1. Response proportions, like survival or fertilization

proportions data. .

2. Counts and measurements, like offspring counts, cystocarp

and algal cell counts, weights, chlorophyll measurements

or turbidity measurements.

2.6.2.2 After the type of analysis for the data is chosen, the

user has the following options:

1. Create a data file

2. Edit a data file

3. Perform analysis on existing data set

4. Stop

2.6.2.3 When Option 1 (Create a data file) is selected for

response proportions, the program prompts the user for ·the

following information:

1. Number of concentrations, including control

2. For each concentration and replicate:

- number of organisms exposed per replicate

- number of organisms responding per replicate (organisms

surviving, eggs fertilized, etc.)

2.6.2.4 After the data have been entered, the user may save the

file on a disk, and the program returns to the main menu (see

below) .

2.6.2.5 Sample data input is shown in Figure C.1.

2.6.3. Program Output

2.6.3.1 When Option 3 (perform analysis on existing data set) is

selected from the menu, the user is asked to select the

transformation desired, and indicate whe~her they expect the

means of the test groups to be less or greater than the mean for

the control group (see Figure C.2) .

596
EMSL Cincinnati Dunnett Software

Version 1.5

What type of data do you wish to analyze?

1) response proportions

(like survival data or fertility proportionda1:.a)

Note: The program calculates a proportion after. prompting for

number of exposed organisms and number of responding

organisms.

2) counts and measurements

(like offspring counts, cystocarps and algal cl~ll counts,

weight;s, chlorophyll measurements, or turbidity measurements)

Enter "1", "2", (or "g" to quit program): 2

Title? Appendix C, Dunnett's Procedure Example - Mysid Data

Output to printer or disk file ? P

1) Create a data file

2) Edit a data file

3) Analyze an existing data set

4) Stop

Your choice ? 1

Number of concentrations, including control ? 4

Number of observations for cone. 1 (the control) ? 5

Enter the data for cone. 1 (the control) one observation at a time.

NO.1? 0.048

NO.2? 0.058

NO.3? 0.047

NO.4? 0.058

NO.5? 0.051

597
Enter the data for conc. 2 one observation at a t-ime.

NO.1? 0.055

NO.2? 0.048

NO.3? 0.042

NO.4? 0.041

NO.5? 0.052

Number of observations for conc. 3? 5

Enter the data for conc. 3 one observation at a time.

NO.1? 0.057

NO.2? 0.050

NO.3? 0.046

NO.4? 0.043

NO.5? 0.045

Number of observations for conc. 4? 5

Enter the data for conc. 4 one observation at a time. _-

NO.1? 0.041

NO.2? 0.040

NO.3? 0.041

NO.4? 0.043

NO.5? 0.040

Do You wish to save the data on disk ? Y

Disk file for output? c:\mysid.dat

598
EMSL Cincinnati Dunnett Software

Version J..5

J.) Create a data file

2) Edit a data file

3) Analyze an existing data set

4) Stop

Your choice ? 3

File name? c:\mysid.dat

Available Transformations

J.) no transform

2) square root

3) 10gJ.0

Your choice ? 1.

Dunnett's test as implemented in this program is

a one-sided test. You must specify the direction

the test is to be run; that is, do you expect the

means for the test concentrations to be less than

or greater than the mean for the control

concentration.

Direction for Dunnetts test L=less than, G=greater truin ? L

Figure C.2. Example of Choosing Option 3 from the Main I~enu of the Dunnett

Program.

599
2.6.3.2 Summary statistics (Figure C.3) for the raw and

transformed data, if applicable, the ANOVA table, results of

Bartlett's Test, the results of the multiple comparison

procedure, and the minimum detectable difference are included in

the program output.

600
EMSL Cincinnati Dunnett Software

Version 1.5

Appendix C, Dunnett's Procedure Example - Mysid Data

Summary Statistics and ANOVA

Transformation None

Conc. n Mean s.d.

1 control 5 .0524 .0053

2 5 .0476 .0061

3 5_ .0482 .0055

4* 5 .0410· .0012

cv%

10.2

12. El

11. ~i

3.0

*) the mean for this conc. is significantly less· than

the control mean at alpha = 0.05 (l-sided) by Dunnett's test

Minimum detectable difference for Dunnett's test -.006974

This difference corres:ponds to -13.31 percent of cOntrol

Between concentrations

sum of squares

Error mean square =

.000333 with 3 degrees of freedom.

.000024 with 16 degrees of freedom.

Bartlett's test p-value for equality of variances =

Do you wish to restart the program ?

.060

Figure C.3. Example of Program Output for the Dunnett's Program Using the

Data in Table C.1.

601
APPENDIX D

t TEST WITH BONFERRONI'S ADJUSTMENT

1. The t test with Bonferroni's adjustment is used as. an

alternative to Dunnett's Procedure when the number of replicates

is not the same for all concentrations. This test sets an upper,

bound of alpha on the overall error ratei in contrast to

Dunnett's Procedure, for which the overall error rate is fixed at

alpha. Thus, Dunnett's, Procedure is a more powerful test.

2. The t test with Bonferroni's adjustment is .based on the same

assumptions of normality of distribution and homogeneity of

variance as Dunnett's Procedure (See Appendix B for testing these

assumptions), and, like Dunnett's Procedure, uses a pooled

estimate of the variance, which is equal to the error value

calculated in an analysis of variance.

3. An example of the use of the t test with Bonferroni's

adjustment is provided below. The data used in the example are a

set of red abalone growth data. Because there are only four

replicates in the highest concentration, Dunnett's Procedure

cannot be used. The length data are presenteq in Table D.l.

TABLE D.1. GIANT KELP, MACROCYSTIS PYRIFERA, GROWTH DATA

Copper Concentration ({J-g/L)

Rep Control 5.60 10.0 18.0 32.0 56.0 100.0 180.0

J. J.9.58 J.8.26 13.31 18.59 12.54 11.44 7.92 6.49

2 J.8.75 16.25 18.92 12.88 10.67 11. 88 7.59 7.25

3 J.9.J.4 16.39 J.5.62 16.28 15.95 11.88 8.25

4 J.6.50 18.70 14.30 15.38 12.54 11. 00 9.1.3 7.63

5 J.7.93 15.62 15.29 19.75 11.66 11.55 8.80 8.13

Yj. J.8.38 17.04 15.49 16.58 12.67 11.55 8.34 7.38

S~ 1.473 1.827 4.498 7.327 3.953 0.133 0.396 0.478

i 1 2 3 4 5 6' 7 8

602
3.1 One way to obtain an estimate of the pooled variance is to

construct an ANOVA table including all sums of f;quares, as

described in Table D.2:

TABLE D. 2. ANOVA TABLE

Source df Sum of Squares Mean Square(MS)

(SS) (SS/df)

2

Between p - 1 SSB S8 = SSB/(p-l)

2

Within N- P SSW Sw = SSW/ (N-p)

Where: p = number of effluent concentrations including

the control

N = the total sample size; i

ni the number of replicates for ,concentration 'i

SST=LYj,/-G 2/N Total Sum of Squares

j,j

SSB=LTj,2/n j,-G 2/N Between Sum of Squares i

SS~SS~SSB Within Sum of Squares

Where: G = The grand total of all sample

p

observations; G=LT!

603
Yij =

The total of the replicate

measurements for concentration i

The jth observation for

concentration i

3.2 For the data in this example:

n l :: n 2 = n 3 = n 4 = n s = n6 = n 7 = 5; n s 4

N = 39

Tl = Yll + Y12 + Y13 + Y14 + Y1S = 91.90

T2 = Y21 + Y22 + Y23 + Y24 + Y2S = 85.22

T3 = Y31 + Y32 + Y33 + Y34 + Y3S = 77.44

T4 = Y41 + Y42 + Y43 + Y44 + Y4S = 82.88

Ts = YS1 + YS2 + 1S3 + YS4 + Yss = 63.36

T6 = Y61 + Y62 + Y63 + Y64 + Y6S = 57.75

T7 = Yn + Yn + Yn + Y74 + Y7S = 41.69

Ts :: YS1 + YS2 + YS3 + YS4 = 29.50

G = T1 + T2 + T3 + T4 + Ts + T6 + T7 + Ts = 529.74

p

SSB", L,T:ln1 -G2 IN

1-1

= 7749.905 - (529.74)2

39

P R1

SST", L,L,Y:j -G2IN

1-1j-1

= 7829.764 - (529.74)2

39

554.406

634.265

SSW. SST-SSB = 634.265 - 554.406 = 79.859

S~ = SSB/(p-1) = 554.406/(8-1) 79.201

S~ = SSW/(N-p) = 79.859/(39-8) = 2.576

604
3.3 Summarize these calculations in the ANOVA table (Table D.3):

TABLE D.3. COMPLETED ANOVA TABLE FOR THE t TEST WITH BONFERRONI'S

ADJUSTMENT EXAMPLE

Source df Sum of Squares Mean Square (MS)

(SS) (SS/df)

Between 7 554.406 79.201

Within 31 79.859 2.576

Total 38 634.265

3.4 To perform the individual comparisons, calculate the t

statistic for each concentration and control combination, as

follows:

t = i

Where: Yi ::::: mean for concentration i.

Y1 ::::: mean for the control

Sw ::::: square root of the within mean square

n 1 = number of replicates in the con1:'rol.

n i = number of replicates for concen1:ration· i.

3.5 Table D.4 includes the calculated t values for each

concentration and control combination.

