United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/R-95/136
August 1995
Short-Term Methods for
Estimating the Chronic
Toxicity of Effluents and
Receiving Waters to West
Coast Marine and Estuarine
Organisms
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EPA/600/R-95-136
August 1995
SHORT-TERM METHODS FOR ESTIMATING THE CHRONIC TOXICITY OP
EFFLUENTS AND RECEIVING WATERS TO WEST COAST MARINE AND ESTUARINE
ORGANISMS
(First Edition)
Edited by
Gary A. Chapman1, Debra L.Denton2,
, and James M. Lazorchak3-
^•National Health and Ecological Effects Research
Laboratory, Newport, Oregon
2EPA Region IX, San Francisco, California
3National Exposure Research Laboratory, Cincinnati, Ohio
NATIONAL EXPOSURE RESEARCH LABORATORY - CINCINNATI
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
Printed on Recycled Paper
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DISCLAIMER
This document has been reviewed by the National Exposure
Research Laboratory-Cincinnati (NERL-Cincinnati) , U. S-.
Environmental Protection Agency (USEPA), and approved for
publication. The mention of trade names or commercial products
does not constitute endorsement or recommendation for use. The
results of data analyses by computer programs described in the
section on data analysis were verified using data commonly
obtained from effluent tpxicity tests. However, these computer
programs may not be applicable to all data, and the USEPA assumes
no responsibility for their use. >
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FOREWORD
Environmental measurements are required to determine the
quality of ambient waters and the character of waste 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, sediment and solid
waste. :
I
• Develop methods and models to detect and quantify
responses in aquatic and terrestrial organisms exposed
to environmental stressors and to correlate the
exposure with effects on chemical and biological
indicators.
The Federal Water Pollution Control Act Amendments of 1972
(PL 92-500), the Clean Water Act (CWA) of 1977 (PL 95-217) and
the Water Quality Act of 1987 (PL 100-4) explicitly state that it
is the national policy that the discharge of toxic substances in
toxic amounts be prohibited. 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 the National
Pollutant Discharge Elimination System (NPDES) permittees.
iii
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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 currently
specified in NPDES permits in one or more of the west coast
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 received 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 Receiving
Waters to Estuarine and Marine Organisms, (Second Edition);."
The exception occurs in chapter 7 where the 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 section.
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 the toxicity o'f '
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
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topsmelt (Atherinops affinis}, giant kelp (Macrocystis pyrifera},
and mysid (Holmes imysis costata} .
2) The EPA West Coast Marine Complex Effluent Program. Started
in 1985, this program provided preliminary work for the topsmelt
(Atherinops affinis), revision of methods for echihoid sperm with
the purple sea urchin (Strongylocentrotus 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 10, and development of yet
unadopted test methods with the mysid (Mysidopsis 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 procedures because it was
considered redundant'with the Holmesimysis costata 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 with the Macrocystis pyrifera
test. It was excluded from this report because the results from
the West Coast Marine Species Chronic Protocol Variability Study
indicated that more experience with the method was needed to
produce acceptable precision. ' •• . '• -
4) West Coast Marine Species Chronic Protocol'Variability Study.
This study was a result of a 1991 settlement agreement among the
Northwest Pulp and Paper Association, the Washington Dept. of
Ecology, Puget Sound Water Quality Authority, and Tulal'ip Tribes
of Washington. The year-long study in 1993-94 included'monthly
or quarterly interlaboratory toxicity test evaluation of tests '
with bivalve molluscs (Crassostrea gigas) and muissels (Mytilus
sp.), echinoid sperm tests with purple sea urchins (Sv
purpuratus) and sand dollar (D. exce'tricus) , sexual reproduction
of kelp (L. saccharina), and the topsmelt (A. affinis).
Following review and recommendations by the PRC to the State" of '
California for use of the procedures in this report, EPA (OR&D '
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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 sediment-
associated contaminants with the bivalve Mulinia lateralis.
VI
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ABSTRACT
This manual describes six short-term (forty minutes to seven
days) estuarine and marine methods for measuring the chronic
toxicity of effluents and receiving waters to eight species: the
topsmelt, Atherinops.af finis; the mysid, Holmesimysis costata;
the sea urchin, Strongylocentrotus purpuratus and sand dollar
Dendraster excentricus; the red aba'lone Haliotis rufescens; the
bivalves Crassostrea gigas and mussel Mytilus spp. and the giant
kelp, Macrocystis 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; dilution water; effluent
and receiving .water sample collection, preservation, shipping,
and holding; test conditions; toxicity test data analysis; report
preparation; and organism culturing, holding, and handling.
Examples of computer input and output for Dunnett's Procedure,
Probit Analysis, Trimmed Speaman-Karber Method, and the Linear
Interpolation Method are provided in the Appendices.
VII
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ACKNOWLEDGEMENTS
The principal authors of this document are: Gary A. Chapman,
OR&D, Newport, Oregon; Debra IJ. Denton, Region 9, San Francisco,
California; and James M. Lazorchak, OR&D, Cincinnati, Ohio.
i
Section 1 through 10 of this manual are only slightly ,
modified from the same sections in the EPA Manual, "Short-term
Methods for Estimating the 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 abalone,
topsmelt, mysids, and kelp were prepared by the following staff
from the University 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 modified from a
method prepared 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
Vlll
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The data analysis and statistical sections and appendices
were the work of Technology Applications, Inc. employee:
Laura Gast
Formal Peer-review comments from the following persons are
gratefully acknowledged:
Amy Wagner, EPA Region 9 Richmond laboratory, who reviewed
all seven methods for technical detail and consistency (any
existing errors sneaked in after her review).
• Paul Dinnel, Dinnel Marine Research
Suzanne Lussier, EPA Narragansett
Doug Middaugh, EPA Gulf Breeze
George, Morrison, EPA Narragansett
Diane Nacci, SAIC
Barry Snyder, Ogden .Environmental and Energy Services
Glen Thursby, SAIC
Southern California Toxicity Assessment.Group (SCTAG) and
its members, especially chairmen Tim Mikel of AB'C Labs, and
Tom Dean of Coastal Resources Associates, Inc.
California Protocol Review Committee !
•Matthew Reeve, California State Water Resources Control
Board (coordinator) i
Business:
Steve Bay, Southern California Coastal Water Research
.Project
Tom Dean, Coastal Resources Associates, Inc.
Andrew Glickman, Chevron Research and Technology
Company :
Dave Cutoff, City of San Diego Marine Laboratory
Timothy Hall, National Council of the Paper Industry
for Air and Stream Improvement
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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, Ltd.
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 Marine
Laboratory, UC Davis, 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
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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
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CONTENTS
Page
Disclaimer ii
Foreword iii
Preface iv
Abstract vii
Acknowledgements ............ viii
Contents xii
Section Number , Page
1. Introduction .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 11
General Precautions . . . . ... . 11
Safety Equipment 11
General Laboratory and Field Operations .... 12
Disease Prevention 12
Safety Manuals ..... 13
Waste Disposal . 13
4. Quality Assurance 14
Introduction • . . [ 14
Facilities, Equipment, and Test Chambers .... 14
Test Organisms 15
Laboratory Water Used for Culturing and
and Test Dilution Water . . . r 15
Effluent and Receiving Water Sampling and, , •
Handling 16
Test Conditions • ' 16
Quality of Test Organisms 16
Food Quality 17
Acceptability of Chronic Toxicity Tests .... 18
Analytical Methods 18
Calibration and Standardization ... 19
xii
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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 ....... 25
/ General Requirements ...... . 25
Test Chambers .......... 26
• Cleaning Test Chambers and Laboratory Apparatus 26
Apparatus and Equipment for Culturing and Toxicity
Tests '. . . '. . 27
Reagents and Consumable Materials ....... 27
Test Organisms ................. 29
Supplies . • . . .' . . 29
6. Test Organisms 30
Test Species 30
Sources of Test Organisms ........... 31
Life Stage ....... 32
Laboratory Culturing /...... 33
Holding and Handling of Test Organisms .... . 33
Transportation to the Test Site ........ 34
Test Organism Disposal ............. 35
7. Dilution Water .......;,.... 36
Types of Dilution Water ....... 36
Standard, Synthetic Dilution Water ....... 36
Use of Receiving Water as Dilution Water .... 38
Use of Tap Water as Dilution Water ....... 41
Dilution Water Holding '. . 42
8. Effluent and Receiving Water Sampling, Sample Handling,
and Sample Preparation for Toxicity Tests . . 43
Effluent Sampling ....... 43
Effluent Sample Types ... 43
Effluent Sampling Recommendations ........ 44
Receiving Water Sampling . . . 46
Effluent and Receiving Water Sample Handling,
Preservation, and Shipping . . . ....... 46
Sample Receiving 48
xiii
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Persistence of Effluent Toxicity During Sample '
Shipment and Holding .... 48
Preparation of Effluent and Receiving Water Samples
for Toxicity Tests • .' 48
Preliminary Toxicity Range-finding Tests .... 52
Multiconcentration (Definitive) Effluent
Toxicity Tests . 53
Receiving Water Tests ........ 53
9. Chronic Toxicity Test Endpoints and Data Analysis -55
Endpoints 55
Relationship between Endpoints Determined by :
Hypothesis Testing and Point Estimation Techniques 56
Precision 58
Data Analysis . 59
Choice of Analysis 61
Hypothesis Tests 63
Point Estimation Techniques 66
10. Report Preparation 68
Introduction 68
Plant Operations ........ 68
Source of Effluent, Receiving Water, and Dilution
Water . ,68
Test Methods 69
Test Organisms 69
Quality Assurance ' :70
Results 70
Conclusions and Recommendations '70
11. Test Method: Topsmelt, Atherinops affinis, 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, Haliotis rufescens,
Larval Development .. 259
xiv
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15. Test Method: Sea Urchin, Strongylocentrotus
purpuratus Embryo-Larval Development Test Method .321
16. Test 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
1009.0 . . . . . . . . . . '. . . . •. . . „ . . 466
Cited References . .... . . . ... . . . . 528
Bibliography 549
Appendices . . 564
A. Independence, Randomization, and Outliers •-. „ . . . 566
B. Validating Normality and Homogeneity of Variance
Assumptions 575
C. Dunnett' s Procedure ........'. ''-—. . „ . . . 587
D. T test with Bonferroni' s Adjustment . . . . „ '. . . 602
E. Steel's Many-one Rank Test ... . . . . . ". ., '. .. . 609
F. Wilcoxon Rank Sum Test . . ". . ". . . 615
G. Single Concentration Toxicity Test - Comparison
of Control with 100% Effluent or Receiving Water- 622
H. Probit Analysis . !. . . . .'627
I. Spearman-Karber Method 631
J. Trimmed Spearman-Karber Method . . . . . . . . . . . 638
K. Graphical Method ;..... 643
L. Linear Interpolation'Method . . .' . 648
xv
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SECTION 1
INTRODUCTION
1.1 This manual describes chronic toxicity tests for use in the
National Pollutant Discharge Elimination System (NPDES) Permits
Program to identify effluents and receiving waters containing
toxic materials in chronically toxic concentrations. The 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, 1988c; USEPA, 1989b; USEPA, 1989c; USEPA,
1989d; USEPA, 1989e; USEPA, 1991a; USEPA, 1991b; 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 Support Document for
Water Quality-Based Toxics Control (USEPA, 1991a).
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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 germ-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;l s
Marine Bioassay Project. The purple 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 oyster
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 Department 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 Molluscs.
ASTM 1989.
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1.7.2 Three of the methods incorporate the chronic enelpoints 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 or estuarine test species or test
conditions other than those described in the methods summary
tables in this manual or in the 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 arid 40 CFR 136.5.
1.10 These methods are restricted to use by or under the
supervision of analysts experienced in the use or conduct of
aquatic toxicity testing and the interpretation of data from
aquatic toxicity testing. Each analyst must demonstrate the
ability to generate acceptable test results with these methods
using the procedures described in this methods manual.
1.11 The manual was prepared in the established NERL-Cincinnati
format (USEPA, 1983). ;
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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 fish and
other aquatic life in the receiving waters. The endpoirits that
have been considered in tests to determine the adverse effects of
toxicants include death and survival, decreased reproduction and
growth, locomotor activity, gill ventilation rate, heart rate,
blood chemistry, histopathology, enzyme activity, olfactory
function, and terata. Since it is not feasible to detect and/or
measure all of these , (and other possible) effects of toxic
substances on a routine basis, observations in toxicity tests
generally have been limited to only a few effects, such as
mortality, growth, and reproduction. ;
2.1.2 Acute lethality is an obvious and easily observed effect
which accounts for its wide use in the early period of evaluation
of the toxicity of pure compounds and complex effluents. The
results of these tests were usually expressed as the
concentration lethal to 50% of the test organisms (LC50) over
relatively short exposure periods (one-to-four days).
2.1.3 As exposure periods of acute tests were lengthened} the
LC50 and lethal threshold concentration were observed to decline
for many compounds. By lengthening the tests to include one or
more complete life cycles and observing the more subtle effects
of the toxicants, such as a reduction in growth and reproduction,
more accurate, direct, estimates of the threshold or safe
concentration of the toxicant could be obtained. However,
laboratory life cycle tests may not accurately estimate the
"safe" concentration of toxicants because they are conducted with
a limited number of species under highly controlled, steady state
conditions, and the results do not include the effects of, the
stresses to which the organisms would ordinarily be exposed in
the natural environment.
2.1.4 An early published account of a full life cycle, fish
toxicity test was that of Mount and Stephan (1967). In this
study, fathead minnows, Pimephales promelas, were exposed to a
graded series of pesticide concentrations throughout their life
cycle, and the effects of the toxicant on survival, growth, and
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reproduction were measured and evaluated. This work was soon
followed by full life cycle tests using other toxicants and fish
species. -.,-..
2.1.5 McKim (1977) evaluated the data from 56 full life' cycle
tests, 32 of which used the fathead minnow, Pimephales promelas,
and concluded that the embryo-larval and early juvenile life
stages were the most sensitive stages. He proposed the use of
partial life cycle toxicity tests with the early life stages
(ELS) of fish to establish water quality criteria.
2.1.6 Macek and Sleight (1977) -found that- exposure of critical
life stages of fish to toxicants provides estinicites of
chronically safe concentrations, remarkably similar to those
derived from full life cycle toxicity tests. They reported that
"for a great majority of toxicants, the concentration which will
not be acutely toxic to the most sensitive life stages is the
chronically safe concentration for fish, and that the most
sensitive life stages are the embryos and fry." Critical life
stage exposure was considered to be exposure of the embryos
during most, preferably all, of the embryogenic (incubation)'
period, and exposure of the fry for 30 days post-hatch for warm
water fish with embryogenic periods ranging from, one-to-fourteen
days, and for 60 days post-hatch for fish with longer embryogenic
periods. They concluded that in the majority of cases, the
maximum acceptable toxicant concentration (MATC) could be
estimated from the results of exposure of the embryos during
incubation, and the larvae for 30 days post-hatch."
2.1.7 Because of the high cost of full life-cycle fish toxicity
tests and the emerging consensus that the ELS test data usually
would, be adequate for estimating chronically safe.concentrations,
there was a rapid shift by aquatic toxicologists to 30- to.90-day
ELS toxicity tests for estimating chronically .safe concentrations
in the late 1970s. In 1980, -USEPA adopted the policy .that ELS
test data could be used in establishing water quality criteria if
data from full life-cycle tests were not available (USEPA,
1980a). , ;
2.1.8 Published reports of the results of ELS tests indicate
that the relative sensitivity of. growth and survival as endpoints
may be species dependent, toxicant dependent, or both. Ward and
Parrish (1980) examined the literature on ELS tests that used
embryos and juveniles of the sheepshead minnow, Cyprinodon
variegatus, and found that growth was not a statistically
sensitive indicator of toxicity in 16 of 18 tests. They
suggested that the ELS tests be shortened to,14 days posthatch
and that growth be eliminated as an indicator of toxic effects.
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2.1.9 In a review of the literature on 173 fish full life-cycle
and ELS tests performed to determine the chronically safe
concentrations of a wide variety of toxicants, such as metals,
pesticides, organics, inorganics, detergents, and complex
effluents, Weltering (1984) found that at the lowest effect .
concentration, significant reductions were observed in fry ;
survival in 57%, fry growth in 36%, and egg hatchability in 19.%
of the tests. He also found that fry survival and growth were
very often equally sensitive, and concluded that the growth
response could be' deleted from routine application of the ELS
tests. The net result would be a significant reduction in the
duration and cost of screening tests with no appreciable impact
on estimating MATCs for chemical hazard assessments. Benoit et
al. (1982), however, found larval growth to be the most
significant measure "of effect and survival to be equally or less
sensitive than growth in early life-stage tests with four organic
chemicals.
2.1.10 Efforts to further reduce the length of partial life-
cycle toxicity tests for fish without compromising their
predictive value have resulted in the development of an ;
eight-day, embryo-larval survival and'teratogenicity test for
fish and other aquatic vertebrates (USEPA, 1981; Birge et al.,
1985), and a seven-day larval survival and growth test (Norberg
and Mount, 1985).
2.1.11 The similarity of estimates of chronically safe
concentrations of toxicants derived from short-term,
embryo-larval survival and teratogenicity tests to those—derived
from full life-cycle tests has been demonstrated by Birge et al.
(1981), Birge and Cassidy (1983), and Birge et al. (1985).
2.1.12 Use of a seven-day, fathead minnow, Pimephales promelas,
larval survival and growth test was first proposed by Norberg and
Mount at the 1983 annual meeting of the Society for Environmental
Toxicology and Chemistry (Norberg and Mount, 1983). This test
was subsequently used by Mount and associates in field
demonstrations at Lima, Ohio (USEPA, 1984), and at many other
locations (USEPA, 1985c, USEPA, 1985d; USEPA, 1985e; USEPA,
1986a; USEPA, 1986b; USEPA, 1986c; USEPA, 1986d). Growth was
frequently found to be more sensitive than survival in
determining the effects of complex effluents.
2.1.13 Norberg and Mount (1985) performed three single toxicant
fathead minnow larval growth tests with zinc, copper, and
DURSBAN®, using dilution water from Lake Superior. The results
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were comparable to, and had confidence intervals that overlapped
with, chronic values reported in the literature for both ELS and
full life-cycle tests. • .
2.1.14 USEPA .(1987b)' and US EPA (1987c) adapted the fathead ''
minnow larval growth and survival test for use with the • •
sheepshead minnow arid the inland silverside, respectively. When
daily renewal 7-day sheepshead minnow larval growth and survival
tests and 28,-day ELS tests were performed with industrial and •
municipal effluents, growth was more sensitive than survival in
seven out of 12 larval growth and survival tests, equally
sensitive in four tests, and less sensitive in only one test. In
four cases, the ELS test may have been three to 10 'times more
sensitive to effluents than the larval growth and survival test.
In tests using'copper, the No Observable Effect Concentrations :
(NOECs) were the same for both types of test, and growth was the
most sensitive endpoint for both. In a four laboratory
comparison, six of seven tests produced identical NOECs for
survival and growth (USEPA, 1987a). Data indicate that the
inland silverside is at least equally s.ensitivei or more sensitive
to effluents and single compounds than the sheepshead minnow, and
can be tested over a wider salinity range, 5-301; (USEPA,
1987a) ., ' . ;
2.1.15 Lussier et al. (1985) and USEPA (1987e) determined that •"
survival and growth are often as sensitive as reproduction in
28-day life-cycle tests with the mysid, Mysidopsis bahia.
2.1.16 Nacci and Jackim (1985) and USEPA (1987g) • compared the _, ''
results from the sea urchin fertilization test,; using organic
compounds, with results from acute toxicity tests using the
freshwater organisms, fathead minnows, Pimphales promelas, and
Daphnia magna. The test was also compared to acute toxicity
tests using Atlantic silverside, Menidia menidia, and the mysid,
Mysidopsis bahia, and five metals. For six'of the eight organic
compounds, the results of the fertilization test and the acute
toxicity test correlated well (r2 = 0.85). However, the results
of the fertilization test with the five metals did not correlate
well with the results from the acute tests.
2.1.17 USEPA (1987f) evaluated two industrial effluents
containing heavy metals, five industrial effluents containing
organic chemicals (including dyes and pesticides), and 15
domestic wastewaters using the two-day red macroalga, Champia
parvula, sexual reproduction test- Nine single compounds were
used to compare the effects on sexual reproduction using a
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two-week exposure and a two-day exposure. For six of the :nine
compounds tested, the chronic values were the same for both
tests.
2.1.18 The use of short-term toxicity tests in .the NPDES Program
is especially attractive because they provide a more direct
estimate of the safe concentrations of effluents in receiving
waters than was provided by acute toxicity tests, at an only
slightly increased level of effort, compared to the fish full
life-cycle chronic and 28-day ELS tests and the 28-day mysid
life-cycle test.
2.2 TYPES OP 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 results of the
tests are expressed in terms of either the highest concentration
that has no statistically significant•• observed effect on those
responses when compared to the controls or the estimated
concentration that causes a specified percent reduction in
responses versus the controls.