605
TABLE 0.4. CALCULATED t VALUES

Concentration (pg/L)

5.6

10.0

18.0

32.0

56.0

100.0

180.0

i

2

3

4

5

6

7

8

1.320

2.847

1.773

5.625

6.728

9.891

10.217

3.6 since the purpose of this test is to detect a'significant

reduction in mean length, a one-sided test is appropriate. The

critical value for this one~sided test is found in Table D.5.

For an overall alpha level of 0.05, 31 degrees of freedom for

error and seven concentrations (excluding the control) the

approximate critical value is 2.597. The mean length for

concentration lIi ll is considered significantly less than the mean

length for the control if t i is greater than the critical value.

Comparing each of the calculated t values in Table D.4 with the

critical value, the 10.0 ~g/L, 32 ~g/L, 56.0 ~g/L, 100.0 ~g/L,

180.0 ~g/L concentrations have significantly lower mean length

than the control. Because the 10.0 ~g/L concentration shows

signigicantly lower mean length that the control while the higher

18.0 ~g/L concentration does ,not, these test results are

considered to have an anomalous dose-res~onse relationship and it

is recommended that the test be repeated. If an NOEC and LOEC

must be determined for this test, the lowest concentration with

significant growth impairment versus the control is considered to

the LOEC for growth. Thus, for this test, the NOEC and LOEC

would be 5.6 ~g/L and 10.0 ~g/L, respectively.

606
TABLE 0.5. CRITICAL VALUES FOR "t" FOR THE t TEST WITH BONFERRONI'S ADJUSTMENT P = 0.05

CRITICAL LEVEL, ONE TAILED

d.f. K= 1 K= 2 K= 3 K = 4 K=5 K=6 K = 7 K = 8 K = 9 K= 10

1 6.314 12.707 19.002 25.452 31.821 38.189 44.556 50.924 57.290 63.657

2 2.920 4.303 5.340 6.206 6.965 7.649 8.277 8.861 9.408 9.925

3 2.354 3.183 3:741 4.177 4.541 - 4~857 5.138 5.392 5.626 5.841

4 2.132 2.777 3.187 3.496 3.747 3.961 4.148 4.315 4.466 4.605

5 2.016 2.571 . 2.912 3.164 3.365 3.535 3.681 3.811 3.927 4.033

m 6 1.944 2.447 2.750 2.969 3.143 3.288 3.412 3.522 3.619 3.708

a 7 1.895 2.365 2.642 2.842 2.998 3.128 3.239 3.336 3.422 3.500 -..J

8 1.860 . 2.307 2.:567 2.752 2.897. 3.016 3.118 3.206 3.285 3.356

9 1.834 2.263 2.510 2.686 2.822 . 2.934 3.029 3.111 3.185 3.250

10 1.813 2.229 2.406 2.634 2.764 2.871 2.961 3.039 3.108 3.170

11 1·.796 2.201 2.432 2.594 2.719 2.821 2.907 2.981 3.047 3.106

12 1.783 2.179 2.404 2.561 2.681 2.778 2.863 . 2.935 2.998 3.055

13 1.771 2.161 2.380 ·2.533 2.651 2.746 2.827 2.897 2.958 3.013

14 1.762 2.145 2.360 2,510 2.625 2.718 2.797 2.864 2.924 2.977

15·· 1.754 2: 132-· 2.343 . 2.490 . 2. 603 ,- 2. 694 . 2.771 2.837 . 2.895- 2.947

16 1.746 2.120 2.329 2.473 \ 2.584 2.674 2.749 2.814 2.871 2:.921

17 1.740 2.110 2.-316 .2.459 2.567 2.655 2.729 2.793 2.849 2.899

18 1.735 2.101 2.305 .2.446 . 2.553 2.640 2.712 2.775 2.830 2.879

19 1.730 2.094 2.295 2.434 2.540 2.626 2.697 2.759 2.813 2.861

20 1.725 2:086 2.206 -2.424 . '2.528 . 2.613 2.684 2.745 2.798 2.846

21 1. 721 2.080 2.278 2.414 2.518 2.602 2.672 2.732 2.785 2.832
TABLE 0.5. CRITICAL VALUES FOR "t" FOR THE t TEST WITH BONFERRONI'S ADJUSTMENT

P = 0.05 CRITICAL LEVEL, ONE TAILED (CONTINUED)

df K= 1 K= 2 K::: 3 K= 4 K:: 5 K:: 6 K= 7 K= 8 K= 9 K= 10

- -- 29 1.700 2.046 2.235 2.364 2.463 2.541 2.607 2.664 2.713 2.757

30 1.698 2.043 2.231 2.360 2.458 2.536 2.602 2.658 2.707 2.750

31 1.696 2.040 2.228 2.356 2.453 2.531 2.597 2.652 2.701 2.745

32 1.694 2.037 2.224 2.352 2.449 .. 2.527 2.592 2.647 2.696 2.739

33 1.693 2.035 2.221 2.349 2.445 2.523 2.587 2.643 2.691 2.734

34 1.691 2.033 2.219 2.346 2.442 2.519 2.583 2.638 2.686 2.729

35 1.690 2.031 2.216 2.342 2.438 2.515 2.579 2.634 2.682 2.724

36 1.689 2.029 2.213 2.340 2.435 2.512 2.575 2.630 2.678 2.720

37 1.688 2.027 2.211 2.337 2.432 2.508 2.572 2.626 2.674 2.716

0 38 1.686 2.025 2.209 2.334 2.429 2.505 2.568 2.623 2.670 2.712 (X)

39 1.685 2.023 2.207 2.332 2.426 2.502 2.565 2.619 2.667 . 2.708

40 1.684 2.022 2.205 2.329 2.424 2.499 2.562 2.616 2.663 2.705

50 1.676 2.009 2.189 2.311 2.404 2.478 2.539 2.592 2.638 2.678

60 1.671 2:001 2.179 2·.300 2.391 2.463 2.324 2.576 2.621 2.661

70 1.667 1.995 2.171 2.291 2.381 2.453 2.513 2.564 2.609 2.648

80 1.665 1.991 2.166 2.285 2.374 2.446 2.505 2.556 2.600 2.639

90 1.662 1.987 2.162 2.280 2.369 2.440 2.499 2.549 2.593 2.632

100 1.661 1.984 2.158 2.276 2.365 2.435 2.494 2.544 2.588 2.626

110 1.659 1.982 2.156 2.273 2.361 2.432 2.490 2.540 2.583 2.622

120 1.658 1.980 2.153 2.270 . 2.358 2.429 2.487 2.536 2.580 2.618

Infinite 1.645 . 1. 960 2.12.9 2.242 2.327 2.394 2.450 2.498 2.540 2.576

d.f. = Degrees of freedom for MSE (Mean Square Error) from ANOVA.
APPENDIX E

STEELIS MANY-ONE RANK TEST

1. Steel's Many-one Rank Test is.a nonparametric test for

comparing treatments witq a control. This test is an alternative

to Dunnett's Procedure, and may be applied to data when the

normality assumption has not been met. Steel'S ITeSt re'quires

equal variances across the treatments and the ccintrol, but it is

thought to be fairly insensitive to deviations f:rom this

condition (Steel, 1959). The tables for Steel's Test require an

equal number of replicates at each concentratior.t. If this is not

the case, use Wilcoxon's Rank Sum Test, with Bonferroni's

adjustment (See Appendix F) .

2. For an analysis using $teel's Test, for each control and

concentration combination, combine the data and arrange the

observations in order of size from smallest to largest. Assign

the ranks to the ordered observations (1 to the 'smallest, 2 to

the next'smallest, etc.). If ties occur. in the ranking, assign

the average rank to the~observation. (Extensive ties would

invalidate this procedure). The sum of the ranks withi.n each

concentration is then calculated. To determine·if the response

in a concentration is signi~icantly less than the. response i? the

control, the rank sum for each concentration is compared to the

significant values of rank sums given later in the section. In

this table, k equals the number of treatments exclu.ding the

control and n equals the number of replicates for each

concentration and the control.

,

3. An example of the use of this test is provided below. The

test employs embryo-larval development data fronl a bivalve 48-

hour chronic test. The data are listed in Table E.1.

4. For each control and concentration·combinati.on, combine the

data and arrange the observations in order of size from smalle~t

to largest. Assign the ranks (1, 2, 3, ... , 8) to the 'ordered

observations (1 to the smallest, 2 to the next smallest, etc.).

If ties occur in the ranking, assign the average.rank to ·each

tied observation.

5. An example of assigning ranks to the combined data for the

control and 0.13 Jlg/L copper concentration is gi.ven in Table,E.2.

609
This ranking procedure is repeated for each control and

concentration combination. The com:plete set of rankings is

listed in Table B.3. The ranks are then summed for each toxicant

concentration, as shown in Table, B.4.

6. For this set of data, determine if the development in any of

the effluent concentrations is significantly lower ,than the

development of the control organisms. If this occurs, ,the rank

sum at that concentration would be significantly lower than the

rank sum of the control. Thus, compare the rank sums for the

development at each of the various effluent concentrations with

some IIminimumII or critical rank sum, at or below which the

survival would be considered to be significantly lower than the

control. At a probability level of 0.05, the critical rank sum

in a test with five concentrations and four replicates per

concentration, is 10 (see Table F.4) .

7. Since the rank sums for the 0.50 ~g/L and 1.00 ~g/L

concentration levels are equal to the critical value, the

proportions of normal development in those concentrations are

considered significantly less than that in the control. Since no

other rank sum is less than or equal to the critical value, no

other concentration has a significantly lower proportion normal

than the control. Because the 0.50 ~g/L concentration shows

signigicantly lower normal development than the control while the

higher 2.00 ~g/L concentration does not, these test results are

considered to have an anomalous dose-response relationship and it

is recommended that the test be repeated. If an NOBC and LOBC

must be determined for this test, the lowest concent~ation with

significant impairment versus the control is considered to the

LOBe for growth. Thus, for this test, the NOBC and LOBC would be

0.25 ~g/L and 0.50 ~g/L, respectively.

610
TABLE E.1. BIVALVE EMBRYO-LARVAL DEVELOPMENT DATA

Copper Concentration (ug!U

Replicate Control 0.13 0.25 0.50 1.00 . 2.00

A 1. 00 0.96 0.92 0.91 0.88 LOO

RAW B 0.96 0.97 0.95 0.93 0.83 0.67

C 1.00· 1. 00 0.90 0.88 0.88 0.75

D 0.97 0.96 0.96 0.93 0.82 0.60

ARC SINE A 1.571 1.369 1.284 1.266 I 1.217 1.571

SQUARE ROOT B 1.369 1.397 1.345 1.303 I 1.146 0.959

TRANSFORMED C 1.571 1.571 1.249 1.217 1.217 1.047

D 1.397 1.369 1.369 1.303 1.133 0.886

Mean (Yi ) 1.477 1.427 1.312 1.272 1.178 1.116

S?, 0.01191 0.00945 0.00303 0.00166 0.00203 0.09644

i 1 2 3 4 5 6

611
TABLE E.2. ASSIGNING RANKS TO THE CONTROL AND 0.13 pg/L CONCENTRATION

LEVEL FOR STEEL'S MANY-ONE RANK TEST

Transformed

Proportion

Rank Normal Concentration

2 1.369 .0.13pg/L

2 1.369 0.13 pg/L

2 1.369 Control

4.5 1.397 0.13 pg/L

4.5 1.397 Control

7 1.571 0.13 pg/L

7 1.571 Control·

7 1.571 Control

612
TABLE E.3. TABLE OF RANKS1

Copper Concentration (pg/l)

Replicate·

1

2

3

4

Control

1.571(7.7.5.7.5.7.5.7)

1.369(2.4.5.5.5.4)

1.571(7.7.5.7.5.7~5.71

1.397(4.5.6.6.6.5)

0.13

1. 369(2)

1.397(4.5)

1.571(7) ,

1. 369(2)i

0.25

1. 284(2)

1. 345(3)

1.249(1)

1.369(4.5)

Copper Concentrati on (P91/l) (Continued)

Replicate 0.50 1.00· 2.00

1 1. 266(2) 1.217(3.5) 1. 571(7)

2 1.303(3.5) 1.146(2) I 0.959(2)

3 1. 217 (1) 1.217(3.5); 1. 047 (3)

4 1.303(3.5) 1.133(1 ) 0.886(1)

,

lControl ranks are given in the order of the concentration with which

they were ranked.

TABLE E.4. RANK SUMS

Concentration

pg/L Copper)

0.13

0.25

0.50

1. 00

2.00

613

Rank Sum

15.5

10.5

10.0

10.0

13.0
TABLE E.5. SIGNIFICANT VALUES OF RANK SUMS: JOINT CONFIDENCE

COEFFICIENTS OF 0.95 (UPPER) and 0.99 (LOWER) FOR

ONE-SIDED ALTERNATIVES (Steel. 1959)

k = number of treatments (excluding control)

n 2 3 4 5 6 7 8 9

4 11 10 10 10 10

5 18 17 17 16 16 16 16 15

15 -- ,-,-

6 27 26 25 25 24 .24 24 23

23 22 21 21

7 37 36 35 35 34 34 33 33

32 31 30 30 29 29 29 29

8 49 48 47 46 46 45 45 44

43 42 41 40 40 40 39 39

9 63 62 61 60 59 59 58 58

56 55 54 53 52 52 51 51

10 79 77 76 75 74 74 73 72

71 69 68 67 66 66 65 65

11 97 95 93 92 91 90 90 89

87 85 84 83 82 81 81 80

12 116 114 112 III 110 109 108 108

105 103 102 100 99 99 98 98 .

13 138 135 133 132 130 129 129 128

125 123 121 120 119 118 117 117

14 161 158 155 154 153 152 151 150

147 144 142 141 140 139 138 137

15 186 182 180 178 177 176 175 174

170 167 165 164 162 161 160 1,60

16 213 209 206 204 203 201 200 199

196 192 190 188 187 186 185 184

17 241 237 234 232 231 229 228 227

223 219 217 215 213 212 211 210

18 272 267 264 262 260 259 257 256

252 248 245 243 241 240 239 238

19 304 299 296 294 292 290 288 287

614
APPENDIX F

WILCOXON RANK SUM TEST

1. wilcoxon I s Rank Sum Test is a nonparametric 'test I to be used

as an alternative to Steel's Many":one Rank Test when the number

of replicates are not the same at each concentration. A

Bonferroni's adjustment of the pairwise error rate for comparison

of each concentration versus the control is used to set an upper

bound of alpha on the overall error rate, in contrast to Steel's

Many-one Rank Test, for which the overall error rate is fixed at

alpha. Thus, Steel's Test is a more powerful test.

2. The use of this test may be illustrated .with devlopment data

from the red abalone test in Table F.1. The control group has

four replicates while each of the concentration levels has five

replicates. Since there is 100% abnormality in all replicates

for the 5.6% and 10.0% concentrations, they are not included in

the statistical analysis and are considered qualitative

abnormality effects.

3. For each concentration and control combinati(~n, combine the

data and arrang~ the values in order of size, from smallest to

largest. Assign ranks to the ordered observations. (a rank of 1

to the smallest, 2 t6 the next smallest~ "etc.) .If ties in rank

occur, assign the average rank to each tied observation.

4. An example of assigning ranks to the combineOldata for the

control and effluent concentration 0.56% is given in Table F.2.

This ranking procedure is repeated for each of the three

remaining control versus test concentration combinations. The

complete set of ranks is listed in Table "F.3. The ranks.are then

summed for each effluent concentration, as shown '.in Table· F. 4.

5. For this set of data, determine if the development in any of

the test concentrations is significantly lower than the

development in the control. If this occurs,. the rank sum at that

concentration would be significantly lower than the rank sum of

the control. Thus, compare the rank sums for fecundity of each

of the various effluent concentrations with some ~'minimum" or

critical rank sum, at or below which the fecundity would be

considered to be significantly lower than the control. At a "

615
probability level of 0.05, the critical rank in a test with four

concentrations (excluding the control), four control replicates,

and five concentration replicates is 15 (see Table F.5, for K =

4) •

616
TABLE F.2. ASSIGNING RANKS TO THE CONTROL AND 0.56% CONCENTRATION LEVEL

FOR THE WILCOXON RANK SUM TEST WITH THE BONFERRONI ADJUSTMENT

Transformed

Proportion

Rank Normal Concentration

1 1.429 0.56 %

4 1.471 0.56 %

4 1.471 0.56 %

4 1.471 Control.

4 1.471 Control

4 1.471 C6ntrol

8 1.521 0.56 %

8 1.521 0.56 %

8 1.521 Control

TABLE F.3. TABLE OF RANKSl

Effluent Concentration (%)

Replicate Control 0.56 1. 00 1.80 3.20

1 1.471(4.3.5,5.5.7) 1.471(4) 1.471(3.5) 1.471(5.5) 0.674(1-)

2 1.471(4.3.5.5.5,7) 1.471(4) 1. 521(8) 1.471(5.5) 0.856(2)

3 1.471(4,3.5.5~5.7) 1.429(1) 1.471(3.5) 1.471(5.5) 0:896(3)

4 ·1.521(8.8.9.9) 1. 521(8) 1. 471(3 ..5) 1. 4,29(2) 0.93B(4)

5 1. 521(8) 1. 521(8) 1.397(1) 1.107(5)

lControl ranks are given in the order of the concentration with which

they were ranked.

617
6. Comparing the rank sums in Table F.4 to the appropriate

critical rank, the rank sum for the· 3 •. 20% concentration level is

equal to the critical value, so the propo~ti6n normal in that

concentration is considered significantly less than that in the

control. Since no other rank sum is less than or equal to the

critical value; no other concentration has a significantly lower

proportion normal than the control. Hence, the NOEC and the LOEC

are ~.80% and 3.20%, respectively.

TABLE F.4.