2.2.3 Use of pass/fail tests consisting of a single effluent
concentration (e.g., the receiving water concentration or RWC)
and a control is not recommended. If the NPDES permit has a
whole effluent toxicity limit for acute toxicity at the RWC, it
is prudent to use that permit limit as the midpoint of a series
of five effluent concentrations. This will ensure that there is
sufficient information on the dose-response relationship.1 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.
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two treatments, a control and the undiluted receiving water, but
may also consist of a series of receiving water dilutions.
2.2.5 A negative result from a chronic toxicity test does not
preclude the presence of toxicity. Also, because of the
potential temporal variability in the toxicity of effluents, a
negative test result with a particular sample does not preclude
the possibility that samples collected at some other time might
exhibit chronic toxicity.
2.2.6 The frequency with which chronic toxicity tests are
conducted under a given NPDES permit is determined by the
regulatory agency on the basis of factors such as the variability
and degree of toxicity of the waste, production schedules, and
process changes.
2.2.7 Tests recommended for use in this methods manual may be
static non-renewal or static renewal. Individual methods specify
which 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
other prescribed interval, either by transferring the te.st
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. ,
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Disadvantages:
1. Dissolved oxygen (DO) depletion may result from high
chemical oxygen demand (COD) , biological oxygen demand
(BOD), or metabolic wastes.
2. Possible loss of toxicants through volatilization and/or
adsorption to the exposure vessels.
3. Generally less sensitive than renewal because the toxic
substances may degrade or be adsorbed, thereby reducing
the apparent toxicity. Also, there is less chance of
detecting slugs of toxic wastes, or other temporal
variations in waste properties.
2.4.2 STATIC RENEWAL, SHORT-TERM TOXICITY TESTS: .'
Advantages: , !
1. Reduced possibility of DO depletion from high COD arid/or
BOD, or ill effects from metabolic wastes from organisms
in the test solutions. ;
2. Reduced possibility of loss of toxicants through :
volatilization and/or adsorption to the exposure vessels,
3. Test organisms that rapidly deplete energy reserves.are
fed when the test solutions are renewed, and are
maintained in a healthier state.
Disadvantages: |
1. Require greater volume of effluent than non-renewal'
tests.
2. Generally less chance of temporal variations in waste
properties.
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SECTION 3
HEALTH AND SAFETY
3.1 GENERAL PRECAUTIONS
3.1.1 Each laboratory should develop and maintain an effective
health and safety program, requiring an ongoing commitment by the
laboratory management and includes: (1) a safety officer with
the responsibility and authority to develop and maintain a safety
program; (2) the preparation of a formal, written, health and
safety plan, which is provided'to the laboratory staff; (3) an
ongoing training program on laboratory safety; and (4) regularly
scheduled, documented, safety inspections.
3.1.2 Collection and use of effluents in toxicity tests may
involve significant risks to personal safety and health.
Personnel collecting effluent samples and conducting toxicity
tests should take all safety precautions necessary for the
prevention of bodily injury and illness which might result from
ingestion or invasion of infectious agents, inhalation or
absorption of corrosive or toxic substances through skin contact,
and asphyxiation due to a lack of oxygen or the presence of
noxious gases.
3.1.3 Prior to sample collection and laboratory work, personnel
should determine that all necessary safety equipment and
materials have been obtained and are in good condition.
3.1.4 Guidelines for the handling and disposal of hazardous
materials must be strictly followed. :
3.2 SAFETY EQUIPMENT \
3.2.1 PERSONAL SAFETY GEAR
3.2.1.1 -Personnel must use safety equipment, as required, such
as rubber aprons, laboratory coats, respirators, gloves, safety
glasses, hard hats, and safety shoes. Plastic netting on glass
beakers, flasks and other glassware minimizes breakage and
subsequent shattering of the glass.
3.2.2 LABORATORY SAFETY EQUIPMENT i-
3.2.2.1 Each laboratory (including mobile laboratories) should
be provided with safety equipment such as first aid kits, fire
extinguishers, fire blankets, emergency showers, chemical spill
clean-up kits, and eye fountains.
11 ;
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3.2.2.2 Mobile laboratories should be equipped with a telephone
to enable personnel to summon help in case of emergency.
3.3 GENERAL LABORATORY AND FIELD OPERATIONS
3.3.1 Work with effluents should be performed 'in compliance with
accepted rules pertaining to the handling of hazardous materials
(see safety manuals listed in Section 3, Health and Safety,
Subsection 3.5). It is recommended that personnel collecting
samples and performing toxicity tests should not work alone.
3.3.2 Because the chemical composition of effluents is usually
only poorly known, they should be considered as potential health
hazards, and exposure to them should be minimized. Fume and
canopy hoods over the toxicity test areas must be used whenever
possible.
3.3.3 It is advisable to cleanse exposed parts of the body
immediately after collecting effluent samples. ;
3.3.4 All containers should be adequately labeled to indicate
their contents.
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
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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
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SECTION 4
QUALITY ASSURANCE
4 .1 INTRODUCTION
4.1.1 Development and maintenance of a toxicity test laboratory
quality assurance (QA) program (USEPA, 1991b) requires an ongoing
commitment by laboratory management. Each toxicity test
laboratory should (1) appoint a quality assurance officer with
the responsibility and authority to develop and maintain a QA
program, (2) prepare a quality assurance plan with stated data
quality objectives (DQOs), (3) prepare written.descriptions of
laboratory standard operating procedures (SOPs) for culturing,
toxicity testing, instrument calibration, sample chain-of-custody
procedures, laboratory sample tracking system, glassware
cleaning, etc., and (4) provide an adequate, qualified technical
staff for culturing and toxicity testing the organisms, and
suitable space and equipment to assure reliable data.
4.1.2 QA practices for toxicity testing laboratories must
address all activities that affect the quality of the final
effluent toxicity data, such as: (1) effluent sampling and
handling; (2) the source and condition of the test organisms; (3)
condition of equipment; (4) test conditions; (5) instrument
calibration; (6) replication; (7) use of reference toxicants; (8)
record keeping; and (9) data evaluation.
4.1.3 Quality control practices, on the other hand, consist of
the more focused, routine, day-to-day activities carried out
within the scope of the overall QA program. For more detailed
discussion of quality assurance and general guidance on good
laboratory practices and laboratory evaluation related to
toxicity testing, see FDA (1978); USEPA (1979d); USEPA (1980b);
USEPA (1980c); USEPA (1991c); DeWoskin (1984); and Taylor (1987).
4.1.4 Guidelines for the evaluation of laboratory performing
toxicity tests and laboratory evaluation criteria are found in
USEPA (1991c).
4.2 FACILITIES, EQUIPMENT, AND TEST CHAMBERS
4.2.1 Separate test organism culturing and toxicity testing
areas should be provided to avoid possible loss of cultures due
to cross-contamination. Ventilation systems should be designed
and operated to prevent recirculation or leakage of air from
chemical analysis laboratories or sample storage and preparation
14
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areas into organism culturing or testing areas, and from testing
and sample preparation areas .into culture rooms.
4.2.2 Laboratory and toxicity test temperature control equipment
must be adequate to maintain recommended test water temperatures.
Recommended materials must be used in the fabrication of the test
equipment which comes in contact with the effluent (see Section
5, Facilities, Equipment, and Supplies;. and specific toxicity
test method). , , . .
4.3 TEST ORGANISMS
4.3.1 The test organisms used .in the procedures described .in
this manual are. the red abalone, Haliotis rufescens; the Pacific
oyster, Crassostrea gigas, and mussel, .-My til us spp.; the.
topsmelt, Atherinops affinis; the mysid, Holmesimysi-$ costata;*
the sea urchin,, Str.ongylocentxptus purpuratus., ,and the s.and
dollar Denstraster excentricus; and the giant kelp, Macrocystis
pyrifera. The. organisms used, should be disease-free, and appear
healthy, behave normally, feed: well, and:, have low mortality in
cultures, during holding, and in test control. Test organisms
should be positively identified to-species ('see Section 6, Test
Organisms) . , '.'.'.• . '
4.4 LABORATORY WATER USED FOR CULTURING AND TEST DILUTION WATER
4.4 il The quality of water used fo'r test- organism culturing and
for dilution water used in toxicity tests is extremely important.
Water for these two uses should come from the same source. The
dilution, water; used'in effluent toxicity tests will depend on the
objectives of the study and Logistical constraints, as discussed
in Section 7,'Dilution Water. The.dilution water'Used in the
toxicity tests may be natural seawater, hypersaline-brine .
(100%;) prepared from natural seawater^ or - artificial seaWater
prepared from commercial sea salts, such as FORTY FATHOMS® or HW
MARINEMIX®,. if recommended in 'the method.. GP2 synthetic
seawater, made from reagent grade chemical salts in conjunction
with natural seawater, may also be-used'if recommended. Types of
water are discussed in Section 5, Facilities, Equipment, and
Supplies. Water used for culturing and test dilution water
should be analyzed for toxic metals and organics at least
annually or whenever difficulty is-encountered in meeting1 minimum
acceptability criteria- for control survival and reproduction or
growth. The concentration of the metals, Al, As, Cr, Go, Cu, Fe,
Pb, Ni, , Zn, expressed as total metal, .should not exceed 1 pg/L
each, and Cd, Hg, and Ag, expressed as total metal, should not
exceed 100 ng/L each. Total organochlorine pesticides plus PCBs
should be less than 50 ng/L (APHA, 1992) . Pesticide
.15
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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 'acute
or short-term chronic toxicity tests are performed with effluents
or receiving waters using test organisms obtained from outside
the test laboratory, concurrent toxicity tests of the same type
must be performed with a reference toxicant, unless the test
organism supplier provides control chart data from at least the
last five monthly short-term chronic toxicity tests using the
same reference 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
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4.7.3 If the laboratory maintains breeding cultures, the
sensitivity of the offspring should be determined in a short-term
chronic toxicity test performed with a reference toxicant at
least once each month (see Section 4, Quality Assurance,
Subsection.4.14, 4.15, 4.16, and 4.17). If preferred, this
reference toxicant test may be performed concurrently- with an
effluent toxicity test. However, if a given species of test : .
organism produced by inhouse cultures is used only monthly, or ...
less frequently in toxicity tests, a. reference toxicant test must
be performed concurrently with each short-term chronic effluent
and/or receiving water toxicity test. '-. . :
- - - _ ' " I . ' "•-_:,-'
4.7.4 If a routine reference toxicant test fails to meet
acceptability criteria, the test must be immediately repeated.-
If the failed reference toxicant test was being performed
concurrently with an effluent or receiving water toxicity test,
both tests must be repeated (For exception, see 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 ;i
quality of the toxicity test data. This is especially true for
the unsaturated fatty acid content of brine shrimp nauplii,
Artemia. Problems with the nutritional suitability of the food
will be reflected in the survival, growth, and reproduction of
the test organisms in cultures and toxicity tests. Artemia cysts
and other foods must be obtained as described in Section 5,
Facilities, Equipment, and Supplies. • : :
4.8.2 Problems with the nutritional suitability of food will be
reflected in the survival, growth, 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 with the
performance of organisms fed with a food of known quality in
side-by-'side tests. If the food is used for culturing, its
suitability should be determined using a short-term chronic test
which will determine the affect of food quality on growth or
reproduction of each of the relevant test species in culture,
using four replicates with each food source. Where applicable,
foods used only in chronic toxicity tests can be compared with a
food of known quality in side-by-side, multi-concentration
chronic tests, using the reference toxicant regularly employed in
the laboratory QA program. For list of commercial sources of
Artemia cysts, see Table 2 of Section 5, Facilities, Equipment,
and Supplies.
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4.17 REFERENCE TOXICANTS
4.17.1 Reference toxicants such as zinc sulfate (ZnS04) , cadmium
chloride (CdCl2) / copper sulfate (CuS04) , and copper chloride
(CuCl2) / are suitable for use in the NPDES Program and other
Agency programs requiring aquatic toxicity tests. NERL-
Cincinnati plans to release USEPA-certified solutions of cadmium
and copper for use as reference toxicants, through cooperative
research and development agreements with commercial suppliers,
and will continue to develop additional reference toxicants for
future release. 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 sheet;'the
original bench sheets for the test organism responses during the
toxicity test(s); chemical analysis data on the sample(s);
detailed records of. the test organisms used in the test(s);, such
as species, source, age, date of receipt, and other pertinent
information relating to their history and health; information on
the calibration of equipment and instruments; test conditions
employed; and results of reference toxicant tests. Laboratory
data should be recorded on a real-time basis to prevent the loss
of information or inadvertent introduction of errors into the
record. Original data sheets should be signed and dated by the
laboratory personnel performing the tests.
4.18.2 The regulatory authority should retain records pertaining
to discharge permits. Permittees are required to retain records
pertaining to permit applications and compliance for a minimum of
3 years [40 CFR 122.41 (j) (2) ].•
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SECTION 5
FACILITIES, EQUIPMENT, AND SUPPLIES
5.1 GENERAL REQUIREMENTS
5.1.1 Effluent toxicity tests may be performed .in a fixed or
mobile laboratory. Facilities must include equipment for rearing
and/or holding organisms. Culturing facilities for test
organisms may be desirable in fixed laboratories which perform
large numbers of tests. Temperature -control can be achieved
using circulating water baths, heat exchangers, :or environmental
chambers. Water used for rearing, holding, acclimating, and ;
testing organisms may be natural seawater or water made up from
hyper-saline 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-l filter,
or equivalent).
5.1.2 The facilities must be well ventilated^and free of fumes.
Laboratory ventilation systems should be checked to ensure that
return air from chemistry laboratories and/or sample handling
areas is not circulated to test organism culture' rooms or
toxicity test rooms, or that air from toxicity test rooms does
not contaminate culture areas. Sample preparation, culturing,
and toxicity testing areas should be separated to avoid cross-
contamination of cultures or. toxicity test solutions with toxic-
fumes. Air pressure differentials between such rooms should.not
result in a net flow of potentially contaminated air to sensitive
areas through open or loosely-fitting doors. Organisms should be
shielded from external disturbances.
5.1.3 Materials used for exposure chambers, tubing, etc., which
come in contact with the effluent and dilution water, should be
carefully chosen. Tempered glass and perfluorocarbon plastics
(TEFLON®) should be used whenever possible to minimize sorption
and leaching of toxic substances. These materials may be reused
following decontamination. Containers made of plastics, such as
polyethylene, polypropylene, polyvinyl chloride, TYGON®, etc.,
may be used as test chambers or to ship, store, and transfer
effluents and receiving waters, but they should not be reused
unless absolutely necessary, because they might carry over
25
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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 more 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 identity of test organisms
is known to the species level, it would be necessary to
examine each organism caught in the wild to confirm its
identity, which would usually be impractical or, at the
least, very stressful to the organisms.
6. Test organisms obtained from the wild must be observed in
the laboratory for a minimum of one week prior to use, to
ensure that they are free of signs of parasitic or[
bacterial infections and other adverse effects. Fish
captured by electroshocking must not be used in toxicity
testing.
6.2.5.2 Guidelines for collection of 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. ;
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6.4 LABORATORY CULTURING
6.4.1 Instructions for culturing, holding and/or handling the
recommended test organisms and broodstock are included in
specified test methods. . i
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 Sfe in salinity in any 12 h
period. '•
6.5.2 Organisms should be handled as little as possible. When
handling is necessary, it should be done as gently, carefully,
and quickly as possible to minimize stress. Organisms that are
dropped or touch dry surfaces or are injured during handling must
be discarded. Dipnets are best for handling larger organisms.
These nets are commercially available or can be made from small-
mesh nylon netting, silk bolting cloth, plankton netting, or
similar material. Wide-bore, smooth glass tubes :(4 to 8 mm ID)
with rubber bulbs or pipettors (such as a PROPIPETTE® or other
pipettor) should be used for transferring smaller organisms such
as mysids, and larval fish. . ' ,
6.5.3 Holding tanks for 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 be piped through
high intensity ultraviolet light sources for disinfection, and to
photo-degrade dissolved organics. ;
6.5.4 Crowding should be avoided because it will stress the
organisms and lower the DO concentrations to unacceptable levels.
The DO must be maintained at a minimum of 4.0 mg/L. The
solubility of oxygen depends on temperature, salinity, and
altitude. Aerate gently if necessary. ' ,
6.5.5 The,, organisms should be observed carefully each day for
signs of disease, stress, physical damage, or mortality. Dead
and abnormal organisms should be removed as soon as observed. It
is not uncommon for some larval fish and mysid mortality (5-10%)
to occur during the first 48 h in a holding tank because of
individuals that failed to feed and die of starvcition.
6.5.6 Organisms in the holding tanks should generally be fed as
in the cultures (see culturing methods in the respective
methods).
33
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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 replacing
the air above the water in the bags with oxygen from a compressed
gas cylinder, and sealing the bags. Another method commonly used
to maintain sufficient DO during shipment is to aerate with an
airstone which is supplied from a portable pump. The DO
concentration must not fall below 4.0 mg/L.
6.6.2 Upon arrival at the test site, organisms are transferred
to receiving water if receiving water is to be used as the test
dilution water. All but a small volume of the holding water
(approximately 5%) is removed by siphoning, and replaced slowly
over a 10 to 15 minute period with dilution water. If receiving
water is used as dilution water, caution must be exercised in
exposing the test organisms to it, because of the possibility
that it might be toxic. For this reason, it is recommended that
only approximately 10% of the test organisms be exposed initially
to the dilution water. If this group does not show excessive
mortality or obvious signs of stress in a few hours, the
remainder of the test organisms are transferred to the dilution
water.
6.6.3 A group of organisms must not be used for a test if they
appear to be unhealthy, discolored, or otherwise stressed, or if
mortality appears to exceed 10% preceding the test. If the
organisms fail to meet these criteria, the entire group must be
discarded and a new group obtained. The mortality may be due to
the presence of toxicity, if receiving 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.
i
34 :
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6.6.5 Saline dilution water can be prepared with deionized water
or a freshwater such as well water or a suitable surface water.
If dry ocean salts are used, care must be taken to ensure that
the added salts are completely dissolved and the solution is
aerated 24 h before the test organisms are placed in the
solutions. The test organisms should be acclimated in synthetic
saline water prepared with the dry salts. Caution: addition of
dry ocean salts to dilution water may result in an increase in
pH. (The pH of estuarine and coastal saline waters is normally
7.5-8.3). ;
6.6.6 All effluent concentrations and the control(s) used in a
test should have the same salinity. The change in salinity upon
acclimation at the desired test dilution should, not exceed 6%.
The required salinities for culturing and toxic'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 test(s) is concluded, all test organisms
(including controls) should be humanely destroyed and disposed of
in an appropriate manner. :
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SECTION 7
DILUTION WATER :
7.1 TYPES OF DILUTION WATER
7.1.1 The type of dilution water used in effluent toxicity tests
will depend largely on the objectives of the study.
7.1.1.1 If the objective of the test is to estimate the chronic
toxicity of the effluent, which is 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 receiving 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
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7.2.2.1 Reagent water is defined as distilled or deionized water
that does not contain substances which are toxic to the test
organisms. Deionized water is obtained from a MILLIPORE
MILLI-Q®, MILLIPORE® QPAK™2 -or equivalent system. It is
advisable to provide a preconditioned (deionized) feed water by
using a Culligan®, Continental®, or equivalent system in front of
the 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 QPAK™2 water system is a sealed .
system which does not allow for the rearranging of the
cartridges. However, the final cartridge is an ORGANEX-Q®
filter, followed'by a final bacteria filter. Commercial
laboratories using this system have not experienced any
difficulty in using the water for culturing or testing.
Reference to the MILLI-Q® systems throughout the remainder of the
manual includes all MILLIPORE® or equivalent systems.
7.2.3 STANDARD, SYNTHETIC SEAWATER .'j-
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 3Its, follow the instructions, below. Other
salinities can be prepared by making the appropriate dilutions..
Larger, or smaller volumes of modified GP2 can be prepared by
using proportionately larger or smaller amounts of salts, and
dilution water.'
1. Place 20 L of MILLI-Q® or equivalent., deionized water in a
properly cleaned plastic carboy.
2. Weigh reagent grade-salts listed in Table 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 1 L/h for 24 h.
4. Check the pH and salinity.
7.2.3.2 Synthetic seawater can also be prepared by adding
commercial sea salts, such as FORTY FATHOMS®, HW MARINEMIX®, or
equivalent, to deionized water. For example,, thirty-one parts
per thousand (31Vo) 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.