Concentration

(% Effluent)

0.56

1. 00

1.80

3.20

6~8

RANK SUMS·

Rank Sum

25.0

26.5

, 19.5

15.0
TABLE F.5.' CRITICAL VALUES fOR WILCOXON'S RANK SUM TIESTWITH .

BONFERRONI'S ADJUSTMENT OF ERROR RATE FOR COMPARISON OF "K"

TREATMENTS VERSU?'A CONTROL FIVE PERCENT CRITICAL LEVEL

(ONE-SIDED ALTERNATIVE: TREATMENT CONTROL~

K No. Replicates No. of Replicates' Per Effluent Concentration

in Control'

3 4 5 6' 7 8 9 10

1 3 6 10 16 23 30 ' '39 49 59

'4 6 11 17 24 ~2 41 51 62

5 7 12 19 26 34 44 54 66

6 8 13 20 28 36 :46 57 . 69

7 8 14 21 29 39 49 60 72

8 9 15 23 31 41 51 63 72

9 10 16 24 33 43 . 54 66 79

10 10 17 26 35 45 56 69 82

2 3 15 22 29 38 47 58

4 10 16 23 31 40 49 . 60

5 6 11 17 24 33 42 52 63

6 7 12 18 26 34 .44 55 66

7 7 13 20 27 36 46 57 69

8 8 14 21 29 38 .49 60 72

9 8 14 22 31 40 51 62 75

10 9 15 23 32 42 53 65 78

3 3 21 29 ,37 46 57

4 10 16 22 30 ,39 48 59

5 11 17 24 32 41 51 62

6 6 11 18 25 33 43 53 65

7. 7 12 19 26 35 45 56 68

8 7 13 20 28 37 47 58 70

9 7 13 21 29 39 49 61 ' 73

10 8 14 22 31 41 51 63 76

4 3 21 28 37 46 56

619
TABLE F.5. CRITICAL VALUES FOR WILCOXON'S RANK SUM TEST WITH BONFERRONI'S

ADJUSTMENT OF ERROR RATE FOR COMPARISON OF "K" TREATMENTS

VERSUS ACONTROL FIVE PERCENT CRITICAL LEVEL (ONE-SIDED

ALTERNATIVE: TREATMENT CONTROL) (CONTINUED)

K No. Replicates 'No. of Replicates Per Effluent Concentration

in Control

3 4 5 6 7 8 9 10

5 3 28 36 46 56

4 15 22 29 38 48 58

5 10 16 23 31 40 50 61

6 11 17 24 32 42 52 63

7 6 11 18 25 34 43 54 66

8 6 12 19 27 35 45 56 68

9 7 13 20 28 37 , 47 59 71

10 7 ' 13 21 29 39 49 61 74

6 3 28 36 45 56

4 15 21 ' 29 38 47 ' 58

5 10 16 22 30 39 49 60

6 11 16 24 32 41 51 63

7 6 11 17 25 33 43 54 65

8 6 12 18 26 35 45 56 68

9 6 12 19 27 37 47 58 70

10 7 13 20 29 38 49 60 73

7 3 36 45 56

4 21 29 37 47 58

5 15 22 30 39 49 60

6 10 16 23 32 41 51 62

7 -- 11 17 25 33 43 53 65

8 6 11 18 26 35 44 55 67

9 6 12 19 27 36 46 58 , 70

10 7 13 20 28 38 48 60 72

620
TABLE F.5. CRITICAL VALUES FOR WILCOXON'S RANK SUM TEST WITH BONFERRONI'S

ADJUSTMENT OF ERROR RATE FOR COMPARISON OF "K" TREATMENTS

VERSUS ACONTROL FIVE PERCENT CRITICAL LEVEL (ONE-SIDED

ALTERNATIVE: TREATMENT CONTROL) (CONTINUED)

K No. Replicates

in Control

No. of Replicates Per Effluent Concentration

3 4 5 6 7 : 8 9 10

9 3 45 55

4 2J 28 . 37 46 57

5 15 22 30 . 39 48 59

6 10 16 23 31 .40 50 62

7 10 17 24 33 42 52 64

8 11 18 25 34 - 44 55 . 66

9 6 11 18 26 35 ·46 57 69

10 6 12 19 28 37 ·47 59 71

10 3 - - 45 55

4 21 28 . 37 46 57

5 15 22 29 38 48 . 59

6 10 16 23 31 40 50 61

7 10 16 24 32 42 52 64

8 11 17 25 34 : 43 54 66

9 6 11 18 26 35 .45 56 68

10 6 12 19 27 37 47 58 71

621.
APPENDIX G

SINGLE CONCENTRATION TOXICITY TEST - COMPARISON OF CONTROL

WITH 100% EFFLUENT OR RECEIVING WATER OR COMPARISON OF

DILUTION AND BRINE CONTROLS

1. To statistically compare a control with one concentration,

such as 100% effluent or the instream waste concentration, a t

test is the recommended analysis. The t.test is based on the

assumptions that the observations are independent and normally

distributed and that the variances of the observations are equal

between the two groups.

2. Shapiro-Wilk's test may be used to test the normality

assumption (See Appendix B for details). For the two sample

case, the datasets must be tested for normality separately. If

either set of data does not meet the normality assumption, the

nonparametric test, Wilcoxon's Rank Sum Test, may be used to

analyze the data. An example of this test is given in ~ppendix

F. Since a control and one concentration are being compared, the

K = 1 section of Table F.S contains the needed critical values

for one-sided tests. An additional reference, such as Snedecor

and Cochran (1980) must be used to determine critical values for

two-sided tests, such as comparing brine ·and dilution controls.

3. The F test for equality of variances is used to test the

homogeneity of variance assumption. When conducting the F test,

the alternative hypothesis of interest is that the variances are

not equal.

4. To make the two-tailed F test at the .0.01 level of

significance, put the larger of the "two variances in the

numerator of F.

5. Compare F with the O. 005 level of a tabled F value with n 1 -

1 and n 2 - 1 degrees of freedom, where n 1 and n 2 are the. number of

replicates for each of the two groups.

622
6. A set of mysid growth data from'a single-concentration

effluent test will be used to illustrate the F t:est. The raw

data, mean and variance for the 'two controls' arElgiven in Table

G.1. The, data from each concentration meets the assumption of

normality. ' .

TABLE G.l. MYSID, HOLMESIMYSIS COSTATA, GROWTH DATA FROM A SINGLECONCENTATION EFFLUENT TEST

RAW

Mean CT;) .

S~

i

Replicate Control Effluent

A 0.048 0.041

B 0.058 0.0:33

C 0.047 0.044

D 0.055 0.040

E 0.051 0.0143

0.052 0.040

0.0000217 '0.0000187

1 2

7. Since the variability' of, the' control is grea.ter than the

variability of the effluent concentration, S2 for the control is

placed 'in the numerator of the F statistic and S? for the

effluent concentration control is placed in the denominator.

0.0000217

F = = 1.160

0.0000187

8. There are 5 replicates for the each groups, so the numerator

and denominator degrees of freedom, ni - i, are :both 4. For a

two-tailed test at the 0.01 level of significance, the critical F

value is obtained from a table of the F distribu.tion (8.nedecor

and Cochran, 1980). The critical F value for this test is 23.16.

Since 2.41 is not greater than 23.16, conclude that the variances

of the brine and dilution controls are homogeneous.

9 '. Equal Variance t Test.

623
9.1 To perform the t test, calculate the following test

statistic:

t •

Where: Y1 = mean for the control

Y2 = mean for the effluent concentration

S a

p

Sf = estimate of the variance for the control

S~ :: estimate of the variance for the effluent

concentration

n 1 = number of replicates for the control

n 2 = number of replicates for tl1e effluent

concentration

9.2 Since we are concerned here with a decrease in response from

the control, a one-tailed test is appropriate. Thus, we will

compare the calculated t with a critical t, where the critical t

is at the 5% level of significance with n1 + n2 - 2 degrees of

freedom. If the calculated t exceeds the critical t, the mean

responses are declared different.

9.3 When comparing brine and dilution controls, the concern is

for any difference between the two cont~ol groups, and'a

two-tailed test is appropriate. In that case, the calculated t

would be compared with a critical t, where the critical t is a

two-tailed value at the 5% level of significance with n 1 + n 2 - 2

degrees of freedom. If the absolute value of the calculated t

exceeds the critical t, the mean responses are declared

different.

624
9.4 Using the data from Table G.1 to illustratE~ the t ·test, the

calculation of t is as follows:

t =

0.052 - 0.040

Jf1.1

0.00449 - + -

5 5

= 4.226

5 = p

( 5 - 1) 0.0000217 + (5 - 1) 0.0000187 = (). 00449

Where:

9.5 For a one-tailed test at the 0.05 level of significance and

8 degrees of freedom, the appropriate critical t value 'is 1.860.

Note: Table D.5 for K = 1 includes the critical t values for

corqpqrip.g two groups in a one-tailed test. Since t = 4.226 is

greater than 1.860, conclude that the growth in the effluent

concentration is significantly less than ,the control group

growth.

9.6 Critical t values for two-tailed tests, such as those needed

in comparing a brine control and a dilution cont:rol, can be found

in a table of the t distribution, such as the one in Snedecor and

Cochran, 1980. Note that the critical t for a t~wo-tailed test is

the upper-tail value at the d./2 leve~ of signifi.cance.

10. UNEQUAL VARIANCE t TEST.

10.1 If the F test for equality of variance fails, the t test is

still a valid test. Howe~er, the den6minator of the t statistic

is adjusted as follows:

Y1 - Y2 t = 2 2

51 52

- +

n1 n2

Where: Y1 = mean for the control

625
Y2 = mean for the effluent concentration

Sf estimate of the variance for the control

S~ = estimate of the variance for the effluent

concentration

n 1 = number of replicates for the control

n2 number of replicates for the effluent

concentration

~0.2 Additionally, the degrees of freedom for the test are

adjusted using the following formula:

(n1-1) (n2-1)

df'----------

(n -1) c 2+ (I-C) 2 (n -1) 2 1

Where:

C z

. n1

2 2

8 1 8 2

-+-

n1 n2

~0.3 The modified degrees of freedom is usually not an integer.

Common practice is to round down to the I!-earest integer.

10.4 The t test is then conducted as the .equal variance t test.

The calculated t is compared to the critical, t at the 0'.05

significance level with the modified degrees of freedom. If the

calculated t exceeds the critical t, the mean responses are found

to be statistically different.

626
APPENDIX H

PROBIT ANALYSIS

1. This program calculates the EC1 and EC50 (or LC1 and LC50) ,

and the associated 95% confidence intervals.

2. The program is written in IBM PC Basic for the IBM compatible

PC by Computer Sciences Corporation, 26 W. Martin Luther King

Drive, Cincinnati, OR 45268. A compiled, executable version of

the program and supporting documentation can be.obtained from

EMSL-Cincinnati by sending a written request to EMSL at 3411

Church Street, Cincinnati, OR 45244.

2.1 A set of mortality data from a mysid survival and growth

test is given in Table R.1. The program's data input routine is

illustrated with this data in Figure R.1. The program begins

with a request for the following information:

1. Desired output of abbreviated (A) or full (F) output?

(Note: only abbreviated output is shown below.)

2. Output designation (P = printer, p = disk file) .

3. Title for the output.

4. The number of exposure concentrations.

5. Toxicant concentration data.

TABLE H.1. DATA FOR PROBIT ANALYSIS

Concentration (%)

No. Dead

No. Exposed

Control

1

25

1.80

o

25

3.20

3

25

5.60

9 '

25 ' .

10.0

24

25

18.0

25

25

2.2 The program output for the abbreviated output options, shown

in Figure R.2, includes the following:

627
· 1. A table of the observed proportion responding and

the proportion responding adjusted for the

controls.

2. The calculated chi-square statistic for

heterogeneity and the tabular value. This test is

one indicator of how well the data fit the model.

The program will issue a warning when the test

indicates that the data do not fit the.model.

3. The estimated LCl and LC50 values and associated

95% confidence intervals.

628
Do you wish abbreviated (A) or full (F) input/output? A I

Output to printer (P) or disk file (D)? P . Titl~ ? Example of Probit Analysis for Appendix H

Number responding in the control group = ? 1

Number of animals exposed in the concurrent control group = ? 25

Number of exposure concentrations. exclusive of controls.? 5

Input data. starting with the lowest exposure concentrati()n

Concentration = ? 1.80

Number responding = ? 0

Number exposed = ? 25

Concentration = ? 3.20

Number responding = ? 3

Number exposed = ? 25

Concentration = ? 5.60

Number responding = ? 9

Number exposed = Z25

Concentration = ? 10.0

Number responding = ? 24

Number exposed = ? 25

Concentration = ? 18.0

Number responding = ? 25

Number exposed = ? 25

Number Number

Number Cone. Resp. Exposed

1 1.8000 0 25

2 3.2000 3 25

3 5.6000 9 25 . 4 10.0000 24 25

629
Example of Probit Analysis for Appendix H

Proportion

Observed Responding

Number Number . Proportion Adjusted for

Cone. Exposed Resp. Responding Controls

Control 25 1 0.0400 0.0000

1.8000 25 0 0.0000 -.0306

3.2000 25 3 0.1200 0.0930

5.6000 25 9· 0.3600 0.3404

10.0000 25 24· 0.9600 0.9588

18.0000 25 25 1.0000 1.0000

Chi - Square for Heterogeneity (calculated)

Chi - Square for Heterogeneity

(tabular value at 0.05 level)

Example of Probit Analysis for Appendix H

= 3.004

= 7.815

Estimated LC/EC Values and Confidence Limits

Point

Exposure

Cone.

95% Confidence Limits

Lower Upper

LC/EC 1.00

LC/EC 50.00

2.642

5.973

1.384

4.998

3.519

6.920 .,

Figure H.2. USEPA Probit Analysis Program used for Calculating LC/EC

Values. Version 1.5. .

630
APPENDIX I

SPEARMAN-KARBER METHOD

1. The Spearman-Karber Method is a nonparametric statistical

procedure for estimating' 'the LC50 and the associated 95%

confidence interval (Finney, 1978). The Spearman-Karber Method

estimates the mean of the distribution of<·the logloof the

tolerance. If the log tolerance distribution is sYmmetric, this

estimate of the mean is equivalent to an estimat.e of the median

of the log tolerance distribution.

2. If the response proportions are not monotonically nondecreasing with increasing concentration' (constant or steadily

increasing with concentration), the data must be smoothed.

Abbott's procedure is used to "adjust" the concentration response

proportions for mortality occurring in the control replicates.

3. Use of the Spearman-Karber Metho<;i is recommended when partial

mortalities occur in the test solutions, but the data do not fit

the Probit model.

4. To calculate the LCSO using the Spearman-Karber Method, the

following must be true: 1) the smoothed adjusted proportion

mortality for the lowest effluent concentration (not including

the control) must be zero, arid 2) the smoothed adjusted

proportion mortality for the highest effluent concentration must

be one.

5. To calculate the 95% confidence interval for the LC50

estimate, one or more of the smoothed adjusted proportion

mortalities must be between zero and one.

6. The Spearman-Karber Method is illustrated bE~low using a set

of mortality data from a Mysid Survival andGro,~th test. These

data are listed in Table I.1.

631
TABLE 1.1. EXAMPLE OF SPEARMAN-KARBER METHOD: MORTALITY DATA FROM

AMYSIDSURVIVAL AND GROWTH TEST (25 ORGANISMS PER

CONCENTRATION) .

Effluent

Concentration

%'

Control

6.25

12.5

25.0

50.0

100.0

Number of

Mortalities

2

2

o

3

16

25

Mortal ity . Proportion

0.08

0.08

0.00

0.12

0.64

1.00

7. Let POt Pl' ... , Pk denote the observed response proportion

mortalities for the control and k effluent concentrations. The

first step is to smooth the Pi if they do not satisfy Po Pl

••• Pk' The smoothing process replaces' any adjacent Pi'S that

do not conform to Po Pl ... Pk with their average .. For

example, if Pi is less than Pi-l then:

Where: p7 the smoothed observed proportion

mortality for ef£luent

.concentration i.

7.1 For the data in this example, because the observed mortality

proportions for the control and the 6.25% effluent concentration

are greater than the observed response p~oportions for .the 12.5%

effluent concentration, the responses for these three groups must

be averaged:

• • • 0.08+0.08+0.00 = 0.16 = 0.053 Po -P1 "P2 -

3 3

632
7.2 Since P3 = 0.12 is larger than p~, set p; = 0.12. Similarly,

P4 = Q.64 is larger than pI, so set P: = 0.64. Finally, Ps = 1.00

is larger than P:, so set p~ = 1.00. Additional smoothing is not

necessary. The smoothed observed proportion mortalities are

shown in Table 1.2.

TABLE 1.2. EXAMPLE OF SPEARMAN-KARBER METHOD: SMOOTHED. ADJUSTED

MORTALITY DATA FROM AMYSID SURVIVAL AND GROWTH TEST

Effluent'

Concentration

%

Control

6.25

12.5

25.0

Mortality

Proportion

0'.08

0.08

0.00

0.12 .

Smoothed

Mortality

Proportion

0.053

0.053

0.053

0.120

.Smoothed.

Adjusted

Mortality

Proportion

0.000

0.. 000

0.000

0.071

8. Adjust the smoothed observed proportion mortality in each

effluent concentration for mo:r:.tality in the control group using

Abbott's formula (Finney, 1971). The adjustment takes the form:

P! = (p~ - p~) / (1 - p~)

Where: ,-p~ = the smoothed observed proportion mortality for the

control

p~ the smoothed observed proportion mortality for

effluent concentration i.

8.1 For the data in this.example, the data for each effluent

concentration must be adjusted for control mortality using .

~bott's formula, as follows:

0.053 - 0.053 = ~_ = 0.0

1 - O. 053 O. 947

633
0.120 - 0.053

1 - 0.053

0.067

--- = 0.071

0.947

0.587 = 0.620

0.947

0.640 - 0.053

1 - 0.053

II

p .. = ---

II

Ps =

s s

P s - Po 1. 000 - 0.053

1 - 0.053

0.947 = 1.000

0.947

The smoothed, adjusted response proportions for the effl~ent

concentrations are shown in Table I.2. A plot of the smoothed,

adjusted data is shown in Fig~re I.l.

9. Calculate the loglo of the estimated LC50, m, as foilows:

i-1 2

Where: pf = the smoothed adjusted proportion mortality at

concentration i

Xi = the loglo of concentration i

k = the number of effluent concentrations tested, not

including the control.

9.~ For this example, the loglo of the estimated LC50, m, is

calculated as follows:

m = [(0.000 -

[(0.071 -

[(0.620 -

[(~.OOO

0.000)

0.000)

0.071)

0.620)

(0.7959 + 1.0969)]/2 +

(1.0969 + 1.3979)]/2 +

(1.3979 + 1.6990)]/2 +

(1.6990 + 2.0000)]/2

= 1.64~47

634
0)

w

U1

1.0

0.9

0.8

>-

.....

- 0.7 -'

-0:

.....

oc 0.8 0

::::;;

z 0.5 0

-

..... 0.4 oc

0

a..

0 0.3 oc

a..

0.2

0.11-1.- * ---

* * * ~11Ec.lMDlIrM"" ---- ClOtIIICl'IHMXmeDIIIIiMW18

call!!CtlnElIIXI'M!D.#IIDU1IlD-'ftWcLLl!l

.

•

• /.,"

'/ "

.;,"

f,l"

,

,

,

,

,

,

,

,

,

,

,

,

,

,

.

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

---_ ...--"

0.0 j j--------------,

0.00 8.25

---

12.50

EFFLUENT (%)

25.00 50.00 100.00

Figure.I.I. Plot of observed. smoothed. and adjusted response proportions for mysid.