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TABLE 2. PREPARATION OF GP2 ARTIFICIAL SEAWATER USING
CHEMICALS1
REAGENT GRADE ____!,2,3
Compound
NaCl
Na2S04
KC1
KBr
Na2B407 • 10 H20
MgCl2 ' 6 H20
Cad, • 2 H2o
SrCln • 6 H2O
NaHC03
Concentration
(g/L)
21
3
0
0
0
9
1
0
0
.03
.52
.61
.088
.034
.50
.32
.02
.17
Amount (g)
Required for
20 L
420.6
70.4
12.2 ;
1.76
0.68 '••
190.0'
26.4
0.400
3.40
, ___— -— r-"
1 Modified GP2 from Spotte et al. (1984).
2 The constituent salts and concentrations were taken from
USEPA (1993a). The salinity is 30.89 g/L. ;
3 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
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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 in the estuary,
it is advisable to collect uncontaminated water from an adjacent
estuary. At times it may be necessary to collect water at a
location closer to the open sea, where the salinity is relatively
high. In such cases, deionized water or uncontaminated
freshwater is added to the saline water to dilute, it to the
.required test salinity. Where necessary, the salinity of a
surface water can be increased by the addition of artificial sea
salts, such as FORTY FATHOMS®, HW MARINEMIX®, or equivalent, GP2,
a 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 um mesh openings 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.
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.
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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 salinity. HSB has
several desirable characteristics for use in effluent toxicity
testing. Br'ine 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 (lOOVo) is used as a
diluent, the maximum concentration of effluent (0°r0) that can be
tested is 66% effluent at 34V« salinity.
7.3.5.3 High quality (and preferably high salinity) seawater
should be filtered to at least 10 urn 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°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) . • . '
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 lOOVo.
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 um 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 heat-
assisted 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
40
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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. . -
7.3.5.5.2 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.
7.3.5.5.3 Seawater should be filtered to at least 10 um 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 lOOVo and that the temperature does not exceed 40°C.
Additional seawater may be added to the brine to,obtain the
volume of brine required. ;
7.3.5.5.4 After the required salinity is attained, the HSB
should be filtered through a 1 um 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.
7.3.5.6 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 1QO'& and the test
is to be conducted at 34t-o, 1001; divided by 34"s-o = 2.94. Thus,
the proportion is one part brine plus 1.94 reagent water.
7.3.5.8 To make 1 L of seawater at 34Vo salinity from a
hypersaline brine of lOOfe,- 340 mL of brine and 660 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 :
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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 thiosulfate-
dechlorinated water.
7.4.2 To be adequate for general laboratory use following
dechlorination, the tap water is passed through a deionizer and
carbon filter to remove toxic metals and organics, and to control
hardness and alkalinity.
7.5 DILUTION WATER HOLDING
7.5.1 A given batch of dilution water should not be used for
more than 14 days following preparation because of the possible
build up of bacterial, fungal, or algal slime growth and the
problems associated with it. The container should be kept
covered and the contents should be protected from light.
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SECTION 8
EFFLUENT AND RECEIVING WATER SAMPLING, SAMPLE HANDLING,
AND SAMPLE PREPARATION FOR TOXICITY TESTS
8.1 EFFLUENT SAMPLING
8.1.1 The effluent sampling point should be the'same as that
specified in the NPDES discharge permit (USEPA, 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 effluent over the
sampling period. Sampling recommendations are provided below
(also see USEPA, 1993a). ;
8.1.3 Aeration during collection.and transfer of effluents
should be minimized to reduce the loss of volatile chemicals.
i
8.1.4 Details of date, time, location, duration,, and procedures
used for effluent sample and dilution water collection should be
recorded.
8.2 EFFLUENT SAMPLE TYPES
8.2.1 The advantages' and .disadvantages of effluent grab and
composite samples are listed below: .
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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: N
1. Sampling equipment is more sophisticated and expensive,
and must be placed on-site for at least 24 h.
2. Toxicity spikes may not be detected because they are
masked by dilution with less toxic wastes. !
8.3 EFFLUENT SAMPLING RECOMMENDATIONS
8.3.1 When tests are conducted on-site, test solutions can be
renewed daily with freshly collected samples.
8.3.2 When 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 (1-gal) CUBITAINER®
will provide sufficient sample volume for most tests.
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8.3.4 THE FOLLOWING EFFLUENT SAMPLING METHODS ARE RECOMMENDED:
8.3.4.1 Continuous Discharges
1. If the facility discharge is continuous, but the
calculated retention time of the continuously discharged
effluent is less than 14 days and the variability of the
effluent toxicity is unknown, at a minimum, four grab
samples or four composite samples are collected over a
24-h period. For example, a grab sample is taken every 6
h (total of four samples) and each sample is used for a
separate toxicity test, or four successive 6-h
composite samples are taken and each is used in a
separate test.
2. If the calculated retention time of a continuously
discharged effluent is greater than 14 days, or if it can
be demonstrated that the wastewater does not vary more
than 10% in toxicity over a 24-h period, regardless of
retention time, a single grab sample is collected for a
single toxicity test.
3. The retention time of the effluent in the wastewater
treatment facility may be estimated from .calculations
based on the volume of the retention basin and rate of
wastewater inflow. However, the calculated retention
time may be much greater than the actual time because of
short-circuiting in the holding basin. Where
short-circuiting is suspected, or sedimentation may have
reduced holding basin capacity, a more accurate estimate
of the retention time can be obtained by carrying out a
dye study.
8.3.4.2 Intermittent Discharges
8.3.4.2.1 If the facility discharge is intermittent, a grab
sample is collected midway during each discharge period.
Examples of intermittent discharges are:
1. When the effluent is continuously discharged during a
single 8-h work shift (one sample is collected), or two
successive 8-h work shifts (two samples are collected).
2. When the facility retains the wastewater during an 8-h
work shift, and then treats and releases the wastewater
as a batch discharge (one sample is collected).
3. When the facility discharges wastewater to an estuary
only during an outgoing tide, usually during the 4 h
following slack high tide (one sample is collected).
4. At the end of a shift, clean up activities may result in'
the discharge of a slug of toxic waste (one sample is
collected).
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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 ,the 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 test
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 residual
chlorine must be measured immediately following sample
collection.
8.5.4 Sample holding time begins when the last grab sample in a
series is taken (i.e., when a series of four grab samples are
taken over a 24-h period), or when a 24-h composite sampling
period is completed. If the data from the samples are to ;be
acceptable for use in the NPDES Program, the 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
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is adequate time to deliver the sample to the laboratories
performing the test in most cases. In the isolated cases, where
the permittee can document that this delivery time cannot be met,
the permitting authority can allow an option for on-site testing
or a variance for an extension of shipped sample holding time.
The request for a variance in sample holding time, directed to
the USEPA Regional Administrator under 40 CFR 136.3(e), 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 containeir 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 h and 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 contents and the lid.
8.5.6 SAMPLES USED IN ON-SITE TESTS •
8.5.6.1 Samples collected for on-site tests should be used
within 24 h. \
8.5.7 SAMPLES SHIPPED TO OFF SITE FACILITIES . j
8.5.7.1 Samples collected for off site toxicity testing are to
be chilled to 4°C during or immediately after collection, and
shipped iced to the performing laboratory. Sufficient ice
should be placed with the sample in the shipping container to
ensure that ice will still be present when the sample arrives at
the laboratory and is unpacked. Insulating material must not be
placed between the ice and the sample in the shipping container.
8.5.7.2 Samples may be shipped in one or more 4-L (1-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 carriers vary with
the carrier.
47
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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 RECEIVING WATER SAMPLES FOR
TOXICITY TESTS
8.8.1 Adjust the sample salinity to the level appropriate1 for
objectives of the study using hypersaline brine or artificial sea
salts.
8.8.2 When aliquots are removed from the sample container, the
head space above the remaining sample should be held to a
minimum. Air which enters a container upon removal of sample
should be expelled by compressing the container before reclosing,
if possible (i.e., where a CUBITAINER® used), or by using an
appropriate discharge valve (spigot).
8.8.3 It may be necessary to first coarse-filter samples through a
NYLON® sieve having 2 to 4 mm mesh openings to remove debris and/or
break up large floating or,suspended solids. If samples contain
indigenous organisms that may attack or be confused with the test
organisms, the samples must be filtered through a sieve with 60 um
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 will indicate a
potential problem in performing the test. Caution: filtration may
remove some toxicity. i
t
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 a: 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, until 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 100 bubbles/min. If aeration is necessary> all test
solutions must be aerated. It is advisable to monitor the DO
closely during the first few hours of the test. Samples with a
potential DO problem generally show a downward trend in DO within 4
to 8 h after the test is started. Unless aeration, is initiated
during the first 8 h of the test, the DO may be exhausted during an
unattended period, thereby invalidating the test. •
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. :
i
8.8.6 Total ammonia is measured in effluent and receiving water
samples where toxicity may be contributed by unionized ammonia
(i.e., where total ammonia >5 mg/L). The concentration (mg/L) of
unionized (free) ammonia in a sample is a function of temperature
and pH, and is calculated using the percentage value obtained from
Table 4,
49
-------
TABLE 3. OXYGEN SOLUBILITY (MG/L) IN WATER AT EQUILIBRIUM
WITH AIR AT 760 MM HG- (AFTER RICHARDS AND CORWIN,
1956)
TEMP
<=•>
0
1
2
3
4
5
6
8
10
12
14
16
18
20
22
24
26
28
30
32
SALINITY (V.)
0
14.2
13.8
13.4
13.1
12.7
12.4
12.1
11.5
10.9
10.5
10.0
9.6
9.2
8.9
8.6
8.3
8.1
7.8
7.6
7.3
5
13.8
13.4
13.0
12.7
12.3
12.0
11.7
11.2
10.7
10.2
9.7
9.3
9.0
8.6
8.4
8.1
7.8
7.6
7.4
7.1
10
13.4
13.0
12.6
12.3
12.0
11.7
11.4
10.8
10.3
9.9
9.5
9.1
8.7
8.4
8.1
7.8
7.6
7.4
7.1
6.9
15
12.9
12.6
12.2 .
11.9
11.6
11.3
11.0
10.5
10.0
9.6
9.2
8.8
8.5
8.1
7.9
7.6
7.4
7.2
6.9
6.7
20
12.5
12.2
11.9
11.6
11.3
11.0
10.7
10.2
9.7
9.3
8.9
8.5
8.2
7.9
7.6
7.4
7.2
7.0
6.7
6.5
25
12.1
11.8
11.5
11.2
10.9
10.6
10.3
9.8
9.4
9.0
8.6
8.3
8.0
7.7
7.4
7.2
7.0
6.8
6.5
6.3
30
11.7
11.4
11.1
10.8
10.5
10.2
10.0
9.5
9.1
8.7
8.3
8.0
• 7.7
7.4
7.2
6.9
6.7
6.5
6.3
6.1
35
11.2
11.0
10.7
10.4
10.1
9.8
9.6
9.2
8.8
8.4
8.1
7.7
7.5
7.2
6.9
6.7
6.5
6.3
6.1
5.9
40
10.8
10.6
10.3
10.0
9.8
9.5
9.3
8.9
8.5
8.1
7.8
7.5
7.2
6.9
6.7
6.5
6.3
6.1
5.9
5.7
43
10.6
10.3
10.0
9.8,
9.5
9.3
9.1
8.7
8.3
7.9
7.6
7.3
7.1
6.8
6.6
6.4
6.1
6.0
5.8
5.6
50
-------
TABLE 4. PERCENT UNIONIZED NH3 IN AQUEOUS AMMONIA SOLUTIONS:
TEMPERATURE 15-26°C AND pH 6.0-8.91
PH
TEMPERATURE (°C)
15
16
17
18
19 .
20
21
22
23
24
25
26
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7,7
7,8
7.9
8.0
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9
0.0274
0.0345
0.0434
0.0546
0.0687
0.0865
0.109
0.137
0.172
0.217
0.273
0.343
0.432
0.543
0.683
0.858
1.08
1.35
1.70
2.13
2.66
3.33
4.16
• 5.18
6.43
7.97
9.83
12.07
14.7
17.9
0.0295
0.0372
0.0468
0.0589
0.0741
0.0933
0.117
0.148
0.186
0.234
0.294
0.370
0.466
0.586
0.736
0.925
1.16
1.46
1.83
2.29
2.87
3.58
4.47
5.56
6.90
8.54
10.5
12.9
15.7
19.0
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0,
0.
0.
1.
1.
1.
2.
3.
3.
4.
5.
7.
9.
11.
13.
16.
20.
0318
0400
0504
0634
0799
1005
127
159
200
252
317
399
502
631
793
996
25
57
97
46
08
85
80
97
40
14
2
8
7
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
2
2
3
4
5
6
7
9
12
14
17
21
.0343
.0431
.0543
.0683
.0860
.1083
.136
.171
.216
.271
.342
.430
.540
.679
.854
.07
.35
.69
.12
.65
.31
,14
.15
.40
.93
.78
.0
.7
.8
.4
0.0369
0.0464
0.0584
0.0736
0.0926
0.1166
0.147
0.185
0.232
0.292
0.368
0.462
0.581
0.731
0.918
1.15
1.45
1.82
2.28
2.85
3.56
4,44
5.52
6.86
8.48
10.45
12.8
15.6
18.9
22.7
0.0397
0.0500
0.0629
0.0792
0.0996
0.1254
0.158
0.199
0.250
0.314
0.396
0.497
0.625
0.786
0.988
1.24
1.56
1,95
2.44
3.06
3.82
4.76
5.92
7.34
9.07
11.16
13.6
16.6
20'. 0
24.0 .
0.0427
0.0537
0.0676
0.0851
0.107
0.135
0.170
0.214
0.269
0.338
0.425
0.535
0.672
0.845
1.061
1.33
1.67
2.10
2.62
3.28
4.10
5.10
6.34
7.85
9.69
11.90
14.5
17.6
21.2
25.3
0.0459
0.0578
0.0727
0.0915
0.115
0.145
0.182
0.230
0.289
0.363
0.457
0.575
0.722
0.908
1.140
1,43
1.80
2.25
2.82
3.52
4.39
5,46
6.78
8.39
10.3
12.7
15.5
18.7
22.5
26.7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
2
3
3
4
5
7
8
11
13
16
19
23
28
.0493
.0621
.0781
.0983
.124
.156
.196
.247
.310
.390
.491
.617
.776
.975
.224
.54
.93
.41
.02
.77
.70
.85
.25
.96
.0
.5
.4
.8
.7
i
. C. :
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
1.
1.
2.
2 .
3.
4.
5.
6.
7,
9.
11.
14.
17.
21.
25.
29.
0530
0667
0901
1134
133
167
210
265
333
419
527
663
833
05
31
65
°7
59
24
04
03
25
75
56
7
*
4
0
1
S.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
1.
1.
2.
2 .
3.
4.
5.
6.
8.
10.
12,
15.
18.
22.
26.
31.
0568
0716
0901
1134
143
180
226
284
358
450
566
711
893
12
41
77
21
77
46
32
38
68
27
f.
5
2
5
2
4
1
o.'oeio
0.0768
0.0966
0.1216
0.153
0.193
0.242
0.305
0.384
0.482
0.607
0.762
0.958
1.20
1.51
1.89
2.37
2.97
3.71
4.62
5.75
7.14
8. 82
10.9
13.3
16.2
19.5
23.4
27.8
32.6
'Table provided by Teresa Norberg-King, 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
(APHA, 1992). Note that the amount of thiosulfate required to
dechlorinate effluents is greater than the amount needed to
dechlorinate tap water, (see Section 7, Dilution Water). Since
thiosulfate may contribute to sample toxicity, a thiosulfate,
control should be used in the test in addition to the normal
dilution water control.
8.8.8 The DO concentration in the samples should be near
saturation prior to use. Aeration will bring the DO and other
gases into equilibrium with air, minimize oxygen demand, and
stabilize the pH. However, aeration during collection, transfer,
and preparation of samples should be minimized to reduce the loss
of volatile chemicals.
8.8.9 Mortality or impairment of growth or reproduction due to pH
alone may occur if the pH of the 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 IN NaOH or IN HC1
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 range-
finding test.
8.9.2 A toxicity range-finding test ordinarily consists of a down-
scaled, abbreviated static acute test in which groups of five
organisms are exposed to several widely-spaced sample dilutions in
a logarithmic series, such as 100%, 10.0%, 1.00%, and 0.100%, and a
control, for 8-24 h. Caution: if the sample must also be used for
the full-scale definitive test, the 36-h limit on holding time (see
Subsection 8.5.4) must not be exceeded before the definitive test
is initiated.
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 MULTICONCENTRATION (DEFINITIVE) EFFLUENT TOXICITY TESTS
8.10.1 The tests recommended for use in determining discharge
permit compliance in the NPDES program are multiconcentration or
definitive tests. 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 non-
renewal .
8.10.2 The tests consist of a control and a minimum of five
effluent concentrations commonly selected to approximate a
geometric series, such as 60%, 30%, 15%, 7.5%, and 3.75%, using a
>0.5 dilution series.
8.10.3 These tests are also to be used in determining compliance •
with permit limits on the mortality of the receiving water
concentration (RWC) of effluents by bracketing the RWC with
effluent concentrations in the following manner. For example, if
the RWC is >25% then, the effluent concentrations utilized in a
test may be: (1) 100% effluent, (2) (RWC + 100)72, (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/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 !
-• I -
CHRONIC TOXICITY TEST ENDPOINTS AND DATA ANALYSIS
9.1 ENDPOINTS
9.1.1 The objective'of chronic aquatic toxicity tests with
effluents and pure compounds is to estimate the highest-"safe" or
"no-effect concentration" of these substances. "For practical
reasons, the responses observed in these tests are usually limited
to 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 purrently used to define the
endpoints employed in the rapid, .chronic and-sub-chronic toxicity
tests have been derived from the terms previously used for full '
life-cycle tests. As shorter chronic tests were'developed, it
became common practice to apply the same terminology :to the -
endpoints. The terms used in this manual are as •'follows:
9.1.1.1 Safe Concentration - The highest concentration-of toxicant
that will 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-effe'ct concentration"
(below): is a statistically defined concentration.
9.1.1.2 No-Observed-Effect-Concentration (NOEC) - The highest
concentration of toxicant to which organisms--are exposed-in a full
life-cycle or partial .life-cycle (short-term) test, that cause's no
observable adverse effects on the test organisms' (i.e., the highest
concentration of toxicant in which the values for-the observed
responses are not statistically significantly different from the'
controls). This value is used, along with "other factors, to
determine toxicity limits in permits. • -• •
9.1.1.3 Lowest-Observed-Effect-Concentration (LOEC) — The lowest
concentration of toxicant to which organisms are-exposed in a life-
cycle or . . " -- - . -r :.
partial life-cycle (short-term) test,:which causes adverse effects
on the test organisms '(i.e., where the values for the observed "
responses are statistically significantly different from the
controls) . -' ••-...[•• '•"'"": ' ,.- :.:•"'.
9.1.1.4 Effective Concentration (EC) - A point estimate of the
toxicant concentration that would cause an observable adverse
affect on a quantal, "all or nothing," response (such as death,
fertilization, germination or, development) in a given percent of
the test organisms, calculated by point estimation techniques. If
55
-------
analysis alone/ unless (I) 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 data sets- included short-term
chronic toxicity tests for the sea urchin, Arbacia punctulata, the
sheepshead minnow, Cyprinodon variegatus, and the red macroalga,
Champia parvula. Birge et al. (1985) reported that LCIs derived
from Probit Analyses of data from short-term embryo-larval tests
with reference toxicants were comparable to NOECs for several
organisms. Similarly, USEPA (1988d) reported that the IC25s were
comparable to the NOECs for a set of daphnia/ Ceriodaphnia dubia :
chronic tests with a single reference toxicant. However, the scope
of these comparisons was very limited, and sufficient information
is not yet available to establish an overall relationship between
these two types of endpoints, especially when derived from effluent
toxicity test data.
9.3 PRECISION
9.3.1 HYPOTHESIS TESTS •.-.•...-.
9.3.1.1 When hypothesis tests are used to analyze toxicity test
data, it is not possible to express precision in terms of a
commonly used statistic. The results of the test are given in
terms of two endpoints, the No-Observed-Effect Concentration (NOEC)
and the Lowest-Observed-Effect Concentration (LOEC). The NOEC and
LOEC are limited to the concentrations selected for the test. The
width of the NOEC-LOEC interval is a function of the dilution
series, and differs greatly depending on whether a dilution factor
of 0.3 or 0.5 is used in the test design. Therefore, USEPA ~
recommends the use of the ^0.5 dilution factor (see Section 4,
Quality Assurance). It is not possible to place confidence limits
on the NOEC and LOEC derived from a given test, and it is difficult
to quantify the precision of the NOEC-LOEC endpoints between tests.
If the data from a series of tests performed with the^ same
58
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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 interpretation of the results of the
analysis of the data from any of the toxicity tests described in
this manual can become problematic because of the inherent
variability and sometimes unavoidable anomalies in biological data.
If the data appear unusual in any way, or fail to meet the
necessary assumptions, a statistician should be consulted.
Analysts who are 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
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by biologists or toxicologists of some low level of adverse effect
that would be deemed acceptable or safe. The statistical methods
contained in this manual have been chosen because they are (1)
applicable to most of the different toxicity test data sets for
which they are recommended, (2) powerful statistical tests, (3)
hopefully "easily" understood by nonstatisticians, and (4) amenable
to use without a computer, if necessary.
9.4.2 PLOTTING THE DATA
9.4.2.1 The data should be plotted, both as a preliminary step to
help detect problems and unsuspected trends or patterns in the
responses, and as an aid in interpretation of the results. Further
discussion and plotted sets of data are included in the methods and
the Appendices.
9.4.3 DATA TRANSFORMATIONS
9.4.3.1 Transformations of the data, (e.g., arc sine square:root
and logs), are used where necessary to meet assumptions of the
proposed analyses, such as the requirement for normally distributed
data.
9.4.4 INDEPENDENCE, RANDOMIZATION, AND OUTLIERS
9.4.4.1 Statistical independence among observations is a critical
assumption in all statistical analysis of toxicity data. One of
the best ways to ensure independence is to properly follow rigorous
randomization procedures. Randomization techniques should be
employed at the start of the test, including the randomization of
the placement of test organisms in the test chambers and
randomization of the test chamber location within the array of
chambers. Discussions of statistical independence, outliers and
randomization, and a sample randomization scheme, are included in
Appendix A.
9.4.5 REPLICATION AND SENSITIVITY
9.4.5.1 The number of replicates employed for each toxicant
concentration is an important factor in determining the sensitivity
of chronic toxicity tests. Test sensitivity generally increases as
the number of replicates is increased, but the point of diminishing
returns in sensitivity may be reached rather quickly. The level of
sensitivity required by a hypothesis test or the confidence
interval for a point estimate will determine the number of
replicates, and should be based on the objectives for obtaining the
toxicity data.
9.4.5.2 In a statistical analysis of toxicity data, the choice of
a particular analysis and the ability to detect departures from the
assumptions of the analysis, such as the normal distribution of the
60
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data and homogeneity of variance, is also dependent on the number
of replicates. More than the minimum number of replicates may be
required in situations where it is imperative to obtain optimal
statistical results, such as with tests used in enforcement cases
or when it is not possible to repeat the tests. For example, when
the data are analyzed by hypothesis testing, the nonparametric
alternatives cannot be used unless there are at least four
replicates at each toxicant concentration.
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
testc.
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-observed-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 significant
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 In a more powerful
and appropriate statistical analysis. In performing the point
estimation techniques recommended in this manual, ian all-data
61
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DATA (SURVIVAL, GROWTH, REPRODUCTION, ETC.)
POINT
ESTIMATION
HYPOTHESIS TESTING
TRANSFORMATION?
ENDPOINT ESTIMATE
LC. EC, 1C
SHAPIRO-WILK'S TEST
NORMAL DISTRIBUTION
NON-NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
BARTLETT'S TEST
HETEROGENEOUS
VARIANCE
NO STATISTICAL ANALYSIS
RECOMMENDED
NO
4 OR MORE
REPLICATES?
YES
EQUAL NUMBER OF
REPLICATES?
NO
YES
EQUAL NUMBER OF
REPLICATES?
YES
NO
T-TESTWITH
BONFERRONI
ADJUSTMENT
DUNNETTS
TEST
STEEL'S MANY-ONE
RANK TEST
. WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC •
Figure 2. Flowchart for statistical analysis of test data,
62 ,
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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, Atherinops 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. Growth is defined as
the change in dry weight of the orginal number of test organisms
when group weights are obtained. When analyzing the data using
point estimating techniques, data from all concentrations are
included in the analysis.
9.5.4 ANALYSIS OF FERTILIZATION, GERMINATION AND DEVELOPMENT DATA
9.5.4.1 Data from the purple urchin, Strongylocentrotus purpuratus
'and the sand dollar, Denstraster excentricus, fertilization test
and development test; the red abalone Haliotis rufescens, the
Pacific oyster, Crassostrea gigas, and mussel, Mytilus spp., larval
development tests; and the giant kelp, Macrocystis pyrifera,
germination test may be analyzed by hypothesis testing 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 Analysis, ^if
appropriate, or other point estimation techniques, (i.e., the
Spearman-Karber Method, the Trimmed Spearman-Karber Method, or the
Graphical Method) (see Appendices 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 1
i
9.6.1.1 Dunnett's Procedure is used to determine the NOEC. The
procedure consists of an analysis of variance (ANOVA) to determine
the error term, which is then used in a multiple comparison
i
63 i
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procedure for comparing each of the treatment means with the
control mean, in a series of paired tests (see Appendix C). Use of
Dunnett's Procedure requires at least three .replicates per
treatment to check the assumptions of the test. In cases where the
numbers of data points (replicates) for each concentration are not
equal, a t test may be performed with Bonferroni's adjustment for
multiple comparisons (see Appendix D)/ instead of using Dunnett's
Procedure.
9.6.1.2 The assumptions upon which the use of Dunnett's Procedure
is contingent are that the observations within treatments are
normally distributed, with homogeneity of variance. Before
analyzing the data, these assumptions must be tested using the
procedures provided in Appendix B.
9.6.1.3 If, after suitable transformations have been carried out,
the normality assumptions have not been met, Steel's Many-one Rank
Test should be used if there are four or more data points
(replicates) per toxicant concentration. If the numbers of data
points for each toxicant concentration are not equal, the Wilcoxon
Rank Sum Test with Bonferroni's adjustment should be used (see
Appendix F).
9.6.1.4 Some indication of the sensitivity of the analysis should
be provided by calculating (1) the minimum difference between means
that can be detected as statistically significant, and (2) the
percent change from the control mean that this minimum difference
represents for a given test. . .
9.6.1.5 A step-by-step example of the'use-of'Dunnett's Procedure
is provided in Appendix C.
9.6.2 T TEST WITH THE BONFERRONI ADJUSTMENT
9.6.2.1 The t test with the Bonferroni adjustment is used as an
alternative to Dunnett's Procedure when the number of replicates is
not the same for all concentrations. This test sets an upper bound
of alpha on the overall error rate, in contrast to Dunnett's
Procedure, for which the overall error rate is fixed at alpha.
Thus, Dunnett's Procedure is a more powerful test.
9.6.2.2 The assumptions upon which the use of the t test with the
Bonferroni adjustment is contingent are that the observations
within treatments are normally distributed, with homogeneity of
variance. These assumptions must be tested using the procedures
provided in Appendix B.
9.6.2.3 The estimate of the safe concentration derived from this
test is reported in terms of the NOEC. A step-by-step example of
the use of a t-test with the Bonferroni adjustment is provided in
Appendix D.
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9.6.3 STEEL'S MANY-ONE RANK TEST '
9.6.3.1 ' Steel's Many-one Rank Test is a multiple comparison
procedure for comparing several treatments with a control. This
method is similar to Dunnett's procedure, except that it is not
necessary to meet the assumption of normality. The data are
ranked/ and the'analysis is performed'on the ranks rather than on
the data themselves. If the data are normally or nearly normally
distributed, Dunnett's Procedure would be more sensitive (would
detect smaller differences between the treatments and control).
For data that are not normally distributed, Steel's Many-one Rank
Test can be much more efficient -(Hodges and Lehmann, 1956).
9.6.3.2 It is necessary to have at least four replicates per
toxicant concentration to use Steel's test. Unlike Dunnett's
procedure, the sensitivity of this test cannot be: stated in terras
of the minimum difference between treatment means and the control
mean that can be detected as statistically significant.
9.6.3.3 The estimate of the safe concentration is reported as the
NOEC. A step-by-step example of the use of Steel's Many-One Rank
Test is provided in Appendix E. '
9.6.4 WILCOXON RANK SUM TEST '
9.6.4.1 The Wilcoxon Rank Sum Test is a nonparametric test for
comparing a treatment with a control. The data are ranked and the
analysis proceeds exactly as in Steel's Test except that
Bonferroni's adjustment for multiple comparisons is used instead of
Steel's tables. When Steel's test can be used (i.e., when there
are equal numbers of data points per toxicant concentration), it
will be more powerful (able to detect smaller differences as
statistically significant) than the Wilcoxon Rank Sum Test with
Bonferroni's adjustment.
9.6.4.2 The estimate of the safe concentration is reported as the
NOEC. A step-by-step example of the use of the Wilcoxon Rank Sum
Test 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. ...
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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 analysis consists of
adjusting the data for mortality in the control, and then using a
maximum likelihood technique to estimate the parameters of the
underlying log tolerance distribution, which is assumed to have a
particular shape.
9.7.1.2 The assumption upon which the use of Probit Analysis is
contingent is a normal distribution of log tolerances. If the
normality assumption is not met, and at least two partial
mortalities are not obtained, Probit Analysis should not be used.
It is important to check the results of Probit Analysis to :
determine if use of the analysis is appropriate. The chi-square
test for heterogeneity provides a good test of appropriateness of
the analysis. The computer program (see discussion, Appendix H)
checks the chi-square statistic calculated for the data set against
the tabular value, and provides an error message if the calculated
value exceeds the tabular value.
9.7.1.3 A discussion of Probit Analysis, and examples of computer
program input and output, are found in Appendix H. ,
9.7.1.4 In cases where Probit Analysis is not appropriate, the
LC50 and confidence interval may be estimated by the
Spearman-Karber Method (Appendix I) or the trimmed Spearman-Karber
Method (Appendix J). If a test results in 100% survival and 100%
mortality in adjacent treatments (all or nothing effect), the LC50
may be estimated using the Graphical Method (Appendix K).
9.7.2 LINEAR INTERPOLATION METHOD
9.7.2.1 The Linear Interpolation Method (see Appendix L) is a
procedure to calculate a point estimate of the effluent or other
toxicant concentration [Inhibition Concentration, (1C)] that causes
a given percent reduction (e.g., 25%, 50%, etc.) in the
reproduction or growth of the test organisms. The procedure was
designed for general applicability in the analysis of data from
short-term chronic toxicity tests. ;
9.7.2.2 Use of the Linear Interpolation Method is based on the .
assumptions that the responses (1) are monotonically non-increasing
(the mean response for each higher concentration is less than or
equal to the mean response for the previous concentration), (2)
follow a 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
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statistically, and no defined statistical procedure is provided to
test the assumption for monotonicity. Where the observed means are
not strictly monotonic by examination, they are adjusted by
smoothing. In cases where the responses at the low toxicant
concentrations are much higher than in the controls, the smoothing
process may result in a large upward adjustment in the control
mean. . ' '-
9.7.2.3 The inability to test.the monotonicity and piece wise
linear response assumptions for this method makes It difficult to
assess when the method is, or is not, producing_reliable results.
Therefore, the method should be used with caution when the results
of a toxicity test approach an "all or nothing" response from one
concentration to the next in the concentration series, and when it
appears that there is a large deviation from monotonicity. See
Appendix L for a more detailed discussion of the use of this method
and a computer program available for performing calculations.
67
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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
1. Effluent Samples
a. Sampling point
b. Collection dates and times
c. Sample collection method
d. Physical and chemical data
e. Mean daily discharge on sample collection date
f. Elapsed time from sample collection to delivery •,
g. Sample temperature when received at the laboratory
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2. Receiving Water Samples •
a. Sampling point
b. Collection dates and times ; •
c. Sample collection method
d. Physical and chemical data .J •
e. Tide stages i
f. Sample, temperature when received at the ilaboratory
g. Elapsed time from sample collection to delivery
3. Dilution Water Samples
a. Source !
b. Collection date and time
c. Pretreatment <
d. Physical and chemical characteristics , :
10.4 TEST METHODS '
1. Toxicity test method used (title, number, source)
2. Endpoint(s) of test
3. Deviation (s) from reference method, if any/; and the
reason(s)
4. Date and time test started \
5. Date and time test terminated
6. Type of volume and test chambers -,!
7. Volume of solution used per chamber
8. Number of organisms used per test chamber '
9. Number of replicate test chambers per treatment
10. Acclimation of.test organisms (temperature and salinity
mean and range)
11. Test temperature (mean and range)
12. Specify if aeration was needed
13. Feeding frequency, and amount and type of food
14. Test salinity (mean and range) . . - ..
10.5 TEST ORGANISMS
1. Scientific name and how determined i
2. Age
3. Life stage
4. Mean length and weight (where applicable) !
5. Source !
6. Diseases and treatment (where applicable) : •
7. Taxonomic key used for species identification
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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, 1C 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,
EC or 1C 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.
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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. England
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
11.1 Scope and Application
11.2 Summary of Method
11.3 Interferences
11.4 Safety
11.5 Apparatus and Equipment :
11.6 Reagents and Supplies
11.7 Effluents and Receiving Water Collection,
Preservation, and Storage
11.8 Calibration and Standardization
11.9 Quality Control ;
11.10 Test Procedures
11.11 Summary of Test Conditions and Test
Acceptability Criteria
11.12 Acceptability of Test Results
11.13 Data Analysis
11.14 Precision and Accuracy
Appendix I Step-by Step Summary
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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 LCSOs).
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
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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 Toxicity
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. i , .
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 10-20°C) , 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.
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11.5.3 Constant temperature chambers or water baths -- for
maintaining test solution temperature and keeping dilution water
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 -- for determining salinity.
11.5.6 Hydrometer(s) -- for calibrating refractometer.
11.5.7 Thermometers, glass or electronic, laboratory grade --
for measuring water temperatures.
11.5.8 Thermometer, National Bureau of Standards Certified (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 Winkler 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 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).
11.5.16 Volumetric flasks -- Class A, borosilicate glass or non-
toxic plastic labware, 10-1000 mL for making test solutions.
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11.5.17 Pipets, automatic -- adjustable, to cover 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 topping 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 waiter cooler jugs
-- for making hypersaline brine. j
11.5.22 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.
11.5.23 Beakers -- six Class A, borosilicate glass or non-toxic
plasticware, 1000 mL for making test solutions. ;
11.5.24 Brine shrimp, Artemia, culture'unit -- see Subsection
11.6.25 and Section 4, Quality Assurance. . j.
11.5.25 Separatory funnels, 2-L -- two-four for culturing
Artemia.
11.5.26 Siphon tubes (fire polished glass) -- for solution
renewals and handling larval fish.
I
11.5.27 Droppers, and glass tubing with fire polished edges, 4
mm ID -- for transferring larvae. • i
11.5.28 Siphon with bulb and clamp•-- for cleaning test
chambers. i
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. i
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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 - U150 ^m, 500 /xm, 3 to 5 mm)
-- for collecting Artemia nauplii and fish larvae. (NITEX® is
available from Sterling Marine Products, 18 Label Street,
MontGlair, NJ 07042; 201-783-9800).
11.5.35 60 £tm 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 marking 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 -- for automatic pipets.
11.6.9 Coverslips -- 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
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11.6.12 Disposable countertop covering -- for .protection of work
surfaces and minimizing spills and contamination.j
11.6.13 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).
11.6.14 Membranes and filling solutions -- for dissolved oxygen
probe (see USEPA Method 360.1, USEPA, 1979), or reagents 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 labwarei(for reference
toxicants) . To avoid contamination from the air cind 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 mm thick).
11.6.17 Ethanol (70%) or formalin (4%) -- for preserving the
larvae. i
11.6.18 Artemia. nauplii -- for feeding test organisms.
11.6.19 Weigh boats or weighing paper -- for weighing reference
toxicants. •
i
11.6.20 Reference toxicant solutions (see Subsection 1.1.10.2.4
and see Section 4, Quality Assurance). :
11.6.21 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). |
'i
11.6.22 Effluent and receiving water -- see Section 8, Effluent
and Surface Water Sampling, and Sample Handling, and Sample
Preparation for Toxicity Tests. • . - • j
77
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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-/mi-filtered natural
seawater. Hypersaline brine should be prepared from dilution
water.
11.6.24 HYPERSALINE BRINES
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 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.
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!r0) is used as a
diluent, the maximum concentration of effluent (Oti) 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 /im before placing into the
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
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11.6.24.4.1 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).
11.6.24.4.2 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
lOOto. It is advisable not to exceed about 70tb brine salinity
unless it is necessary to test effluent concentrations greater
than 50%.
11.6.24.4.3 After the required salinity is attained, the HSB
should be filtered through a 1 /im 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.
11.6.24.5 Heat Preparation of Brine
11.6.24.5.1 The ideal container for making brine using heat-
assisted 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 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, heater, and
any other materials that will be in direct contact with the
brine. A good quality biodegradable detergent should be used,
79 ;
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followed by several (at least three) thorough reagent water
rinses.
11.6.24.5.3 Seawater should be filtered to at least 10 /-im 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.
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
Salinity
&
0
1
2
3
4
5
10
15
20
25
Brine
60
to
43.33
44.07
44.83
45.61
46.43
47.27
52.00
57.78
65.00
74.29
Brine
70
&
51.43
52.17
52.94
53.73
54.55
55.38
60.00
65.45
72.00
80.00
Brine
80
fc
57.50
58.23
58.97
59.74
60.53
61.33
65.71
70.77
76.67
83.64
Brine
90
fc
62.22
62.92
63.64
64.37
65.12
65.88
' 70.00
74.67
80.00
86.15
Brine
100
& '
66.00
66.67
67.35
68.04
68.75
69.47
73 .33
77.65
82.50
88.00
11.6.24.5.4 After the required salinity is attained, the HSB
should be filtered through a 1 /im filter and poured directly into
portable containers (20-L cubitainers or polycarbonate water
80
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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 from topsmelt larval tests using sea salts
or artificial seawater (e.g., GP2) are available for evaluation
at this time, and their 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 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. ; ' ,
11.6.24.7.2 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 lOOti and .the test is to be
conducted at 341i, lOOSi divided by 34li = 2.94. The proportion
of brine is 1 part plus 1.94 reagent water. To
-------
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 68f<>; the required brine volume is then always
equal to the effluent volume needed for each 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: .salinity
of the brine (SB, in &>) , the salinity of the effluent (SE, in
to) , 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)
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 prepare 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 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 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
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TABLE 2. EXAMPLES OF EFFLUENT DILUTION SHOWING VOLUMES OF
EFFLUENT (xfc), 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§i and, unless natural seawater is used
for dilution water, also a brine-based dilution water of 34%i.
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 .
(%)
40
Effluent
Xib
800 mL
Brine
(68-x)& '
800 mL
Dilution
Water* 34&
400 mL
Step 2. Use either serially prepared dilutions of the 'highest
test concentration or individual dilutions of 100% effluent.
Effluent Cone. (%)
20
10
5
2.5
Control
Effluent Source
1000 mL of 40%
1000 mL of 20%
1000 mL of 10%
1000 mL of 5%
none
Dilution Water*
(34fc)
1000 mL
1000 mL
10 00 mL
1000 mL
1000 mL
INDIVIDUAL PREPARATION
Effluent Cone .
(%)
40
20
10
5
2.5
Control
Effluent xfe
400 mL
200 mL
100 mL
50 mL
25 mL
none
Brine (68-x)&>
400 mL <
200 mL
100 mL i
50 mL :
25 mL
i
none
Dilution Water*
34& • ' ' • '-'• •
200 mL
• 600 mL
800 mL .
900 mL
950 mL
1000 mL
*May be natural seawater or brine-reagent water\equivalent
83
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1-liter volumetric flask. Then, assuming an effluent salinity of
2lo 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 £P., NAUPLII -- for feeding
cultures and test organisms.
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 has been found to be
acceptable is Aquarium Products, 18OL 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 al., 1985, Leger, et al.,
1986) against known suitable reference cysts by performing a
side-by-side larval growth test using the "new" and "reference"
cysts. The "reference" cysts used in the suitability test may be
a previously tested and acceptable batch of cysts, or may be
obtained from the Quality Assurance Research Division, EMSL,
84
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Cincinnati, OH 45268, 513-569-7325. A sample of newly-hatched
Artemia nauplii from each new batch of cysts should be chemically
analyzed. The Artemia cysts should not be used if the
concentration of total organochlorine pesticides 0.15 ug/g wet
weight or that the total concentration of organochlorine
pesticides plus PCBs exceeds 0.30 ^tg/g wet weight (For analytical
methods see USEPA, 1982). ;
11.6.25.3 Artemia nauplii are obtained as follows:
1. Add 1 L of seawater, or an aqueous unionized salt :
(NaCl) 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, 1985a; USEPA, 1993a; ASTM,
1993) . i . •
3. After 24 h, cut off the air supply in the separatory
funnel. Artemia nauplii are phototactic, and will
concentrate at the bottom of the funnel if it is
covered for 5-10 minutes with a dark cloth or paper
towel. To prevent mortality, do not1 leave the
concentrated nauplii at the bottom of the funnel more
than 10 min without aeration. j
4. Drain the nauplii into a funnel fitted with a sl50 /mi
NITEX® or stainless steel screen, and rinse with .
seawater or equivalent before use. i ,
11.6.25.4 Testing Artemia nauplii as food for, toxicity test
organisms. .. j , , ;
11.6.25.4.1 The primary criteria for acceptability of each new
supply of brine shrimp cysts is adequate survival, and growth of
the larvae. The larvae used to evaluate the acceptability of the
brine shrimp nauplii must be the same geographical origin and,
stage of development (9 to 15 days old) as those,used routinely
in the toxicity tests. Two 7-day chronic tests are performed
side-by-side, each consisting of five replicate test vessels
containing five larvae (25 organisms per test, total .of 50
organisms). The juveniles in one set of test chambers is fed.