Ho7mesimysis costata. survival data.
10. Calculate the estimated variance of m as follow$:

Where: Xi = the loglo of· concentration i

ni = the number of organisms tested at effluent

concentration i

p~ = the smoothed adjusted observed proportion mortality

at effluent concentration i

k = the number of effluent concentrations tested, not

including the control.

10.1 For this example, the estimated variance of m, V(m), is

calculated as follows:

V(m) = (0.000) (1.000) (1.3979 - 0.7959)2/4(24) +

(0.071) (0.929) (1.6990 - 1.0969)2/4(24) +

(0.620) (0.380) (2.0000 - 1.3979)2/4(24)

= 0.0011388

11. Calculate the 95% confidence interval for m:

m ± 2.0 vv(m)

11.1 For this example, the 95% confidence interval for m is

calculated as follows:

1. 64147 ± 2 vo. 0011388 = (1. 57398, 1. 70896)

12. The estimated LC50 and a 95% confidence interval for the

estimated LCSO can be found by taking base10 antilogs of the

above values.

636
12.1 For this example, the estimated LC50 is calculated as

follows:

LC50 = ant{log(m) = antilog(l.64147) = 43.8%.

12.2 The limits of the 95% confidence interval for the estimated

LC50 are calculated by taking the antilogs of tl~e upper and lower

limits of the 95% confidence in"terval for m as ":Eollows : , '

lower limit:

upper limit:

antilog(l.57398)

'antilog(l.70896) =

637

,

3'7.5% ,

I

51.2%

I,

I
APPENDIX J

TRIMMED SPEARMAN-KARBER METHOD

~. The Trimmed Spearman-Karber Method is a modification of the

Spearman-Karber Method, ,a nonparametric statistical procedure for

estimating the LC50 and the associated 95% confidence interval

(Hamilton, et aI, 1977). The Trimmed Spearman-Karber Method

estimates the trimmed mean of the distribution of the loglo of

the tolerance. If the log tolerance distribution is symmetric,

this estimate of the trimmed mean is equivalent to an estimate of

the median of the log tolerance distribution.

2. If the response proportions are not monotonically nondecreasing with increasing concentration (constant or steadily

increasing with concentration), the data must be smoothed.

Abbott's procedure is used to lIadjustll the concentration,response

proportions for mortality occurring in the control replicates.

3. Use of the Trimmed Spearman-Karber Method is recommended only

when the requirements for the Probit Analysis and the SpearmanKarber Method are not met.

4. To calculate the LC50 using the Trimmed Spearman-Karber

Method, the smoothed, adjusted,' observed proportion mortalities

must bracket 0.5.

5. To calculate the 95% confidence inte~val for the LC50

estimate, one or more of the smoothed, adjusted, observed

proportion mortalities must be between zero and one.

6. Let POI Pl' ... , Pk denote the observed proportion mortalities

for the control and the k effluent concentrations. The first'

step is to smooth the Pi if they do not satisfy Po i!;; Pl .:. Pk.

The smoothing process replaces any adjacent Pi'S that do not

conform to Po Pl ... Pk' with their average. For example,

if Pi is less than Pi-l then:

Where: p~ = the smoothed observed proportion mortality for

effluent concentration i.

638
, ,

7. Adjust the smoothed observed proport~on 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~ ) / (1 - p~ )

Where: p~ = the smoothed ,observed proportion mortality for the

control

= the smoothed observed proportion mortality for, I

effluent concentration i.

8. Calculate the amount of trim to use in the estimation of the

LC50 as follows:

Trim = max (pr, ;L -p~)

Where: pr the smoothed, adjusted proportion mortality for

the lowest effluent concentration, exclusive of

the control

p~ = the smoothed, adjusted prqportion mortality for'

the highest effluent concentration

k the number of effluent concentrajt:ions, exclusive

of the control. I

The minimum trim should be calculated fo!? each. d(1ta se,t rather

than using a fixed amount of trim for each, data, se,t.

9. Due to the intensive nature of the calculation for'the

estimated LC50 and the calculp.tion of the associated 95.%

confidence interval' using the Trimmed Spearman-Karber Method, ,,it

is recommenqed that the data be analyzed by computer.

. ,

10. A, computer program which estimates the LC50 and associated

95% confidence interval using the Trimmed Spearman-Karber Method,

can be obtained through EMSL, 3411 Church Stree1:, Cincinnati, OH

45244. The program can be obtained ,from EMSL-C:Lncinnati by

sending a written request to the above address.

639
11. The Trimmed Spearman-Karber program automatically performs

the following functions:

a. Styl0othing.

b. Adjustment for mortality in the control.

c. Calculation of the necessary trim.

d. Calculation of the LCSO.

e. Calculation of the associated 95% confidence interval.

12. To illustrate the Trimmed Spearman-Karber method using the

Trimmed Spearman-Karber computer program, a set of data from a

Topsmelt Larval Survival and Growth ,test will be used. The data

are listed in Table J.1.

TABLE J.1. EXAMPLE OF TRIMMED SPEARMAN-KARBER METHOD: MORTALITY

DATA FROM ATOPSMELT LARVAL SURVIVAL AND GROWTH TEST (25

ORGANISMS PER CONCENTRATION)

Effluent

Concentration

%

Control

6.25

12.5

25.0

50.0

100.0

Number of

Mortalities

o

2

1 .

.s

25

25

Mortality

Proporti'on

0.00

0.08

0.04

0.20

1.00 ,

1. 00

12.1 The program requests the following input (Figure J.l) :

a. Output destination (D = disk file or P = print~r) .

b. Control data.

c. Data for each toxicant concentration.

12.2 The program output includes the following (Figure J.2) :

a. A table of the concentrations tested, number of

organisms exposed, and the mortalities.

b. The amount of trim used in the calculation.

c. The estimated LCSO and the associated 9S% confidence

interval.

640
Ii

THE NUMBER OF MORTALITIES AT EACH EXPOSURE CONCENTRATION:

25 25

YOU LIKE THE AUTOMATIC TRIM CALCULATION(Y/N)?

A: >TSK

TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5

ENTER DATE OF TEST:

1

ENTER TEST NUMBER:

2

WHAT IS TO BE ESTIMATED?

(ENTER "L" FOR LC50 AND nEn FOR EC50)

L

ENTER TEST SPECIES NAME:

Topsmelt

ENTER TOXICANT NAME:

effluent

ENTER UNITS FOR EXPOSURE CONCENTRATION OF TOXICANT

%

ENTER THE NUMBER OF INDIVIDUALS IN THE CONTROL:

25

ENTER THE NUMBER OF MORTALITIES IN THE CONTROL:

o

ENTER THE NUMBER OF CONCENTRATIONS

(NOT INCLUDING THE CONTROL; MAX = 10) :

5 I

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:

25

ENTER UNITS FOR DURATION OF EXPERIMENT

(ENTER "H" FOR HOURS, "D" FOR DAYS, ETC.):

Days

ENTER DURATION OF TEST:

7

ENTER

2 1 5

WOULD

Y

Figure J.1. Example input for Trimmed Spearman-Karber Method.

641
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5

DATE: J.

TOXICANT:

SPECIES:

effluent

Topsmelt

TEST NUMBER: 2 ;DURATION: 7 Days

RAW DATA: Concentration

--- ---- (%)

.00

6.25

J.2.50

25.00

50.00

J.OO.OO

SPEARMAN-KARBER TRIM:

Number

Exposed

25

25

25

25

25

25

6.00%

Mortalities

o

2

J.

5

25

25

SPEARMAN-KARBER ESTIMATES: LC50:

95% LOWER CONFIDENCE:

95% UPPER CONFIDENCE:

30.98

27.J.7

3"5.32

NOTE: MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING.

ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION."

Figure J.2. Example output for Trimmed "Spearman-Karber Method.

642
APPENDIX K

GRAPHICAL METHOD

1. The Graphical Method is used. to calculate the LCSO. It is a

mathematical procedure which estimates the LCSO by linearly

interpolating between points of a plot of observ~d percent

mortality versus the base 10 logarithm (loglO) of percent

effluent concentration. This method does not provide a

confidence interval for the LCSO estimate and its use is only

recommended when there are no partial mortalities after the data

is smoothed and adjusted for control mortality. The only

requirement for the Graphical Method is that the observed percent

mortalities bracket 50%.

2. For an analysis using the Graphical Method the data must

first be smoothed and adjusted for mortality in the control

replicates. The procedure ,for smoothing and adjusting the data

is detailed in the following steps.

3. The Graphical Method is illustrated below using, a set of

mortality data from a Topsmelt Larval Survival and Growth test.

These data are listed in Table K.l.

, TABLE K.1. EXAMPLE OF GRAPHICAL METHOD: MORTALITY DATA FROM A

,TOPSMELT LARVAL SURVIVAL AND GROWTH TEST (25

ORGANISMS PER CONCENTRATION)

Effluent

Concentration

%

Control

6.25

12.5

25.0

50.0

100.0

Number of

Mortalities

1

9

o

o

25

25

643

Mortal i.ty I Proportion

0.04

0.00

0.00

0.00

1.00

1.00
4. Let Po, Pl' •.• , Pk denote the observed proportion m?rtalities

for the control and the k effluent concentrations. The first

step is to smooth the Pi if they do not satisfy Po,;!: Pl ;!: ... ;!: Pk'

The smoothing process replaces any adjacent Pi'S that do not

conform to Po;!: Pl ;!: •.. ;!: Pk with their average. For example, if

Pi is less than Pi-l then:

Where: P~ = the smoothed observed proportion mortality for

effluent concentration i.

4.1 For the data in this example, because the obser-ved mortality

proportions for the 6.25%, 12.5%, and 25.0% effluent

concentrations are less than the observed response proportion for

the control, the values for these four groups must be averaged:

& S S s

Po z P1 z Pz z P3

O. 04 + O. 00 + O. 00 + O. 00

4

0.04

4

0.01

4.2 Since P4 = Ps = 1.00 are larger then 0.01, set p~ = p~

1.00. Additional smoothing is not necessary. The smoothed

observed proportion mortalities are shown in Table K.2.

5. Adjust the smoothed observed proportion mortality in each

effluent concentration for mortality in the control group using

Abbott's formula (Finney, 1971). The adjustment takes the form:

Where: p8 = the smoothed observed proportion mortality for the

control

pr = the smoothed observed proportion mortality for

effluent concentration i.

644
5.1 Because the smoothed observed proportion mortality for the

conj:rol group is greater than zero, the responses must be

adjusted using Abbott's formula, as follows:

0.01 - 0.01 = ~~ = 0.0

1 - 0.0125 0.99

1. 00 - 0.01 = 0.99 = ]~. 00

1 - 0.01 0.99

Ii a

P4 = Ps = ---

A table of the smoothed, adjusted response propoJrtions for the

effluent concentrations is shown in Table K.2.

TABLE K.2. EXAMPLE OF GRAPHICAL METHOD: SMOOTHED. ADJUSTED

MORTALITY DATA FROM ATOPSMELT LARVAL SURVIVAL AND

GROWTH TEST

Smoothed.

Effluent Smoothed Adjusted

Concentration Mortality Mortality Mortality

% Proportion Proportion Proportion

Control 0.04 0.01 0.00

6.25 0.00 0.01 0.00

12.5 0.00 0.01 0.00

25.0 0.00 0.01 . O. 00

50.0 1.00 1. 00 1. 00

100.0 1. 00 1. 00 1. 00

5.2 Plot the smoothed, adjusted data on 2-cycle'semi-~og graph

paper with the logarithmic axis (the y axis) used for percent

effluent concentration and the linear axis (the x axis)" used for

observed percent mortality. A plot of the smoothed, adjusted

data is shown in Figure K.1.

645
100

50

w

:E

:::>

-'

g

IZ

10

-'

U.

LL

W

I- 5 z

w

U

tt:

w

Q.

1

---- _l....--"" """"""'"

:r-I-"""'"

I""'" ----- 4r" - -,

0

n

c

o 10 20 30 40 50 60 70 80 90 100

PERCENT MORTALITY

Figure K.1. Plot of the smoothed adj usted response proportfons for

topsmelt. Atherinops affinis. survival data.

646
6. Locate ~he two points on the graph which bracket 50%

mortality and connect them with a straight line.

7. On the scale for percent effluent concentration, read the

value for the point where the plotted line .and the 50% mortality

line intersect. This value is the estimated LC50expressed as a

percent effluent concentration.

7.1 For this example, the two points on the graph which bracket

the 50% mortality line (0% mortality at 25% effluE~nt, and 100%

mortality at 50% effluent) are connected with a sl:raight line;

The point at which the plotted line intersects the 50% mortality

line is the estimated LC50. The estimated LC50 = 35% effluent.

647
APPENDIX L

LINEAR INTERPOLATioN METHOD

1. GENERAL PROCEDURE

1.1 The Linear Interpolation Method is used to calculate a point

estimate of the effluent or other toxicant concentration that

causes a given percent reduction (e.g., 25%, 50%, etc.) in the

reproduction or growth of the test organisms (Inhibition

Concentration, or IC). The procedure was designed for general

applicability in the analysis of data from short-term chronic

toxicity tests, and the generation of an endpoint from a

continuous model that allows a traditional quantitative

assessment of the precision of the endpoint, such as confidence

limits for the endpoint of a single test, and a mean and

coefficient of variation for the endpoints of multiple tests.

1.2 The Linear Interpolation Method assumes that the responses

(1) are monotonically non-increasing, where the mean response fo~

each higher concentration is less than or equal to the mean

response for the previous concentration, (2) follow a piecewise

linear response function, and (3) are from a random, independent,

and representative sample of test data. If the data are not

monotonically non-increasing, they are adjusted by smoothing

(averaging). In cases where the responses ,at the low toxicant

concentrations are much higher than in the controls, the

smoothing process may result in a large upward adjustment in the

control mean. Also, no 'assumption is made about the di.stribution

of the data except that the data within a group being resampled

are independent and identically distributed.

2 • DATA SUMMARY AND PLOTS

2.1 Calculate the mean responses for the control and each

toxicant concentration, construct a summary table, and plot the

data.

3 • MONOTONICITY

3.1 If the assumption of monotonicity o~ test results is met,

the observed response means (Yi ) should stay the same or decrease

648
as the t9xicant concentration increases. If the means do not

decrease monotonically, the responses are "smoothed" by averaging

(pooling) adjacent means.

\

3.2 Observed means at each concentration are cbnsidered in' order

of increasing concentration, starting'with the control mean (Y1 ).

If the mean observed response at the lowest toxicant

concentration (Y2 ) 'is equal to or smaller tllan the control mean

(Y1 ), it is used as the response. If it is lar9'er than the

control mean, it is averaged with the control, and this average

is used for both the control response (M1 ) and t:he lowest

toxicant concentration response (M2 ). This mean is then compared

to the mean observed response for the next higher toxicant

concentration (Y3 ). Again, if the mean observed response for the

next higher toxicant concentration is smaller than the mean of

the control and the lowest toxicant concentration, it is used as

the responsj=. If it is higher than the mean of the first two, it

is averaged with the first two, and the mean is, used as the

response for the control and two lowest concentrations of

toxicant. This process is continued for data from the remaining

toxicant concentrations. A numerical example of smoothing the

data is provided below. (Note: Unusual 'patterns in the

deviations from monotonicity may require an additional step of

smoothing). Where Y i decrease monotonically, the Y i be~ome Mi

without smoothing.

4. LINEAR INTERPOLATION METHOD

4.1 The method assumes a linear response from one concentration

to the next. Thus, the ICp is estimated by linear interpolation

between two concentrations whose responses bracket the response

of interest, the (p) percent reduction from the controY.

4.2 To obtain the estimate, determine the-concentrations CJ and

CJ +1 which bracket the response M1 (1 - p/100), where M1 is the

smoothed control mean response and p is ~he percent reduction in

response relative to the control response. These calculations

can easily be done by hand or with a computer p;rogram as

described below. The linear interpolation estimate is

calculated as follows:

649
ICp • Col + [ M1 (1 - pl1DD) - Mol ]

(Cold-C)

(Mol. 1 - M)

Where:

p

ICp

=

=

=

=

=

=

=

tested concentration whose observed mean

response js greater th~n M1(1 - p/1DD).

tested concentration whose observed mean

response is less than M1(1 - p/lDO).

smoothed mean response for the control.

smoothed mean response for concentration

J.

smoothed mean response for concentration

J + 1.

percent reduction in response relative

to the control response.

estimated concentration at which there

is a percent reduction from the smoothed

mean control response. The ICp is

reported for the' test, together with the

95% confidence interval calculated by

the ICPIN.EXE program described below.

4.3 If the CJ is the highest concentration tested, the ICp would

be specified as greater than CJ • If the response at the lowest

concentration tested is used to extrapolate the ICp'value, the

ICp should be expressed as a less than the lowest test

concentration.

5. CONFIDENCE INTERVALS

5.1 Due to the use of a linear interpolation technique to

calculate an estimate of the ICp, standard statistical methods

for calculating confidence intervals are not applicable,for the

ICp. This li~itation is avoided by use a technique known as the

bootstrap method as proposed by Efron (1982) for deriving point

estimates and confidence intervals.

650
5.2 In the Linear Interpolation Method, the smoothed response

means are used to obtain the ICp estimate reported for the test.

The bootstrap method is used to obtain the 95% confidence

interval for the ~rue mean. In the bootstrap method, the test

data Yji is randomly resampled with replacement t.O produce a new

set of data Yji*, that is. statistically equivalent to the

original data, but a new and slightly different 'estimate of the

ICp (ICp*) is obtained. This process ,is repeated at least 80

times (Mar<;:l..ls.and Holtzman, 1988) resulting in multiple "data"

sets, each with an associate ICp* estimate. The distribution of

the ICp* estimates derived from the sets of resampled data'

approximp.tes the sampling distribution of the Iep estimate. The

standard." error of the ICp is estimated' by the standard deviation

of the individual ICp* estimates.. Empirical confidence intervals

are d~rived'from the quantiles of the ICp* empirical

distribution. For example, if the test data are resampled a

minimum of 80 times, the empirical ,2.5% and the 197.5% c.onfidence

limits are approximately the second smallest and secorid largest

ICp* estimates (Marcus and Holtzman, 1988).

5.3 The wi.dth 'of the confidence intervals calcu.lated by the

bootstrap method is related to the variability of the data. When

confidence int~rvals are. wide, the reliability of the I,C estimate

is in question. However, narrow. intervals do not necessarily

indicate, that the estimate is highly reli~ble, because 'of

undet~cted violations ;.of as'sumptions, and the fact that the ._

confidence limits based on the empirical quantiles of a bootstrap

distribution, of 80 samples may be unstable.

5.4 Th,e bootstrapping method of calculating confidence intervals

is computationally intensive. For this reason, ,all. of the

calculations associated with. determining the confidence' ,intervals

for the ICp estimatt: have been incorporated intq a computer

program. Computations are most eas'ily done with a computer

program such as the revision of the BOOTSTRP prdgram (USEPA,

1988; USEPA, 1989) which is now called "ICPIN" Olnd is desc!ibed

below in ,subsection 7.