85
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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 larvae of 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 species 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, Borthwick 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 (Anderson 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 jacksmelt, the insertion of
86
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the first dorsal fin occurs well in advance of the origin of the
anal fin. In tdpsmelt, the origin of the anal fin is under the
insertion of the first dorsal fin. Consult 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 permit 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 transport fish;
100-liter covered plastic trash cans have been used 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
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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' (e'g. ,
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 18°C
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 one-
thirtieth (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&; tank salinity should be 34 ± 2lo.
11.6.26.4.2 Adult topsmelt in each tank are fed twice daily (at
0900 and 1500 hrs) approximately 0.3g of Tetramin™ 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
/iE/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°C and to provide temperature spikes. For
88
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spiking, the temperature is raised from 18°C to 21°C over a 12 h
period, then allowed to return to 18°C overnight. The temperature
should be checked to the nearest 0.1°C at 1 to 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 mortality 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 i
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 re-
use . r • '
11.6.26.6 Test Organisms
11.6.26.6.1. Newly fertilized embryos should be placed in screen
tubes set in aquaria and equipped with gently flowing seawater at
20 ± 1°C. 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 post-
hatch; supply gently flowing seawater, and aeration. .Larvae
aged 9 to 15 days are used in toxicity tests (McNulty et al.,
1994). For information regarding topsmelt larva suppliers call
the Marine Pollution Studies Laboratory (408) 624-0947.
11.6.26.6.2 Larvae can be transported in 1-liter ziplock plastic
bags (double-bagged). No more than approximately 100 larvae
should be transported in any one 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 I •
-------
should be held between 15 and 18°C. 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 £35tr0) 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
9.0
-------
with samples used directly as collected or with samples passed
through a 60 pm 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 per day. ;
11.10.2.2 Effluents :
11.10.2.2.1 '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 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 instrearn
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 lOOli HSB is used as a diluent, the maximum concentration of
effluent that can be tested will be 66% at 34ti salinity.
11.10.2.2.2 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%).
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 i
11.10.2.3.1 Dilution water should be uncontaminated l-/mi-
filtered 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.
91
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11.10.2.4 Reference Toxicant Test
11.10.2.4.1 Reference toxicant tests should be conducted as
described in Quality Assurance (see Section 4.7).
11.10.2.4.2 The preferred reference toxicant for topsmelt is
copper chloride (CuCl2°2H2O). 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 /xg/L copper stock solution
by adding 0.0268 g of copper chloride (CuCl2o2H2O) 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 0 (control), 56, 100, '180, and 320 /^g/L 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 Test).
92
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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 test temperature (20
± 1°C) and maintained at that temperature during 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°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 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
larvae have been examined at the end of the test.
11.10.3.1.5 Note: Loss of the randomization sheet would
invalidate the test by making it impossible to ainalyze 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. ;
11.10.3.1.6 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.
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 1-liter
beaker by slow 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 are test chambers
(including reference toxicant chambers). These cups should
93 i
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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 /iE/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 should 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 + 2ti 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.
i
15.10.4.4 Rooms or incubators with high .volume ventilation
should be used with caution because the volatilization of the
94 :
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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. ;
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, such as a 1 mL KIMAX® serological pipet No. 37033, or
equivalent. Care should be taken to ensure that (turbulence
resulting from aeration does not cause undue stress to the fish.
11.10.6 FEEDING
11.10.6.1 Artemia nauplii are prepared as described below.
11.10.6.2 The test larvae are fed newly-hatched .(less than 24-h-
old) Artemia. nauplii once a day from Day 0 through Day 6; larvae
are not fed on Day 7. Equal amounts of Artemia. nauplii must be
fed to each .replicate test chamber to minimize the variability of
larval weight. Add 40 newly hatched Artemia nauplii 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 Artemia 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 daily renewal of test solutions, uneaten
and dead brine shrimp, dead larvae, and other debris are removed
95
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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 larvae
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 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.
11.10.8.1.3 Record all the measurements on the data sheet.
11.10.8.2 Routine Biological Observations
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 LC50s.
11.10.8.2.2 Protect the larvae from unnecessary disturbances
during the test by carrying out the daily test observations,
solution renewals, and removal of dead larvae, carefully. Make
sure the larvae remain immersed at all times during the
performance of the above operations.
96
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11.10.9 TEST SOLUTION RENEWAL
j
11.10.9.1 The test solutions are renewed daily using freshly
prepared solutions, immediately after cleaning the test chambers.
The old solution is carefully siphoned 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 in an incubator or refrigerator at 4PC. Plastic
containers such as 8-20 L cubitainers have proven suitable for
effluent collection and storage. For on-site toxicity studies no
more than 24 h should elapse between collection of the effluent
and use in a toxicity test (see Section 8, Effluent and Receiving
Water Sampling, Sample Handling, and Sample Preparation for
Toxicity Tests).
11.10.9.4 Approximately 1 h before test initiation, a sufficient
quantity of effluent or receiving water sample is warmed 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 i
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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. Note:
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 /xm mesh screen in a large beaker 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 forceps, to a 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 original
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
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percent survival for the entire test. Average weights should be
expressed to the nearest 0.01 mg.
11.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
11.11.1 A summary of test conditions and test acceptability
criteria is listed in Table 3 .
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY
CRITERIA FOR THE TOPSMELT, ATHERINOPS AFFINIS, LARVAL
SURVIVAL AND GROWTH TEST WITH EFFLUENTS AND RECEIVING
WATERS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Test type :
Salinity:
Temperature :
Light quality:
Light intensity:
Photoperiod:
Test chamber size:
Test solution volume:
Renewal .of test
solutions :
Age of test organisms:
No. larvae per test
chamber :
No . replicate chambers
per concentration:
Source of food :
Feeding regime :
Static -renewal
5 to 34li (± 2& of the
selected test salinity)
20 ± 1°C
Ambient laboratory illumination
10-20 /uE/m2/s (Ambient
laboratory levels)
16 h light, 8 h darkness
600 mL
200 mL/replicate
Daily
9-15 days post -hatch
5 !
5 ' !
Newly hatched Artemia nauplii
Feed 40 nauplii per larvae
twice daily (morning arid night)
99
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15. Cleaning:
Siphon daily., immediately
before test solution renewal
and feeding
16. Aeration:
None, unless DO concentration
falls below 4.0 mg/L, then
aerate all chambers. Rate
should be less than 100
bubbles/min.
17. Dilution water:
Uncontaminated l-/zm-filtered
natural seawater or hypersaline
brine prepared from natur'al
seawater
18. Test concentrations:
Effluent: Minimum of 5' and a
control
Receiving waters: 100%
receiving water and a control
19. Dilution factor:
Effluents: 2:0.5 • .
Receiving waters: None, or
20. Test duration:
7 days
21. Endpoints:
Survival and growth (weight)
22. Test acceptability
criteria:
:>80% survival in controls, 0.85
mg average weight of control
larvae (9 day old), LC50 with
copper must be s205 /zg/L, <25%
MSD for survival and <50% MSB
for growth
100
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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 three 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 f or : Toxicity Tests)
2 L per day
11.12 ACCEPTABILITY OF TEST RESULTS
I
11.12.1 Tests results are acceptable 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 controls; 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
(4)
The minimum significant difference (%MSD) of <25%
relative to the control for survival for the reference
toxicant test. The (%MSD) of <50% relative to the
control for growth for the reference toxicant test .
11.13 DATA ANALYSIS
101
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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 estimation
techniques (see Section 9, Chronic Toxicity Test Endpoints and
Data Analysis). LOEC and NOEC values, for survival and growth,
are obtained using a hypothesis testing approach such as
Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank Test
(Steel, 1959; Miller, 1981)(see Section 9). Separate analyses
are performed for the estimation of the LOEC and NOEC endpoints
and for the estimation of the LC50 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 the LC50
and IC25. See the Appendices for examples of the manual :
computations and examples of data input and program output.
11.13.1.3 The statistical tests described here must be used with
a knowledge of the assumptions upon which the tests are
contingent. Tests for normality and homogeneity of variance are
included in Appendix B. The assistance of a statistician is
recommended for analysts who are not proficient in statistics.
11.13.2 EXAMPLE OF ANALYSIS OF 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 LC50
endpoint. Concentrations at which there is no survival in any of
the test chambers are excluded from statistical analysis of the
NOEC and LOEC, but included in the estimation of the 1C, EC, and
LC endpoints.
11.13.2.2 For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and
LOEC endpoints is made via a parametric test, Dunnett's
102
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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 arid homogeneity of
variance, are formally tested. The test for normality is the
Shapiro-Wilk's Test, and Bartlett's Test is used to test for
homogeneity of variance. If either of these tests fails, the
nonparametric test, Steel's Many-one Rank Test, is used to
determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the
parametric procedure. .
11.13.2.3 If unequal numbers of replicates occur among the
concentration levels tested, there are parametric and
nonparametric alternative analyses. The parametric analysis is a
t test with the Bonferroni adjustment (see Appendix D). The
Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
nonparametric alternative.
i
11.13.2.4 Probit Analysis (Finney, 1971; see Appendix H) is used
to estimate the concentration that causes a specified percent
decrease in survival from the control. In this analysis, the
total mortality data from all test replicates at1 a given
concentration are combined. If the data do not fit the Probit
Analysis, the Spearman-Karber Method, the Trimmed Spearman-Karber
Method, or the Graphical Method may be used to estimate the LC50
(see Appendices H-K). :
11.13.2.5 Example of Analysis of Survival 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 transformed 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 concentration and control
are listed in Table 5. A plot of the survival proportions is
provided in Figure 5. Since there was 100% mortality in all five
replicates for the 100 jug/L and 180 /xg/L concentrations, they are
not included in the statistical analysis and are considered
qualitative mortality effects. i
103
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TABLE 4. SUMMARY OF SURVIVAL AND GROWTH DATA FOR TOPSMELT,
ATHERINOPS AFFINIS, LARVAE EXPOSED TO COPPER FOR
SEVEN DAYS1
Copper
Cone.
0.0
32.0
56.0
100.0
180.0
Cone.
0.0
32.0
56.0
100.0
180.0
Replicate Survival Proportions
A B
1.0 0.8
1.0 1.0
0.0 0.6
0.0 0.0
0.0 0.0
Replicate
A B
0.00134 0.00153
0.00146 0.00142
0.00147
__
— —
C
1.0
1.0
0.2
0.0
0.0
Averaae Dry
C
0.00134
0.00150
0.00170
--
—
D E
1.0 1.0
1.0 1.0
1.0 0.6
0.0 0.0
0.0 0.0
Weicrhts (ma)
D E
0.00146 0.00144
0.00138 0.00128
0.00124 0.00130
__
— —
Mean
Proportion
Survival
0.96
1.00
0.48
0.00
0.00
.Mean Dry
Wgt (mg)
0.00142
0.00141
0.00114
.
—
replicates of 5 larvae each.
104
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•^"•^. ^m^C^m^^^J^'^^^&&
rrvflrpr > . -fir-- - . :?.•'•»-A ;.j?'\*.*-. .":-: ' "'" --."-' @£i»-.'^?X:K.!
HOMOGENEOUS VARIANCE
EQUAL NUMBER OF
'-*. MR.ICATCS1?/
Figure 1. Flowchart for statistical analysis of the topsmelt,
Atherinis affinis, larval survival data by hypothesis testing.
105
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STATISTICAL ANALYSIS OF TOPSMELT lMv$C
SURVIVAL AND GROWTH TEST
SURVIVAL POINT ESTIMATION
MORTALITY DATA
#DEAD
TWO OR MORE
PARTIAL MORTALITIES?
NO
YES
IS PROBIT MODEL
APPROPRIATE?
(SIGNIFICANT X2TEST)
1
ONE OR MORE
ARTIAL MORTALITIES?
YES
T
NO
YES
PROBIT METHOD
ZERO MORTALITY IN THE-
LOWEST EFFLUENT CONC.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONG.?
T
YES
SPEARMAN-KARBER .
METHOD
LG5JB
NO
TRIMMED SPEAR
KARBER METHOD
LC50AND95%
CONFIDENCE
INTERVAL
11 #
Figure 2. Flowchart for statistical analysis pf the topsmelt,
Atherinis affinis, larval survival data by point estimation.
106
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TABLE 5. TOPSMELT, ATHERINOPS AFFINIS, SURVIVAL DATA
Replicate Control
Copper Concentration
32.0
56.0
RAW
ARC SINE
SQUARE
ROOT
TRANSFORM
ED
Mean (Yj
S2
i1
A
B
C
D
E
A
B
C
D
E
1
0
1
• 1
1
1
1
1
1
1
1
0
1
:0
.8
.0
.0
.0
.345
.107
.345
.345
.345
.297
.0113
1
1
1
1
1
1
1
'1
1
1
1
.0
2
.0
.0
.0
.0
.0
.345
.345
.345 '
.345. :
.345
.345
.000
0
0
0
1
0
0
0
0
1
0
0
0
3
.0
.6
.2
.0
.6
.225
.886
.464
.345
.886
.761
.187
11.13.2.6 Test for Normality
11.13.2.6.1 The first step of the test for normality is to
center the observations by subtracting the mean of all
observations within a concentration from each observation in that
concentration. The centered observations are summarized in Table
6. • ,
107
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8
09
CP
00
O
m
o
CJ
o
p
o
NOIlHOdOHd
in
0)
•H
PI
-H
nJ
4-i
g
-H
o
81
•H
CQ
§
M-l
o
-,
o
tH
cu
n
0)
g.
•H
108
-------
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Copper Concentration
(/zg/L)
Replicate
A
B
C
D
E
Control
0.
-0.
0.
0.
0-
048
190
048
048
048
32
0.
0.
0.
0.
0.
.0
000
000
000
000
000
56
-0.
o.
~0.
0.
0.
.0
536
125
297
584
125
11.13.2.6.2 Calculate the denominator, D, of the statistic
Where: XA = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
11.13.2.6.3 For this set of data,
n = 15
X = 1 (0.003) = 0.000
15 . ' ';
D = 0.793
11.13.2.6.4
largest
Order the centered observations from smallest to
X(2)
X(n>
109
-------
where X(i) denotes the ith ordered observation. The ordered
observations for this example are listed in Table 7.
TABLE 7. ORDERED CENTERED OBSERVATIONS. FOR THE SHAPIRO-WILK1S
EXAMPLE
1
2
3
4
5
6
7
8
-0
-0
-0
0
0
0
0
0
.536
.297
.190
.000
.000
.000
.000
.000
9
10
11
12
13
14
15
0
0
0
0
0
0
0
.048
.048
.048
.048
.125
.125
.584
11.13.2.6.5 Prom Table 4, Appendix B, for the number of
observations, n, obtain the coefficients a17 a2, ... ak where k is
n/2 if n is even and (n-l)/2 if n is odd. For the data in this
example, n = 15 and k = 7. The a± values' are listed in Table 8.
11.13.2.6.6 Compute the test statistic, W, as follows:
W. -[Ea (X"1-1'11-*1*')]
D 1.1 *
The' differences x(n~i+1) - X(i) are listed in Table 7. For the data
in this example,
(0.817)2 = 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 data 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 wil.1 be used
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK1S
EXAMPLE ' , "
a. • xtn-i+i) _ Xd)
1 ' 0
2 0
3 0
4 0
5 0
6 0
7 0
.5150
.•3306
.2495
.1878
.1353
.0880
.0433
1
0
0
0
0
0
0
.12.0
.422
.315
.048
.048
.048
. 048
X(1S)
XU4)
X(13)
: x(l2>
: x(ii)
X(10)
! X(9)
- X(1>* '
- X(2>
- X'3'
- X(4>
- X(5)
- X(6>
- X<7)
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 in 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
observation, etc. If ties occur when ranking, assign the average
rank to each tied observation.
11.13.2.7.2 An example of assigning ranks to the combined data
for the control and 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 for each
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 (J.g/Is copper
concentration is equal to the critical value, the proportion
surviving in the 56.0 /ig/L concentration is considered
significantly less than that in the control. 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 pig/L
and 56.0 fJ.g/Ij concentrations, respectively.
11.13.2.8 Calculation of the LC50
11.13.2.8.1 The data used for the calculation of the LC50 is
summarized in Table 12. For estimating the LC50, the data for
the 100 /ig/L and 180 ptg/L copper concentrations with 100%
mortality are included.
112
-------
TABLE 9. ASSIGNING RANKS TO THE CONTROL AND 32.0
COPPER CONCENTRATION FOR STEEL'S MANY-ONE RANK
TEST . •- ; -.
Rank
Transformed
Proportion
Surviving
Copper
Concentration
(/ig/L)
1
6
6
6
6
6
6
1.107
1.345
1.345
1.345
1.345
1.345
1.345
I
Control
32.0
32.0
32.0
32.0
32.0
Control
TABLE 10. TABLE OF RANKS
CoiDTDer
Replicate
A
B
C
D
E
Control
1
1
1
1
1
.345
.107
.345
.345
.345
(6,
(1,
(6,
(6,
(6,
8)
5)
8)
8) .
8)
1
1
1
1
1
32.
.345
.345
.345
.345
.345
Concentration (ucr/L)
0 -
(6)
(6)
(6)
(6)
(6)
. : o
1°
0
1
0
56.0
.225
.886
.464
.345
.886
(1)
X3.5
(2)
(8)
(3.5
)
)
113
-------
TABLE 11. RANK SUMS
Copper Concentration . Rank Sum
(Aig/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 adjusted, 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 fj-g/i- 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 0.040+0.000 „ nnn
P0 -Pj. • = 0.020
Where: pf = the smoothed observed mortality proportion'for
effluent concentration i.
11.13.2.8.3.1 Because the rest of the responses are monotonic,
additional smoothing is not necessary. The smoothed observed
proportion mortalities are shown in Table 12.
11.13.2.8.4 Because the smoothed observed proportion mortality
for the control is now greater than zero, the data in each
effluent concentration must be adjusted using Abbott's formula
114
-------
(Finney, 1971) . The adjustment takes the form;;
Pi = (P? - PS) / (1 - PS) - ;
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:
Pi-Po 0.020-0.020 0.000
Po • Pi = = = :— = 0.0
i • 1-0.020 0.980
a Pz -Po 0.520-0.020 0.500
p. = = = = 0.510
lps 1-0.02-0 0.980
P3-P0 1.000-0.020 0.980
p. = p, = - = - = — - = 1 . 000
-.* 1-0.020 0.980
The smoothed, adjusted response proportions for the -effluent
concentrations are shown in Table 12.
11.13.2.8.5 Calculate the Iog10 of the estimated LC50, m, as
follows :
m
Where: pf = the smoothed adjusted proportion mortality at
concentration i
Xi = the Iog10 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
Concentration
%
Control
32.0
56.0
100.0
180.0
Number
of Deaths
1
0
13
25
25
Number of
Organisms
Exposed
25
25
25
25
25
Smoothed
Mortality
Proportion
0.040
0.000
0.520
,1.000
1.000
Adjusted
Mortality
Proportion
0.020
0.020
0.520
1.000
1.000
Mortality
Proportion
0.000
0.000
0.510
1.000
1.000
11.13.2.8.5.1 For this example, the Iog10 of the estimated LC50,
m, is calculated as follows :
m * [(0.510 - 0.000)
[(1.000 - 0.510)
[(1.000 - 1.000)
(1.5051 + 1.7482}]/2 +
(1.7482 + 2.0000)]/2 +
(2.0000 + 2.2553)]/2 +
11.13.2.8.6 Calculate the estimated variance of m as follows
T ,.
V(m)
Where: Xi = the Iog10 of concentration i
HI SB 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 estimated variance of m,
V(m) , is calculated as follows:
116
-------
V(m) = (0.510) (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 V V (m) • . ; ,
11.13.2.8.7.1 For this example, the 95% confidence interval for
m is calculated as follows:
1.7479 ± 2 ^/0. 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 . ,
i
11.13.2.8.8.1 For this example, the estimated LC50 is calculated
as follows : .
i
LC50 = antilog(m) = antilog (-1 . 7479) = 56.0 /xg/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: antilog (1 . 6974) = 49.; 8
upper limit: antilog (1 . 7984) = 62,9 /xg/L
11.13.3 EXAMPLE OF ANALYSIS OF TOPSMELT, ATHERINOPS AFFINIS,
GROWTH DATA !
11.13.3.1 Formal statistical analysis of the growth data is
outlined in Figure 4 .
The response used in the statistical analysis is mean weight per
surviving organism for each replicate. The IC25 can be
calculated for the growth data via a point estimation technique
(see Section 9, Chronic Toxicity Test Endpoints and Data
Analysis) . Hypothesis testing can be used to obtain an NOEC and
LOEC for growth. Concentrations above the NOEC for survival are
excluded from the hypothesis test for growth effects.
117
-------
11.13.3.2 The statistical analysis using hypothesis testing
consists of a parametric test, Dunnett's Procedure, and a
nonparametric test, Steel's Many-one Rank Test. The underlying
assumptions of the Dunnett's Procedure, normality and homogeneity
of variance, are formally tested. The test for normality is the
Shapiro-Wilk1s Test and Bartlett's Test is used to test for
homogeneity of variance. If either of these tests fails, the
nonparametric test, Steels' Many-one Rank Test, is used to
determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are determined by the
parametric test.
11.13.3.3 Additionally, if unequal numbers of replicates occur
among the concentration levels tested there are parametric, and
nonparametric alternative analyses. The parametric analysis is a
t test with the Bonferroni adjustment. The Wilcoxon Rank Sum
Test with the Bonferroni adjustment is the nonparametric
alternative. For detailed information on the Bonferroni
adjustment, see Appendix D.
11.13.3.4 The data, mean and variance of the observations at
each concentration.including the control are listed in Table 13.
A plot of the mean weights for each treatment is provided in
Figure 5. Since there is no survival in the 100 /xg/L and 180
pig/L copper concentrations, they are not considered in .the
growth analysis. Additionally, since there is significant
mortality in the 56.0 JKJ/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
-------
NORMAL DlSTRI.BUTiOW
ENDPQINT ESTIMATES
Figure 4. Flowchart for statistical analysis of the topsmelt,
Atherinops affinis, larval growth data.
119 !
-------
TABLE 13. TOPSMELT, ATHERIttOPS AFFINIS, GROWTH DATA
Replicate
A
B
C
D
E
Mean (XL)
sj
i
Control
0.00134
0.00153
0.00134
0.00146
0.00144
0.00142
0.000000006
1
Copper Concentration (^g/L)
32.0 56.0 100.0 180.0
0.00146 - - -
0.00142 - -
0.00150 - - -
0.00128 - - -
0.00141 -' - -
0.00141 - - -
0.000000007 - - -
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 ftg/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
D = E(X± - X)2
i-1
Where: X± = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations.
120
-------
to
0)
4J
4J
I
M
tn
E
n)
to
•H
CO
•H
q
a
i
w
8-
NV3W
•a
-H
M-l
O
4J
O
iH
CM
in
-H
Pn
121
-------
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 :
X'2>
X(n)
Where X(i) 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 aif a2, . . ., ak where k
is n/2 if n is even and (n-l)/2 if n is odd. For the data in
this example, n = 10 and k = 5. The ai values are listed in
Table 16.
TABLE 15. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK' S
EXAMPLE
i
1
2
3
4
5
. -0
-0
-0
-0
0
X(i)
.00013
.00008
.00008
.00003
.00001
i
6.
7
8
9
10
0
0
0
0
0
x«i)
.00002
.00004
.o'ooos
.00009
.00011
122
-------
TABLE 16. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S EXAMPLE
1
2
3
4
5
0.5739
0.3291
0.2141
0.1224
0.0399
•0.00024
0.00017
0.00013
0.00007
0.00001
X(10)
X'9>
X(a>
X(7>
X.(«>
- X'1?
- X(2>
- X(3>
- X'41
" X'5'.
11.13.3.5.5 Compute the test statistic, W, as follows:
1 * 2
*** ~Dli**i(X )] •
The differences x(n-i+1) - X(i) are listed in Table 16. For this set
of data:
W =
0.000000055
(0.0002305)2 = 0.966
11.13.3.5.6 The decision rule for this test is to compare W with
the critical value found in Table 6, Appendix B. If the computed
W is less than the critical value, conclude that the data are not
normally distributed. For this example, the critical value at a
significance level of 0.01 and 10 observations (n) is 0.781.
Since W = 0.966 is greater than the critical value, the conclude
that the data are normally distributed.
v
11.13.3.6 Test for Homogeneity of Variance
11.13.3.6.1 The test used to examine whether the variation in
mean dry weight is the same across all effluent concentrations
including the control, is Bartlett's Test (Snedecor and Cochran,
1980). The test statistic is as follows:"
InS2 -
123
-------
Where: VA = degrees of freedom for each effluent
concentration and control, Vd = (ni - 1)
ni = the number of replicates for concentration i
p = number of levels of effluent concentration
including the control
In = loge
i = 1,2, ..., p where p is the number of
concentrations including the control
Ev
p p
i i
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 (n^. = 5 for all i) . Thus, Vt = 4 for all i
11.13.3.6.3 Bartlett ' s statistic is therefore:
f
B = [(8) ln(6.5xl(r9) - 4 Eln( S* } ] / 1.125
= [8(-18.851) - 4(-37.709)
= 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, at 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 df Sum of Squares Mean Square(MS)
(SS) . " ' (SS/df)
Between
Within
p - 1
N - p
SSB
SSW
2
SB
2
sw
= SSB/Cp-1)
= SSW/ (N-p)
Total N - 1 . SST
Where: p = number of concentration levels including-the
control
N = total number of observations na -t- n2 ... + n^
n = number of observations in concentration i
SSB
^/fl -G2/N Between Sum of Squares
SST = EZr2 -G2/N T°tal Sum °f Squares
125
-------
ssw = SST-SSB Within Sum of Squares
G = the grand total of all sample observations,
1
-*-1
Ti = the total of the replicate measurements for
concentration i
YiD- = 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:
H! = n2 = 5
N « 10
TI = Y13. + Y12 -I- Y13 + Y14 + Y15 = 0.-00711 •
T2 = Y21 + Y22 + Y23 + Y24 + Y25 = 0.007.04
G = T! + T2 = 0.01415
SSB -
i-i
_!_(!• 001137 x 10'4) - (0.01415)2 = 4.90 x 10'10
5 10
SST
0.0000201 - (0.01415)2 = 7.775 x IQ'8
10
126
-------
SSW = SST-SSB =7.775 X l.CT8 - (4.9 X lO'10) .= 7.726 X lQ-82
-10
SB = SSB/(p-l) = (4.9 X 10-10)/(2-l) = 4.9 x 10
S« = SSW/(N-p) = 7.726 x 10-8/(10-2) = 9.658 x 10
-9
11.13.3.7.3 Summarize these calculations in the! ANOVA table
(Table 18) .
TABLE 18. ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
Between
Within
Total
df
1 • ..-
8
9
Sum of Squares
(SS) .
4.90- X lO'10.
7. 725 x ID'8
7.775 X 10-"
Mean Square (MS)
(SS/df)
4.9 x ID'10 •
9.658 x ID'9
1
11.13.3.7.4 To perform the individual comparisons, calculate the
t statistic for each concentration, and control combination as
follows: !
fci -
Where: Yi = mean dry weight for effluent concentration i
Y! = mean dry weight for the control
Sw = square root of the within mean square
nx = number of replicates for the control
ni = number of replicates for concentration i.
11.13.3.7.5 Table 19 includes the calculated t values for each
concentration and control combination. In this example there is
only one comparison, of the 32.0 /ng/L copper concentration with
the control. The calculation is as follows:
127
-------
(0.00142 - 0.00141)
t = ' = 0.161
TABLE 19. CALCULATED t VALUES
Copper Concentration (ptg/L)
32.0 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 tA is greater than the critical value.
Since t2 is less than 1.86, the 32.0 fJ-g/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 (MSB) that can be statistically detected
may be calculated:
MSD = d sJd/n + (l/.n)
Where: d = the critical value for Dunnett's Procedure
Sw = the square root of the within mean square
n = the common number of replicates at each
concentration
(this assumes equal replication at each
concentration)
nx = the number of replicates in the control.
128
-------
11.13.3.7.8 In this example:
MSD =1.86 (9.828xl(T5) ^(1/4) + (1/4)
= 1.86 (9.828 X 1CT5) (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 weight
from the control.
11.13.3.8 Calculation of the ICp ;
i
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 1C. In the following discussion,, the observed
means are represented by Y± and the smoothed means by Mi.
11.13.3.8.2 Since Y5 = 0 < Y4 = 0 < Y3 = 0.00114 < Y2 - 0.00141
< Y! = 0.00142, set M! = 0.00142, M2 = 0.00141, M3 = 0.00114, M4 =
0 and M5 = 0.
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 using 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
Mid-p/100) = 0.00142(1-25/100). Examining the smoothed means
and their associated concentrations (Table 20) , the response,
0.001065 mg, is bracketed by C3 = 56.0 /xg/L copper and C4 = 100.0
copper.
129 • • ,
-------
11.13.3.8.5 Using the equation from Section 4.2 of Appendix M,
the estimate of the IC25 is calculated as follows:
rep
IC25 « 56.0 + [0.00142(1 - 25/100) - 0.00114] (100.0 -56.0)
(0.0 - 0.00114)
= 58.9
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 /*g/L. The empirical 95% confidence interval for the true
mean was 44.2778 ^tg/L to 67.0000 ^tg/L. The computer program.
output for the IC25 for this data set is shown in Figure 7 . •
TABLE 20. TOPSMELT, ATHERINOPS AFFINIS, MEAN GROWTH
RESPONSE AFTER SMOOTHING
Copper
Cone. (jig/L)
Control
32
56
100
180
.0
.0
.0
.0
i
1
2
3
4
5
Response
Means
(mg) Yi
0
0
0
0
0
.00142
.00141
.00114
.0
.0
Smoothed '
Means
(mg) Mi
0
0
0
0
0
.00142
.00141
.00114
.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 the
topsmelt larval survival and growth test using copper chloride as
the reference toxicant are provided in Tables 21 and 22. In the
five copper tests presented here, the NOECs for survival were 100
fig/L for all tests but one; this test had a NOEC of 180 /xg/L.
The coefficient of variation for copper based on the LC25 is
17.3% for survival; the coefficient of variation for copper based
130
-------
on the LC50 is 9.7% for survival. The weight endpoint was less
sensitive than survival in all but one test. An IC25 could be
calculated for three of five tests and the coefficient of
variation for these three tests was 60.69%, the coefficient of
variation based on the IG50 for these three -tests was 4.75%.
11.14.1.2 Multilaboratory Precision
14.11.1.2.1 Data 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/Kj/L). The coefficient of variation, based on LCSOs
for survival was 36%. In the second comparison the NOEC for
effluent was 20% at both laboratories. The coefficient of
variation, based on the LC50s for survival was 19%. In the third
comparison the NOEC for copper was 32 £tg/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 toxicity tests cannot be determined.
131
-------
a
tn
CD
•H
R
3
•X- -X-
to
•H
_R
0)
•u
0)
I
81
4J
m
o,
0) TJ
01 Pi
X) ns
O
(fl 01
4J 0)
ni i-i
T) fi
(&"O iHO I 3M
NV3W
n e
IW 1-1
O M-l
jJ n)
O 4->
H a!
fn -a
VD
d)
I
132
-------
Cone . ID
Cone . Tested
Response
Response
Response
Response
Response
1
2
3
4
5
1
0
.00134
.00153
.00134
.00146
.00144
2
32
.00146
.00142
.00150
.00138
. 00128
3
56
0
.00147
.00170
.00124
.00130
4
100
0
0
0
0
0
5
, 180
! 0
0
: 0
0
i
0
*** 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: we aa.i25
Cone.
ID
1
2
3
4
5
Number
Replicates
5
5
5
5
5
Concentrat ion
ug/L
0.000
32.000
56.000
100.000
180.000
Response
Means
0.001
0.001
0.001
0.000
0.000
Std.
Dev.
0.000
0.000
0.001
0.000
0.000
Pooled
Response Means
0,001
0.001
0.001
0.000
0.000
The Linear Interpolation Estimate: 58.9089 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean: 58.1571 Standard Deviation: 7..9299
Original Confidence Limits: Lower: 44.2778 Upper: 67.0000
Expanded Confidence Limits: Lower: 36.9622 Upper: 71.0455
Resampling time in Seconds: 0.11 Random_Seed: -498847050
Figure 7. ICPIN program output for the IC25
133
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TABLE 21. SINGLE LABORATORY PRECISION OP THE TOPSMELT, ATHERINOPS AFFINIS
SURVIVAL ENDPOINT WITH COPPER (CU ,uG/L) CHLORIDE AS A REFERENCE
TOXICANT
Test Number
1
2
3
4
5
NOEC
100
100
100
180
100
LC25
142.1
NC3
151.7
181.0
119.2
# of Tests Statistic LC25
5 Mean 148.5
SD 25.6
CV (%} 17.3
LC50
187.4
162.4
165.6
190.6
204.0
LC50
182.0
17.6
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
1
2
3
4
5
# Of Tests
5
NOEC
180
180
>180
56
>180
Statistic
LC25
222.1
NC4
NC4
47.6
NC4
LC25
LC50
264.2
NC4
NC4
NC4
NC4
LC50
Mean 156.8
SD 95.2
CV (%) 60.7%
*Data from Anderson et al. 1994; point estimates calculated using probit
analysis, except where noted.
2Five replicate exposure chambers with five larvae per chamber were used for
each treatment.
3LC50 calculated using Spearman-Karber method, this method does not calculate
an LC25.
*Point estimate not calculated because the response was less than either 25 or
50%.
134
-------
TABLE 23. MULTI-LABORATORY PRECISION OF THE TOPSMELT,
ATHERINOPS AFFINIS, GROWTH AND SURVIVAL TEST
CONDUCTED WITH COPPER (CU AiG/L) CHLORIDE AS A
REFERENCE TOXICANT
Test
Number
^
1
2
3
Toxicant
Copper*
Copper3
CV
Effluent
Effluent
CV
Coppera
Copper3
CV
Laboratory
lb
2d
lb
2e
lb
le
Survival
NOEC LC50
100 162.0
100 274.0
36%
20 31.4 .
20 23.9
19%
32 5.5.7
32 5.8.4
3%
Growth
NSC
NS
NS
10
NS
NS
Two separate interlaboratory comparisons were conducted, in
August 1990 and August 1991. . j . •"'
aThe August 1990 copper test was conducted at 34&> salinity; the
August 1991 copper test was conducted at 20& salinity.
bMarine Pollution Studies Laboratory, Monterey County,
California.
°Not Significant.
dVantuna Research Group, Occidental College, California.
eChevron Research and Technology Co., Environmental Research
Group.
135
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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
p.g/L) by adding 0.0268 g of copper chloride (CuCl2o2H2O) to 1
liter of reagent water.
D. Prepare zinc reference toxicant solution of 0 (control) 56,
100, 180, and 180 /zg/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 for
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
A. Obtain 9-15 day old larvae from a commerical supplier or in-
house cultures.
B. 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.
C. Remove all dead larvae daily, and add 40 newly hatched
Artemia nauplii per larva twice daily; once in the morning .
and once in the afternoon. Adjust feeding to account for
larva mortality.
D. Renew test solutions daily using freshly prepared solutions,
immediately after cleaning the test chambers.
E. 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 test concentration
to each chamber. Remove all dead larvae.
F. The surviving larvae in each test chamber are 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.
G. 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 6, hours. Weigh
each weigh boat on a microbalance (accurate to 1 £tg) .
Record the chamber number, larvae weight, weigh boat weight
(recorded previously), and number of larvae per weigh boat :
(replicate) on the data sheet. j
H. Analyze the data.
I. 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:
End Time:
Fish Age at Start:
Teat
Cent.
H
Concentration
Numer
Alive
Day
1
Day
2
Day
3
Day 4
Day
5
Day
6
Day
7.
Total
Number
Alive
Total
Number
at
Start
Notes
Computer Data Storage
Disk
File
Note: See larval weight data on separate sheet.
138
-------
Data Sheet for Weighing Larval Fish
Test Start Date: Start Time:
Test End Date: End
Time:
Toxicant:
Sample Source:
Sample Type: Sediment Elutriate Porewater
Water
Fish Species :
Collection/Arrival
Date:
Fish Age at Start:
Test
Container
Number
I
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
l9
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Site Code
or
Concentration
•
Foil
Number
Foil Weight
(mg)
Total Weight
(mg)
Weight of
Larval
Fish i
(mg)
,
1
Number
of Fish
Larvae
Weight per ,
Larval Fish
(mg)
Computer Data Storage Notes
Disk:
File: ' ;
Note: See larval mortality data on separate sheet.
139
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SECTION 12
MYSID, Holmesimysis costata
SURVIVAL AND GROWTH TEST 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'l, Monterey, CA 93940
TABLE OP CONTENTS
12.1 Scope and Application
12.2 Summary of Method
12.3 Interferences
12.4 Safety
12.5 'Apparatus and Equipment
12.6 Reagents and Supplies
12.7 Effluents and Receiving Water Collection,
Preservation, and Storage
12.8 Calibration and Standardization
12.9 Quality Control
12.10 Test Procedures
12.11 Summary of Test Conditions and Test
Acceptability Criteria
12.12 Acceptability of Test Results
12.13 Data Analysis
12.14 Precision and Accuracy
Appendix I Step-by Step Summary
140
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SECTION 12
MYSID, HOLMESIMYSIS COSTATA
SURVIVAL AND GROWTH TEST
12.1 SCOPE AND APPLICATION
12.1.1 This method estimates the chronic toxicity of effluents
and receiving waters to the mysid, Holmesimysis costata, using
three-to-four day old juveniles in a seven-day,,static-renewal
exposure. The effects include the synergistic, antagonistic,
and additive effects of all 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 periods (i.e., 24-
h, 48-h, 96-h LCSOs).
12.1.3 Detection limits of the toxicity of an effluent 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 aqucitic toxicity
testing. Specific experience with any toxicity test is usually
needed before acceptable results become routine.
12.2 SUMMARY OP 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 the effluent, 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 aerating 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 suspensions at test
temperature (13 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 -- for determining salinity.
12.5.6 Hydrometer(s) -- for calibrating refractometer.
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 calibrate laboratory
thermometers.
12.5.9 pH and DO meters -- for routine physical and chemical
measurements.
I
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 accurately weighing to
0.0001 g (for weighing reference toxicants). '
12.5.13 Microbalance -- Analytical, capable of accurately
weighing to 0.000001 g (for weighing mysids). '
12.5.14 Fume hood -- to protect the analyst from effluent or
formaldehyde fumes. , ,
12.5.15 Glass stirring rods -- for mixing test solutions.
i
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 non-
toxic plastic labware, 10-1000 mL for making test solutions.
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 equivalent.
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 fire-
polished edges) for handling adult mysids.
12.5.31 Siphon tubes (fire polished glass with attached silicone
tubing) -- for test solution renewals.
144
-------
12.5.32 Fire-polished wide-bore 10 mL pipet -- for handling
juveniles. i •
12.5.33 Forceps with fine points -- for transferring 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 ptm, 500-lOOOpim) -- for
concentrating organisms. (NITEX® is available from Sterling
Marine Products, 18 Label. Street, Montclair, NJ 07042; 201-783-
9800). .
12.5.38 60 /itn NITEX® filter - for filtering receiving 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.Sample
Handling, 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 chambers and
containers.
12.6.4 Markers, water-proof -- for marking containers, etc.
12.6.5 Parafilm --to cover graduated cylinders and vessels.
12.6,6 Gloves, disposable --for personal protection from
contamination. . ;
12.6.7 Pipets, serological -- 1-10 mL, graduated.
12.6.8 Pipet tips -- for automatic pipets. :
12.6.9 Coverslips -- for microscope slides.
145
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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 spills 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 mm 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, Hypersaline Brines.' The
dilution water should be uncontaminated 1-jiim-f iltered 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 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 waiter (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. ;
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 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 (IQOto) is used as a
diluent, the maximum concentration of effluent ;(0t») that can be
tested is 66% effluent at 34§b 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 collected 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 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).
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
lOOSo. 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 /im 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.
12.6.23.5 Heat Preparation of Brine
12.6.23.5.1' The ideal container for making brine using heat-
assisted 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
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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'(%) THAT CAN BE TESTED
AT 34li WITHOUT THE ADDITION OF DRY SALTS GIVEN THE
INDICATED EFFLUENT AND BRINE SALINITIES.
Effluent
Salinity
tb
0
1
2
3
4
5
10
15
20
25
Brine
60
&
43.33
44.07
44.83 •
45.61
46.43
47.27
52.00
57.78
65.00
74.29
Brine
70
%b
51.43
52.17
52.94
53 .73
54.55 '
55.38
60.00
65.45
72.00
80.00
Brine
80
So
57.50
58.23
58.97
59. .74
60.53
61.33
65.71
70 . 77
76.67
83.64
Brine
90
lo
62.22
62.92
63.64
64.37
65.12
65.88
70.00
• *74. 67
80.00
86.15
Brine
100
l-o
66.00
66.67
67.35
68.04
68.75
69.47
73.33
77.65
82.50
88.00
12.6.23.5.3 Seawater should be filtered to at least 10 /zm 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 lOOli and that the temperature does not 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
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should be filtered through a 1 /itn 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.
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 brine is 100& and the test
is to be conducted at 34%b, lOOtb divided by 34li = 2.94. The
proportion of brine is 1 part, plus 1.94 parts reagent water. To
make 1 L of dilution water at 34t-0 salinity from a HSB of lOOii,
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
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12.6.23.8.1 Table 2 illustrates the preparation of test
solutions (up to 50% effluent) at 34li 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 68tb; the required brine volume is then always
equal to the effluent volume needed for each effluent
concentration as in the example in Table 2.
12.6.23.8.2 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(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 of 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 ± 2ti.
12.6.23.9 Preparing Test Solutions
12.6.23.9.1 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
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 it, and shake to mix.