6 . MANUAL CALCULATIONS

6.1 DATA SUMMARY AND PLOTS

651
6.1.1 The data used in this example are the mysid growth data

used in the example in Section 14. The data is presented as the

mean weight per surviving organism. Table L.1 includes the raw

data and the mean growth for each concentration. A plot of the

data is provided in Figure L.1.

6.2 MONOTONICITY

6.2.1. As seen in the table, the observed means are

monotonically non-increasing with respect to concentration.

Therefore, the smoothed means will be simply the corresponding

observed mean. The observed means are represented by Yi and the

smoothed means by Mi' Table L.2 contains the smoothed means and

Figure L.1 gives a plot of the smoothed response curve.

6.3 LINEAR INTERPOLATION

6.3.1 An estimates of the IC25 can be calculated using the

Linear Interpolation Method. A 25% reduction in mean weight,

compared to the controls, would result in a mean weight of 0.039,

where MJ. (1-p/100) = 0.052 (1-25/100). Examining the. smoothed

means and their associated concentrations (Table L.2), the

response, 0.039 mg, is bracketed by C4 = 5.60% and and Cs =

10.0%.

TABLE L.1. MYSID. HOLMESIMYSIS COSTATA. GROWTH DATA

Concentration (%)

Replicate Control 1.80 3.20 5.60 10.0

1 0.048 0·.055 0.057 0.041 0.033

2 0.058 0.048 0.050 0.040 0.000

3 0.047 0.042 0.046 0.041 0.000

4 0.058 0.041 0.043 0.043 0.000

5 0.051 0.052 0.045 0.040 0.000

MeaneVi ) 0.052 .0.048 0.048 0.041 0.007

i 1 2 3 4 5

652
0.08 J. ***----

* --- ClDIICISS-......Wt&.-

* ------ ---=raM............

0.05 "tk * *

* 0-

o.04 J * E

I-

:c * <.:> \ * -

w

:;= 0.03

>-

a:::

U1 Cl

W z· 0-02 <t:

w

:::1<

0.01

0.00', i , [ l' T

0.0 1.8 3.2 5.8 10.0 18.0

EFFLUENT CONCENTRATION (%)

Figure L.l. Plot of raw data. observed means, and smoothed means for the mysid,

Ho7mesimsyscostata, growth data.
TABLE L.2. MYSID. HOLMESIMYSIS COSTATA. MEAN

GROWTH RESPONSE AFrER SMOOTHING

Toxicant Response Smoothed

Cone. Means Means

(%) i Yi (mg) Mi (mg)

Control 1 0.052 0.052

1.80 2 0.048 0.048

3.20 3 0.048 0.048

5.60 4 0.041 0.041

10.00 5 0.007 0.007

18.00 6 0.000 0.000

6.3.2 Using the equation from section 4.2, the estimate 9f the

IC25 is calculated as follows:

(CJ • 1 - C)

(M;.l - M)

IC25.5.60+ [0.052 (1-25/100) -0.041]

= 5.86%

6.4 CONFIDENCE INTERVALS

(10.0 - 5.60)

(0.007 - 0.041)

6.4.1 Confidence intervals for the ICp are derived using the

bootstrap method. As described above, this method involves

randomly resampling the "individual observations and recalculating

the ICp at least 80 times, and determining the mean ICp, standard

deviation, and empirical 95% confidence ~ntervals. For this

reason, the confidence intervals are calculated using a computer

program called ICPIN. This program is descri~edbelow and is

available to carry out all 'the calculations of both the"

interpolation estimate (ICp) and the confidence intervals.

654
7. COMPUTER CALCULATIONS

,

7.1 The computer program, ICPIN, prepared for the Linear

Interpolation Methods was written in TURBO PASC1~L for IBM

compatible PCs. The program (version 2.0) has been modified by

Computer Science Corpor'ation, Duluth, MN with fll,riding p'rovided 'by

the Environmental Research Laboratory, Duluth, ~1N (Norberg-King,

1993). The program was originally developed by Battelle

Laboratories, Columbus, OR through a government contract

supported by the Environmental Research Laboratory, Duluth, MN

(USEPA, 1988)'. A compiled, executable version of the program and

supporting documentation can be obtained by sending a written

request to EMSL-Cincinnati, 3411 Church Street, ,Cincinnati, OR

45244.

7.2 The ICPIN.EXE program performs the following functions: 1)

it calculates the observed response means (Yi ) (response means) ;

2) it calculates the standard, deviations; 3) checks the

responses for monotonicity; 4) calculates smoothed means (MJ

(pooled response means) if necessary; 5) uses the means, Mi , to

calculate the initial ICp of choice by linear interpolation; 6)

performs a user-specified number of bootstrap n~samples between

80 and 1000 (as multiples of 40) i 7) calculates the mean and

standard deviation of the bootstrapped'ICp estimates; and 8)

provides an original 95% confidence intervals tp be used with the

initial ICp when the number of replicates per concentration is

over six and provides both original and expanded confidence . ,

intervals when the number of replicates per concentration are

less than seven (Norberg-King, 1993).

7.3 For the ICp calculation, up to twelve treatments can be

input (which includes the control). There can be up to 40

replicates per concentration, and the program does not require an

equal number of replicates per concentration. ~he value of p can

range from 1% to 99%.

7.4 DATA INPUT

7.4.1 Data is entered directly into the ,program onscreen. A

sample data entry screen in shown in Figure L.2. The program

~ocumentation provides guidance on the entering and analysis of

data for the Linear Interpolation Method.

655
rep Data Entry/Edit Screen

Cone. ID 1

Cone. Tested

Response 1

Response 2

Response 3

Response 4

Response 5

Response 6

Response 7

Response 8

Response 9

Response 10

Response 11

Response 12

Response 13

Response 14

Response 15

Response 16

Response 17

Response 18

Response 19

Response 20

2 3

Current File:

4 5 6

FlO for Command Menu Use Arrow Keys to Switch Fields

Figure L.2. ICp data entry/edit screen. Twelve concentration identifications can be

used. Data for concentrations are entered in columns 1 through 6. For

concentrations 7 through 12 and responses 21-40 the data is entered in

additional fields of the same screen.

656
7.4.2 The user selects the lCp estimate desired_(e.g., lC25 or

le50) and the number of· resamples to be taken for the bootstrap

method of calculating the confidence intervals. The program has

the capability of performing any number of resamples from 80 to

1000 as multiples of 40. However, Marcus and Holtzman (1988)

recommend a minimum of 80 resamples for the bootstrap method be

used and at least 250 resamples are better (Norberg-I<;ing, 19.93).

7.5 DATA OUTPUT

7.5.1 The program output includes the. following (see Figure L.3)

1. A table of the. concentration identificat:ion, the

concentration tested and raw data response for each

replicate and concentration. .'

2. A table of test concentrations, number of replicates,

concentration (units), response means (YJ, standard

deviations for each re~ponse mean·, and t:he pooled

response means (smoothed means; Mi ).

3. The linear interpolation estimate Of the lep using the

means (Mi ). Use this value for the rep estimate.

4. The mean lep and standard deviation from the.bootstrap

resampling.

5. The confidence intervals calculated by t:he bootstrap

method for the lCp. Provides an original 95% confidence

intervals to be used with the initial rep when the number

of replicates per concentration is over six and. provides

both original and expanded confidence intervals when the

number of replicates per concentration are less than

seven.

7.6 lCPlN program output for the analysis of the mysid growth

data in Table L.1 is provided in Figure L.3.

7.6.1 ·When the lCPlN program was used to analyze this ·set of

data, requesting 80 resamples, the estimate of the lC25 was

5.8174%. The empirical 95% confiden~e intervals for the true

mean was 4.9440% to 6.2553%.

657
Cone. ID

Cone. Tested

J.

o

2

1.80

3'

3.20

4

5.60

5

J.O.O

6

J.8.0

Response J. .048 .055 .057 .04J. .033 0

Response 2 .058 .048 .050 .040 0 0

Response 3 .047 .042 .046 .041 0 0

Response 4 .058 .041 .043 .043 0 0

Response 5 .05J. .052 .045 .040 0 0

*** Inhibition Concentration Percentage Estimate *** Toxicant/Effluent: Effluent

Test Start Date: Test Ending Date:

Test Species: mysid, Holmesimysis costata

Test Duration: 7 days

DATA FILE: mysid.icp

OUTPUT FILE: mysid.i25

Cone.

ID

Number

Replicates

Concentration

%

Response

Means

Std.

Dev.

Pooled

Respo.nse Means

---------------,--------------------------------------------------------

J. 5 0.000 0.052 0.005 0.052

2 5 1.800 0.048 0.006 0.048

3 5 3.200 0.048 0.006 0.048

4 5 5.600 0.041 O.OOJ. , 0.04J. "

5 5 J.O.OOO 0.007 . 0.0J.5 0.007

6 5 ;L8.000 0.000 0.000 0.000

The Linear Interpolation Estimate: 5.8J.74 Entered P Value: ·.25

Number of Resamplings: 80

The Bootstrap Estimates Mean:

Original Confidence Limits:

Expanded Confidence Limits:

Resampling time in Seconds:

5.8205 Standard Deviation: 0.2673

Lower: 4.9440 Upper: 6.2553

Lower: 4.5073 Upper: 6.4743

0.22 Random Seed: 52680543,5

Figure L.3. Example of ICPIN program output for the'IC25.

658
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