Distribute equal volumes into the replicate test chambers.
12.6,. 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
2ti and a brine salinity of 66&>, add 400 mL of brine (see
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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 Oti, 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 SP., 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 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.
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
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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 organochlorine pesticides 0.15 ug/g wet
weight or that the total concentration of organochlorine
pesticides plus PCBs exceeds 0.30 /*g/g wet weight (For analytical
methods see U'SEPA, 1982) . !
12.6.24.3 Artemia nauplii are obtained as follows:
1. Add 1 L of seawater, or an aqueous unionized salt
(NaCl) 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, 1985a; USEPA, 1993a; ASTM,
1993) .
3. After 24 h, cut off the air supply In the separatory
funnel. Artemia nauplii are phototactic, and will
concentrate at the bottom of the funnel if it is
covered for 5-10 minutes 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 s!50 /im
NITEX® or stainless steel,screen, and rinse with
seawater or equivalent before use. •'
i
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 used routinely in
the toxicity tests. Two 7-day chronic tests are performed side-
by-side, each consisting of five replicate test vessels
containing five juveniles (25 organisms per test, total of 50
153
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TABLE 2. EXAMPLES OF EFFLUENT DILUTION SHOWING VOLUMES OF
EFFLUENT (x&) , BRINE, AND DILUTION WATER NEEDED FOR
ONE LITER OF EACH TEST SOLUTION.
STEP; Combine brine with deionized water or natural seawate.r 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.
1 Effluent Cone.
(%)
40
Effluent
x&
800 mL
Brine
(68-x)&
800 mL
Dilution Water*
34&
400 mL
Step 2. Use either serially prepared dilutions of the highest test
concentration or individual dilutions of 100% effluent.
Effluent Cone. (%)
20
10
5
2.5
Control
Effluent Source
1000 mL of 40%
1000 mL of 20%
1000 mL of 10%
1000 mL of 5%
none
Dilution Water* (34&)
1000 mL
1000 mL
1000 mL
1000 mL
1000 mL
INDIVIDUAL PREPARATION
Effluent Cone.
(%)
40
20
10
5
2.5
Control
Effluent x&
400 mL
200 mL
100 mL
50 mL
25 mL
none
Brine (68-x)&
400 mL
200 mL
100 mL
50 mL
25 mL
none
Dilution Water*
34&
200 mL
600 mL
800 mL
900 mL ,
950 mL
1000 mL
*May be natural seawater or brine-reagent water equivalent.
154
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organisms). The juveniles in one set of test chambers is fed
reference (acceptable) nauplii and the other set 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. I
12.6.-24.4.3 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. • ; • '•
12.6.25 TEST ORGANISMS : '....''
i , . - .
12.6.25.1 The test organisms for this method are 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 Macrocystis
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
forest habitats and is considered to be an important food 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
release 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, 1985; Davidson et
al., 1986; Machuzac and Mikel, 1987; Reish and Lemay, 1988;
Asato, 1988; Martin et al., 1989; Singer et al., 1990; 1991; Hunt
et al., In Press). Mysids are useful as toxicity test organisms
because of their widespread availability, ecological importance,
sensitivity to toxicants, and amenability to laboratory culture
155
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(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 verification 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 collection
site, and should be gently aerated or oxygenated to maintain
dissolved oxygen above 60% saturation. Transport temperatures
should remain within 3°C 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
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fill the plastic bag with one liter of dilution water seawater,
2) saturate the seawater with oxygen by bubbling 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
nauplii per mysid, for 100 juveniles add a pinch (10 to 20 mg) of
ground Tetratnin® flake food and 200 newly-hatched Artemia
nauplii, 5) seal the bag securely, eliminating any 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 1-liter blue ice block for every five 1-liter bags
of mysids (a temperature range of 12 to 16°C is tolerable). Wrap
the ice in newspaper and a plastic bag to insulate it from the
mysid bags. Pack the bags tightly to avoid shifting 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. Initial flow rates should be adjusted so that any
temperature change occurs gradually (0.5°C per hour) . The water
temperature should be held at 15 + 1°C. Note: Mysids 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 ranging from 4 to
1000 liters: Tanks should be equipped with gentle aeration and
blades of Macrocystis to provide habitat. Static culture tanks
can be 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 Artemia nauplii per
mysid 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 monitored arid rations adjusted to
157
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prevent overfeeding and fouling of culture water. Refer to
section 12.6.19 for details.of Artemia culture and quality .
control.
12.6.25.6 Culture Materials
12.6.25.6.1 Refer to Section 5, Facilities and Equipment, 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 before re-
use.
12.6.25.7 Test Organisms ' - • -
12.6.25.7.1 Approximately 150 gravid female mysids should be
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. Marsupia appear.
distended and gray when females are ready to release young, due
to presence of the juveniles.
12.6.25.7.2 Gravid females are easily isolated from other mysids
using the following technique: (1) use a small dip net to
capture about 100 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)lift 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
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inadvertently mixing them with the soon-to-be released -juveniles...
used in testing. Provide the gravid females with newly hatched
Artemia nauplii (approximately 200 per mysid) to. help stimulate
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 placed within
the screened compartment so that as the juveniles are released,
they -can swim through the mesh into the bottom of the aquarium.
Outflows on flow-through aquaria should be screened (150-/mi-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 screened 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-/im-mesh screen-bottom tube (25 cm diam.) immersed in a bucket
filled with clean seawater. Once the release aquarium is
emptied, it should be washed with hot fresh water to eliminate
stray juveniles that might mix with the next cohort.
12.6.25.7.5 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 conduct the necessary tests,
they can be mixed with juveniles from one previous or subsequent
release so that the test is initiated with three iand/or,four-day
old juveniles. Initial experiments indicate that 'mysids 2-days- '
old and younger survive poorly in toxicity tests and that 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-liter
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 Arteraia 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 held in each
screen-tube (200 juveniles per static 4-liter beaker). Juveniles
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 day 0
(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 effluent
concentrations plus a dilution water control. 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
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12.10.2.1 Receiving waters !
I
12.10.2.1.1 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 ^im NITEX® filter and compared without dilution,
against a control. Using five replicates chambers per test, each
containing 200 rhL would require approximately 1 L or more of
sample per test per renewal. j
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
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 (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 lOQti HSB is used as a diluent, the maximum concentration of
effluent that can be tested will be 66% at 34lo salinity.
12.10.2.2.2 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%). ,
I
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 mL of test
solution, is approximately 370 mL. ,
12.10.2.2.4 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.
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12.10.2.3 Dilution Water
12.10.2.3.1 Dilution water should be uncontaminated l-/Ltm-
filtared 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°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 ^ug/L zinc stock solution by
adding 0.0440 g of zinc sulfate (ZnSO4°7H2O) 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 /xg/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 ± 2St.
12.10.3 START OF THE TEST
12.10.3.1 Prior to Beginning the Test
162
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12.10.3.1.1 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 Test),.
12.10.3.1.2 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 test 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 water bath, .room, or
incubator to the required .test temperature (13 or 15 ± 1°C) .
12.10.3.1.4 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 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
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 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.
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 cani be arranged in
numerical order for"convenience, since this will also ensure
random placement of treatments.
163
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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 juveniles
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 accomplished 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 /iE/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
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12;. 10.4.2 The water temperature in the test chambers should be-
maintained at 13 or 15 ± 1°C. It is critical that the test water
temperature be maintained at 13 ± 1°C (for mysids collected north
of Pt. Conception, California) or 15 ± 1°C (for mysids collected',
south of Pt. Conception, California) . If a wateir 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. ,
12.10.4.3 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..
12*10.4.4 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.
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 th€>. 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. . i . •
12.10.6 FEEDING ' .'
12.10.6.1 Artemia nauplii are prepared as 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
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solutions. Add 40 newly hatched Artemia nauplii per mysid per
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-/im-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 experiment, the
ration should be reduced proportionally. The mysids 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 hatched nauplii. By placing
the test chambers on a light box, inadvertent removal of live
mysids can be greatly reduced because they can be more easily
seen. 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
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12.10.8.2.1 The number of live mysids are counted and recorded
each day. Dead animals and excess food should be 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 removal of the dead mysids, 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. Because effluent
toxicity may change over short time periods in test chambers, the
test solutions must be renewed after 48 h and 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 damaging mysids. This
can be done by siphoning with a small-bore (2 to 3 mm) fire-
polished glass tube or pipet. Attach the glass tube to clear
plastic tubing fitted with a pinch clamp so that the siphon 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. If 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 highest 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.siphoning new
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 minimize
turbulence. i
i
12.10.9.3 The effluent or receiving water used in the test is
stored in an incubator or refrigerator at 4°C. Plastic chambers
such as 8-20 L cubitainers have proven suitable for effluent
collection and storage. For on-site toxicity studies no more
than 24 h should elapse between collection of the effluent and
167 •!•
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use in a toxicity test (see Section 8, Effluent and Receiving
Water Sampling, Sample Handling, 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 end of the
exposure period, remove any remaining dead mysids, then carefully
pour the contents of the test chamber through a small mesh screen
(<300ptm) . Count the mysids before screening, and take 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
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foil weigh boats over the mysids to avoid loss while drying test
organisms.
12.10.10.2.2 To prepare weigh boats prior to testing, write the
test chamber number on each with a fine felt-tipped 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 (pig) 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 use for weighing
mysids. The juvenile mysids-are very small, and light (60 jug)
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 or other small open
chamber, and place them in a clean drying oven. Dry for at least
24 hours at 60°C 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 /xg) . Record the
chamber number, mysid weight, weigh boat weight (recorded
previously), and number of mysids per weigh boat (replicate) on
the data sheet.
12.10.10.3 Endpoint
12.10.10.3.1 Growth is measured as dry weight of surviving
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.
at the end of the test will be used for subsequent statistical
analysis. :
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12.11 SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
12.11.1 A summary of test conditions and test acceptability
criteria is listed in Table 3.
12.12 ACCEPTABILITY OP 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 p.<3 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 /KJ in the
reference toxicant test.
(5) Both the mortality NOEC and LC50 must be less than 100
ptg/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
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performed for the estimation 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 the LC50 and IC25.
See the Appendices 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 OP MYSID, HOLMESIMYSIS COSTATA,
SURVIVAL DATA
12.13.2.1 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 LC50
endpoint. Concentrations at which there -is no survival in any of
the test chambers are excluded from statistical analysis of the
NOEC and LOEC, but included in the estimation of the LC,""EC, and
1C endpoints*
12.13.2.2 For the case of equal numbers of replicates across all
concentrations and the control, the evaluation of the NOEC and
LOEC endpoints is made via a parametric test, Dunnett's
Procedure, or a nonparametric test, Steel's Many-one Rank Test,
on the arc sine square root transformed data. Underlying
assumptions of Dunnett's.Procedure, normality and homogeneity of
variance, are formally tested. The test for normality is the
Shapiro-Wilk's Test, and Bartlett's Test is used to test for
homogeneity of variance. If either of these tests fails, the
nonparametric test, Steel's Many-one Rank Test, is used to
determine the NOEC .and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the
parametric .procedure.
12.13.2.3 If equal numbers of replicates occur among the
concentration levels tested, there are parametric and
nonparametric alternative analyses. The parametric analysis is a
171
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TABLE 3. SUMMARY OF :TEST CONDITIONS AND TEST ACCEPTABILITY
CRITERIA FOR THE MYSID, HOLMESIMYSIS COSTATA, GROWTH
AND SURVIVAL TEST WITH EFFLUENTS AND RECEIVING,WATERS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Test type:
Salinity:
Temperature :
Light quality:
Light intensity:
Photoperiod :
Test chamber:
Test solution volume :
Renewal of test
solutions :
Age of test organisms :
No . organisms per test
chamber :
No. replicate chambers
per concentration:
No. mysids per
concentration :
Source of food :
Feeding regime:
Static -renewal
34 ± 2to
13 + 1°C (mysids collected north
of -Ft. Conception)
15 ±, 1°C (mysids collected south
of Pt . Conception)
Ambient laboratory illumination
10-20 /iE/m2/s (Ambient
laboratory illumination)
16 h light, 8 h darknes's
1000 mL
200 mL
75% renewal at 48 and 96 hours
3 to 4 days post-hatch
juveniles
5
5
25
Newly hatched Artemia nauplii
(less than 24 h old)
Feed 40 nauplii per larvae
daily (dividing into morning
and evening feedings)
172
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16. Cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution factor:
21. Test duration:
22. Endpoints:
23. Test acceptability
criteria :
24. Sampling requirements:
25. Sample volume required:
Siphon during test solution
renewal
None unless DO falls below 4.0
mg/L, then gently aerate in all
cups
Uncontaminated l-/im- filtered
natural seawater or hyper saline
brine prepared from natural
seawater
Effluents : Minimum of 5 and a
control
Receiving waters: 100%
receiving water and a control
Effluents: ^0.5 series
Receiving waters: None,- or ^0.5
7 days
Survival and growth
^75% survival, average dry
weight ^ 0.40 /KJ in the
controls; survival MSD <40%;
growth MSD <50 /Ltg; and both
survival and growth NOECs must
be less than 100 ^g/L with zinc
For on- site tests, samples must
be used within 24 h of the time
they are removed from the
sampling device (see Section 8,
Effluent and Receiving Water
Sampling, Sample Handling, and
Sample Preparation for Toxicity
Tests)
2 L per renewal ;
173
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TABLE 4. DATA FOR HOLMES IMYSIS COSTATA
Treatment Repl i cate
Chamber
Control, Brine 1
2
3
4
5
Control. Dilution 1
2
3
4
5
1.80*
2
3
4
5
3.20*
2
3
4
5
Total
My s ids
5
5
5
5
5
5
5
5
5
5
1
5
5
5
5
1
5
5
5
5
7 -DAY SURVIVAL AND GROWTH' TEST1
No.
Alive
5
5
5
5
5
5
5
5
5
5 '•
5
5
5
4
5
5
• 4
5
5
4
Prop .
Alive
1.00
1.00
1,00
1.00
1.00
1.00
1.00
1.00
1.00
. 1.00
, 5
"-'
1.00
1.00
0.80
1.00
5
0.80
1.00
1.00
0.80
Mean
Weight
0.051
0.050
0.040
0.064
0.039
0.048
0.058
0.047
0.058
0.051
1.00
0.055
0.048
0.042
0.041
0.052
1.00
0.057
0.050
0.046
0.043
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;- 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 given
concentration are combined. If the data do not fit the Probit
model, the Spearman-Karber method, the Trimmed Spearman-Karber
174
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method, or the Graphical method may be 'used to estimate the LC50'
(see Appendices H-K) . ,
12.13.2.5 The proportion of survival in each replicate must
first be transformed by the arc sine square root transformation
procedure described in Appendix B. The raw and transformed data,
means and variances of the transformed observations at each
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 observation in that
concentration. The centered observations are listed in Table 6.
12.13.2.6.2 Calculate the denominator, D, of the test statistic:
Where: XL = the ith centered observation :
X = the overall mean of the centered observations
n = the total number of centered observations. '
12.13.2.6.3 For this set of data, n == 25 '
. ..X = 1 (Q.OQ1) = 0. 00
' ' i ' 25 ' . !
'. D = 0.227 ;
12.13.2.6.4 Order the centered observations from smallest to
largest :
X(1) <: X(2) s . :. <:, X(n>
Where X!i) is the ith ordered observation. These ordered
observations are listed1 in Table 7. '.','-
175.
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TABLE 5. MYSID, HOLMESIMYSIS COSTATA, SURVIVAL DATA
Concentration (%)
Replicate Control
1.80
3.20
5.60
10.00
RAW
ARC SINE
SQUARE
ROOT
TRANS-
FORMED
Mean(Yt)
sf
i
1
2
3
4
5
1
2
3
4
5
' 1.00
1.00
1.00
1.00
1.00
1.345
1.345
1.345
1.345
1.345
1.345
0.000
1
1.00
1.00
1.00
0.80
1.00
1.345
1.345
1.345
1.107
1.345
1.297
0.011
2
1.00
0.80
1.00
1.00
0.80
1.345
1.107
1.345
1.345
1 . 107
1.250
0.017
3
0.80
1 . 00
1.00
0.80
0.80
1.107
1.345
1.345
1.107
1.107
1.202
0.017
4
0.20
0.00
0.00
0.00
0.00
0.464
0.225
0.225
0.225
0.225
•0.273
0.011
5
12.13.2.6.5 From Table 4, Appendix B, for the number of
observations, n, obtain the coefficients al7 a2, . . . . , ak where k
is n/2 if n is 'even and (n-1) /2 if n is odd. For the data in
this example, n = 25 and k = 12. The a± values are listed in
Table 8.
12.13.2.6.6 Compute the test statistic, W, as follows:
If (.11, u, 2
W = —[Da (X( •* '-XUI) ]
D 1.1
The differences x(n-i+1) - X(i) are listed in Table 8.
176
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|P ?l 7 >'^-: '""
Ss!gJ3«f .T ' !. ~jt \ss t.f * 1 * —• 3 *'" ™ rf '•'"^tiife •"* i ^"M* % **K - "-^P" - •'•MSr^S S ^ ~a ~x :4 ^ " =- ?
Bid- •. >H :;r':^fe^5^iF^\«l»POTililfie^ L:-'^
HOMOGENEOUS VARIANC
EQUAL NUMBER OF
f TEST WITH
BONFERRONI
ADJUSTMENT
STEEL'S MANY-ONE
"RANK TEST
ENDFK3INT ESTIMATES
NOEC,I.OEq
Figure 1. Flowchart for statistical analysis of mysid,
Holmesimysis costata, survival data by hypothesis testing.
177 :
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«¥=*»>
fe-._,^
,.,..,....„ T^T." fJH-T€§f"
MORTM1TYDATA
#DEAD
TWO OR MORE ,
PARTIAL MORTALITIES?
YES
IS PROBIT MODEL
APPROPRIATE?
(SIGNIFICANT X2TEST)
YES
PROBIT METHOD
ONE OR MORE
PARTIAL MORTALtfIES?
ZERO MORTALITY M
LOWEST EFFLyENT,C0Nc",
HIGHEST EFFLUENT CON&?:'
X1 ' •*•*" •"->/* J1^"1" ^J1f~! X-* !: ^li'1""
1i"ll/n:r]>;;5,' vk;--1 ,»,J' 'ftV
r/,r_ -<^-i;-'---%,yi-.| --;-.' ^ ^^
SPEARMAN-KARBER
1 =' V J -£&
-------
6Z.T
to U
SURVIVAL PROPORTION
o o o o o o
-------
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
Concentration
Replicate Control 1.80
3.20
5.60
10.00
(Dilution)
1 0
2 0
3 0
4 0
5 0
.000 0.048
.000 0.048
.000 0.048
.000 -0.190
.000 0.048
0.095
-0..143
0.095
0.095
-0.143
-0.095'
0.143
0.143
-0.095
-0.095
0.191
-0.048
-0.048
-0.048
-0.048
=
TABLE 7. ORDERED
i
1
2
3
4
5
6
7
8
9
10
11
12
13
CENTERED OBSERVATIONS
x«)
-0.190
-0.143
-0.143
-0.095
-0.095
-0.095
-0.048
-0.048
-0.048
-0.048
0.000
0.000
0.000
FOR SHAPIRO-WILK'S
1
14
15
16
17
18
19
20
21
22
23
24
25
x<»
0.000
0.000
0.048
0.048
0.048
0.048
0.095
0.095
0.095
0.143
0.143
0.191
EXAMPLE "
180
-------
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-MILK'S-EXAMPLE
1 a, X(n-1+1) - XC1)
1 0.4450
2 0.3069
3 0.2543
4 0.2148
5 0.1822
6 0 . 1539
7 0.1283
8 0.1046
9 0.0823
10 0.0610
11 0.0403
12 0.0200
0.381
0.286
0.286
0.190
0.190
0.190
0.096
0.096
0.096
0.096
0.000
0.000
X(25)
XC24)
X(23)
X(22)
: X(2i)
X(20)
XU9)
: Xd8)
. •: X(17)
X(16)
X(15)
X(14)'
.- X(1)
- x(2)
- X(3)
- x(4)
- x(5)
- X(6)
- x(7)
- x(8)
- XC9)
- x(10)
- x(11)
_ Xa2) •••••
'
For this data in this example: ;
W = 1 (0.4708)2 = 0.976
0.227 ' "
i
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 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 = 25 observations is 0.888. Since W == 0.976 is
greater than the critical value, conclude that the data are
normally distributed. ;
12.13.2.6.8 Since the variance of the control giroup 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 the assumption of
homogeneity of variance, Steel's Many-one Rank Test will be used
to analyze the survival data. •:.,'.•••• , -.•••„
181
-------
12.13.2.7 Steel's Many-one Rank Test
12.13.2.7.1 For each control and concentration combination,
combine the data and arrange the observations in order of size
from smallest to largest. Assign the,ranks (1, 2, ... , '10) to
the ordered observations with a rank of i assigned to the
smallest observation, rank of 2 assigned to the next larger
observation, etc. If ties occur when ranking, assign the average
rank to each tied observation.
12.13.2.7.2 An example of assigning ranks to the combined data
for the control and 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 the control.
Thus compare the rank sums for the survival at each of the
various concentration levels with some "minimum" or critical rank
sum, at or below which the survival would be .considered
significantly lower than the control. At a significance level of
0.05, the minimum rank sum in a test with four concentrations
(excluding the control) 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
LC50 for this dataset.
12.13.2.8.2 For the Trimmed Spearman-Karber analysis, run,the
USEPA Trimmed Spearman-Karber program, TSK. An example of the
program output is provided in Figure 4. ,
182
-------
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 Proportion
of Total Mortality
1.107
.1.345
1.345
1.345
1.345
1.345
1.345
1.345
1.345
1.345
Concentration
1 .80%
Control
Control
Control
: Control
Control
1.80%
: 1.80%
1.80%
1.80%
• - •
•:
TABLE 10. TABLE OF RANKS1 :
Concentration (%)
Repli- Control
cate
1 1.345(6,6.5.7.8)
2 1.345(6,6.5.7,8)
3 1.345(6,6.5,7,8)
4 1.345(6,6.5,7,8)
5 1.345(6,6.5,7,8)
1.80 3.20
1.345(6) 1.345(6.5)
1.345(6) 1.107(1.5)
1.345(6) 1.345(6.5)
1.107(1) 1.345(6.5)
1.345(6) 1.107(1.5)
5.60 10.0
1.107(2) 0.464(5)
1.345(7) 0.225(2.
1.345(7) 0.225(2.
1.107(2) 0.225(2.
1.107(2) '0.225(2.
5)
5).
5)
5)
Control ranks are given in the order of the concentration with which
they were ranked. !
183
-------
TABLE 11. RANK SUMS
Concentration Rank Sum
1.80 25.0
3.20 22.5
5.60 20.0
10.00 15.0
TABLE 12. DATA FOR TRIMMED SPEARMAN-KARBER ANALYSIS
Concentration (%)
Control 1.80 3.20 5.60 10.0 18.0
No Dead
No Exposed
0
25
1
25
2
25
3
25
24
25
25
25
12.13.3 EXAMPLE OF ANALYSIS OF MYSID, HOLMESIMYSIS COSTATA
GROWTH DATA
12.13.3.1 Formal statistical analysis of the growth 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:
TOXICANT
SPECIES:
RAW DATA:
TEST NUMBER: 1
DURATION:
7 days
: Effluent
Holmesimysis costata
Concentration
U)
.00
1.80
3.20
5.60
10.00
18.00
SPEARMAN-KARBER TRIM:
Number
Exposed
25
25
25
25
25
25
4.00%
SPEARMAN-KARBER ESTIMATES: LC50:
95% LOWER CONFIDENCE:
9b% UPPER CONFIDENCE:
Mortalities
i
0
1
2
3
• 24
25
6.95
6.22
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 testing can be used to
obtain an NOEC and LOEC for growth. Concentrations above the
NOEC for survival are excluded from the hypothesis test for
growth effects. '
12.13.3.2 The statistical analysis using hypothesis tests
consists of a1parametric test, Dunnett's Procedure, and a
nonparametric test, Steel's Many-one Rank Test. ;The underlying
185
-------
SURVIVAL AND GROWTH TEST
";, :!• ,,, :,•" - •..;.,'..-;' ^i^v-^a',;
GROWTH :•.;,: N,..,.,. u „ wtfj»A.i*V.ffi
^ff^W-iS^iWK
^-^S^^^^AyfW^VfS'W
•i' >• k.' r- r i i, i i it j< n (li {i ii 11 11 ,ni}», 't i nil i -1 .
r J~J,"S | HH.! "A" Si,! " ' '" „'''•« ' ***' I 'l
WEAN WEIGHT
POINT ESTIMATION
(EXCLUDING CONCENTRATIONS
AlOVE NOEC FORSMVIVAL)
ENDPO1NT ESTIMATE
IC25
SHAPlRO-WILIgSTEST
NORMAL DISTRIBUTION
BARTl-El-STEST
HOMOGENEOUS VARIANCE
EQUAL NUMBER OF
REPLICATES?
tTESTWITH
BONFERRONI
ADJUSTMENT
DUNNETTS
TEST ,
STEEL'S MANY-ONE
RANK TEST
ENDPOINf fIMATES
~
Figure 5. Flowchart for statistical analysis of mysid,
Holmesimysis costata, growth data.
186
-------
assumptions of the Dunnett's Procedure, normality and homogeneity
of variance, are formally tested. The test for, normality is the
Shapiro-Wilk1s 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 observations 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
D = E(XJ - X)2
i-1 , •:...•
187
-------
TABLE 13. MYSID, HOLMESIMYSIS COSTATA, GROWTH DATA
Replicate Control
1.80
Concentration (%)
3.20
5.60
10.0
1
2
3
4
5
0.048
0.058
0.047
0.058
0.051
0.055
0.048
0.042
0.041
0.052
0.057
0.050
0.046
0.043
0.045
0 . 041
0.040
0.041
0.043
0.040 .
0.033
0.000
0.000
0.000
0.000
MeanCY,)
sf
i
0.052
0.0000283
1
0.048
0.0000373
2
0.048
0.0000307
3
0 . 041
0.0000015
4
0.007
0.000218
5
TABLE 14. CENTERED OBSERVATIONS FOR S.HAPIRO-WILK'S EXAMPLE
Concentration (%)
Replicate
Control
1.80
3.20
5.60
1
2
3
4
5
-0.004
0.006
-0.005
0.006
-0.001
0.007
0.000
-0.006
-0.007
0.004
0.009
0.002
-0.002
-0.005
-0.003
0.000
-0.001
0.00.0
0.002
-0.001
188
-------
ta
0)
4J
n)
4J
n)
4J
w
o
o
ta
Q)
E
•a
•H
w
-a
(5uj) 1HDI3M AHQ NV3W
189
-------
Where: X± = 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 = 1 (0.001) = 0.000
20
D = 0.000393
I
12.13.3.5.4 Order the centered observations from smallest to
largest
X'1' * X(2) <: ... s X(n>
where X(i) denotes the ith ordered observation. The ordered
observations for this example are listed in Table 15.
TABLE 15.
i
1
2
3
4
5
6
7
8
9
10
ORDERED CENTERED
xo)
-0.007
-0.006
-0.005
-0.005
-0.004
-0.003
-0.002
-0.001
-0.001
-0.001
OBSERVATIONS
i
11
12
13
14
15
16
17
18
19
20
FOR. SHAPIRO-MILK'S 'EXAMPLE
xu)
0.000
0.000
0.000
0.002
0.002
0.004
0.006
0.006 . . . . •
0.007
0.009
190
-------
12.13.3.5.5 From Table 4, Appendix B, for the number of
observations, n, obtain the coefficients alf a2, ... ak where k is
n/2 if n is even and (n-l)/2 if n is odd. For the data in this
example, n = 20 and k = 10. The a± values are listed in
Table 16. . . . : •
12.13.3.5.6 Compute the test statistic, W, as follows:
W- -[£a fX1"1*11-*"1)]
D i.i
The differences x(n-i+1) - 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-MILK'S EXAMPLE
1 0.4734
2 0.3211
3 0.2565
4' 0.2085
5 0.1686
6 0.1334
.7 0.1013
8 0.0711
9 0.0422
10 0.0140
0.016
0.013
0.011
0.011
0.008
0.005
0.004
0.001
0.001
0.001
:X(2o)
X(19)
; x
X(13)
X(12)
. X(ll)
- x(1)
- x(2)
- x(3)
- x(4)
- x(5)
- x(6)
- x(7)
- x(8)
- x(9>
- X(10)
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 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
191 i
-------
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 _ f
[(Svp In S2 - El^ In
B= —^ i2
Where :Vi = degrees of freedom for each concentration and the
control, Vi = (n.j. - 1)
p = number of concentration,levels including the
control
In = loge
i = 1,2, ..., p where p is the number of
concentrations including the control
nj. = the number of replicates for concentration i.
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, V± =4 for all i.
192
-------
12.13.3.6.3 Bartlett's statistic is therefore:
B= [(16)ln(0.0000245)-4Eln(Si2)]/1.104
[16(-10.617) - 4 (-44.470) ]•/!. 104
= [-169.872 - (-177.880)]/I.104
i
= 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 level of 0.01 with three degrees of freedom, is
9.210. Since B = 7.254 is less 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 as described in
Table 17.
TABLE 17. ANOVA TABLE :
Source
Between
Within
df Sum of Squares
(SS)
p - 1 SSB
N - p SSW
Mean Square (MS)
(SS/df)
SB = SSB/Cp-1)
Sw = SSW/(N-p)
Where: p = number of concentration levels including the
control i
N = total number of observations nt + n2 ... + np
193
-------
n.i = number of observations in concentration i
e
SSB - ErVn -G2/N Between Sum of Squares
i-i
p ai
SST = £Er2 -GZ/N Total Sum of Squares
i-ij-i
SSW « SST-SSB Within Sum of Squares
G = the grand total • of all sample observations,
e ' .
G = E^
i-i ' • •
Ti = the total of the replicate measurements for
concentration i
Yij = the jth 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 example:
nx = n2 = n3 = n4 = 5
N = 20
TI = YU + Y12 + ... + Y15 = 0.262
T2 = Y21 + Y22 + '. . . + Y25 = 0.238
T3 = Y31.+ Y32 + ... + Y35 = 0.241
T4 = Y41 + Y42 + ... + Y45 = 0.205
G = T! + T2 + T3 + T4 = 0.946
J_(0.225) - (0.946)2 = 0.000254
5 . 20
SSB =
1-1
p
SST =
= 0.0455 - (0.946)2 = 0.000754
20
194
-------
SSW = SST-SSB
= 0.000754 - 0.00.0254 - 0.000500
S| = SSB/(p-l) = 0.000254/(4-l). = 0.0000847
Sj''a SSW/{N-p) = 0.000500/(20-4) = 0.0000313
12.13.3.7.3 Summarize these calculations in the ANOVA table
(Table 18). . '
TABLE
Source
Between
Within
Total
18. ANOVA TABLE FOR DUNNETT'S
df Sum of Squares
(SS)
3 -0.000254
16 0.000500
19 0.000754
PROCEDURE EXAMPLE
1
Mean Square (MS)
(SS/df)
0.0000847
0.0000313
12.13.3.7.4, To perform the individual comparisons, calculate the
t statistic for each concentration, and control combination as
follows:.
ti-
Where: YA = mean weight for concentration i
Yx =' mean weight for the control
• Sw = square root of the within mean square
nt = number of replicates for the control
195 :
-------
Hi = number of replicates for concentration i
12.13.3.7.5 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:
(0.052-0.048)
[0.00559/71/5)7(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
12.13.3.7.6 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, 16
degrees of freedom for error and three concentrations (excluding
the control) the approximate critical value is 2.23. The mean
weight for concentration "i" is considered significantly less
than the mean weight for the control if t^ is greater than the
critical value. Therefore, the 5.60% concentration has
significantly lower mean weight than the control. Hence the NOEC
and the LOEC for growth are 3.20% and 5.60%, respectively.
12.13.3.7.7 To quantify the sensitivity'of the test, the minimum
significant difference (MSD) that can be detected statistically
may be calculated.
MSD
196
-------
critical value for Dunnett's Procedure
square root of the within mean square
common number of replicates at'each
nentration
.ssumes equal replication at .each concentration)
number of replicates in the control.
his example:
MSD = 2.23(0.00559)^/(l/5)
i
= 2.23 (0.00559) (0.632)
= 0.00788
t . •.
•efore, for this set of data, thle minimum
:an be detected as statistically significant is
s represents a 15.2% reduction in mean weight
, '.*.'"., - f~ f
ation of the ICp
i
growth data from Table 13. are utilized in. this
L in the table, the observed means are
L-increasing with respect to concentration.
loothed means will be simply the> corresponding
'he observed means are represented by Y± and the
r Mi. Table 20 contains the smoothed means and
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:
ICp
IC25 = 5.60 + [0.052(1 - 25/100) - 0.041] (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
Toxi cant
Cone.
U)
Control
1.80
3.20
5.60
10.00
18.00
1
1
2
3
4
5
6
Response
Means
Y, (mg)
0.052
0.048
0.048
0.041
0.0066
0.000
Smoothed
Means
M,. (mg)
0.052
0.048
0.048
0.041
0.0066
0.000
12.14 PRECISION AND ACCURACY
12.12.1 PRECISION
12.12.1.1 Single-Laboratory Precision
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 £ig/L Zn. There was also good agreement among LGBOs, 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. IC50 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 used 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 concentration 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 iboth
laboratories. Growth was the more sensitive indicator 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
-------
•a
•H
s.
-X-
NV3W
200
-------
Cone. ID
Cone. Tested
Response
Response
Response
Response
Response
1
2
3
4
5
1
0
.048
.058
.047
.058
.051
2
1.80
.055
.048
.042
.041
.052
3
3.20
.057
.050
.046
.043
.045
4
5.60
.041
.040
.041
.043
.040
5
10.0
.033
0
0
0
0
6
18.0
0
0
0
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
1
2
3
4
5
6
Number
Replicates
5
5
.5
5
5
5
Concentration
%
0.000
1.800
3.200
5.600
10.000
18.000
Response
Means
0.052
0.048
0.048
0.041
0.007
0.000
Std. Pooled
Dev. Response 'Means
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: 5.8205 Standard Deviation: 0.2673
Original Confidence Limits: Lower: 4.9440 Upper: 6.2553
Expanded Confidence Limits: 'Lower: 4.5073 Upper: 6.4743
Resampling time in Seconds: 0.22 Random_Seed: 526805435
Figure 8. Output for USEPA Linear Interpolation Program'for the IC25.
201
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TABLE 21. SINGLE LABORATORY PRECISION DATA FOR THE MYSID,
HOLMESIMYSIS COSTATA GROWTH AND SURVIVAL TEST WITH
ZINC (ZN /-iG/L) SULFATE AS THE REFERENCE TOXICANT
Test
NOEC
1 32
2 32
3 56
4 56
N 4
Mean 44
SD
CV (%)
Survival
LC50
47
59
62
65
4
58
7.9
14
Growth
NOEC
>32
>32
>56
>56
4 '
>44
No growth effect was observed in zinc concentrations below those
causing significant mortality (10, 18, 32, 56 and 100 /i.g/L) .
All tests were conducted at MPSL.
202
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TABLE 22. MULT I-LABORATORY PRECISION DATA FOR THE MYSID,
HOLMESIMYSIS COSTATA GROWTH AND SURVIVAL TEST WITH
SPLIT EFFLUENT (%) ON THE SAME DATE.
Test
1
1
2
2
3
3
4
4
Effluent
Type
BKME
BKME
POTW
POTW
POTW
POTW
POTW
POTW
Lab
OSU
MPSL
ATL
MPSL
SRH
MPSL
SRH
MPSL
Survival
NOEC LC50
1.0 1.8
1.0 1.3
CV=26%
3.2 4.1
3.2 5.1
CV=14%
10.0 12.8
10.0 11.7
CV=6%
10.0 15.8
5.6 9.1
CV=38%
Growth
NOEC
0.5L
0.5L
>3.2L
>3.2L
na
3.2"
5 . 6W
3.2W
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
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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
jig/L) by adding 0.0440 g of zinc sulfate (ZnSO4°7H2O) to 1
liter of reagent water.
D. Prepare zinc reference toxicant solution of 0 (control) 10,
18, 32, 56 and 100 /Kj/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 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
continuously.
I. At the end of the test, measure salinity, pH, and dissolved
oxygen concentration from each test concentration.
204
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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 80-liter
aquarium (15°C) . Add a surplus of Artemia nauplii (200 per
tnysid, static; 500 per mysid, flow-through) to stimulate
overnight release of juveniles. Add" blades of kelp as
habitat.
B. Isolate the newly released juveniles by slowly siphoning
into a screen-tube (150-/zm-mesh, 25 cm diarrt.) immersed in a
bucket of clean seawater. Transfer juveniles into
additional screen-tubes or static 4-liter beakers at a .
density of approximately 50 juveniles per liter.
Juveniles should be fed five to ten 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.
Maintain the juveniles for three days at 13; to 15 °C,
changing the water at least once in static chambers.
C. After three days, begin randomized introduction 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
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
Artemia. nauplii/mysid/day, adjusting feeding 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 test chamber through a
small mesh screen (<300/itn) . 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 jug) . 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
-------
Date Sheet for Juvenile Holmesimysis Toxicity Test
Test Start Date:
Test End Date:
Reference Toxicant:
Sample Source:
Start Time:
End Time:
Mysid Source
Collection/Arrival Date:
Mysid Age at Start:
Test
Cont.
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
. l4
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Toxic
Cone.
Number Alive ;
Day 1
Day 2
Day 3
Day 4
Day5
Day 6
Day 7
Total
Niimber
Alive
••
- '
Total
Number
at Start
Notes and
Initials
Computer Data Storage
Disk: !
File: ;
Note: See juvenile growth data on separate sheet. '
207
-------
Data Sheet for Weighing Juvenile Mysids
Test Start Date:
Start Time:
Mysid Source :
Test End Date:
End Time:
Collection/Arrival Date:
Reference Toxicant:
Sample Source:
Sample Type:
Mysid Age at Start:
Test
Container
Number
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
5.1
28
29
30
31
32
33
34
35
Site Code
or
Concentration
Foil
Number
Foil
Weight
fog)
Total
Weight
fog)
Mysid Wt
(Total - Foil)
(mg)
Number of
Mysids
Weight per
Mysid
fog)
Computer Data Storage
Disk:
File:
Note: See mysid mortality data on separate sheet.
208
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SECTION 13
PACIFIC OYSTER, Crassostrea gigas
AND MUSSEL, Mytilus sp.
EMBRYO-LARVAL DEVELOPMENT TEST METHOD
Adapted from a method developed by
Gary A. Chapman, U.S. EPA, ORD Newport, OR
and Debra L. Denton, U.S. EPA, Region IX
TABLE OF CONTENTS
13.1 Scope and Application i
13.2 Summary of Method
13.3 Interferences
13.4 . Safety
13.5 Apparatus and Equipment :
13.6 Reagents and Supplies ,!
13.7 Effluents and Receiving.Water Collection,
Preservation, and Storage
13.8 Calibration and Standardization
13.9 Quality Control ;- -.
13.10 Test Procedures , - '
13.11 Summary of Test Conditions and Test :
Acceptability Criteria
13.12 Acceptability of Test Results
13.13 Data Analysis ,
13.14 Precision and Accuracy
Appendix I Step-by Step Summary
209
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SECTION 13
PACIFIC OYSTER, CRASSOSTREA GIGAS, 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. galloprovineialls, 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 not 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-by-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
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substances in marine and estuarine waters. The test endpoint is
normal shell development and should include mortality 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 Supplies).
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 acclimating
adult pacific oysters and mussels, e.g., standard salt water
aquarium 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' solutions 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 -- for determining salinity.
13.5.6 Hydrometer(s) -- for calibrating refractometer.
211
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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 non-
toxic 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
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13.5.21 20-liter cubitainers or polycarbonate water cooler jugs
-- for making hypersaline brine. ... • • .
13.5.22 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.
13.5.23 Beakers, 50 mL -- for pooling surrogate water samples
for chemistry 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 (formalin or
glutaraldehyde) during the counting of larvae and reduces sample
examination time. Alternatively, a Sedgewick-Rafter 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. " i
13.5.29 Nytex screens, ca. 75 /im and ca. 37 /on, -- for rinsing
gametes to separate individual gametes from larger material; for
retaining eggs, embryos, or larvae.
13.5.30 60 /im NITEX® filter -- for filtering receiving water.
13.6 REAGENTS AND SUPPLIES
13.6.1 Sample containers -- for sample shipment and storage (see
Section 8, Effluent and Receiving Water Sampling, and Sample
Handling, and Sample Preparation for Toxicity Tests).
213
-------
13.6.2 Data sheets (one set per test) -- for data recording (see
Figure 1).
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 -- for automatic pipets.
13.6.9 Coverslips -- 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), 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 larvae fo± 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, Facilities> 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-/zm-filtered natural
seawater. Hypersaline brine should be prepared from dilution
water. ;
13.6.23 HYPERSALINE BRINES . j
13.6.23.1 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. [
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 (lOOli) is used as a
diluent, the maximum concentration of effluent (0&) that can be
tested is 66% effluent at 34&> salinity (see Table 1) .
13.6.23.3 High quality (and preferably high salinity) seawater
should be filtered to at least 10 /xm before 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
lOOio. It is advisable not to exceed about 70&> brine salinity
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 /im 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
-------
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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 for making brine using heat-
assisted 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.
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 /xm 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 lOOti 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 jum 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.
218
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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 Brine1
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 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.
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 water to brine.
For example, if the salinity of the brine is 100&> and the test
is to be conducted at 30lro, 10.0& divided by 30%> ='3.33. The
proportion of brine is 1 part in 3.33 (one part brine to 2.33
parts reagent water) . To make 1 L of dilution water at 30li
salinity from a HSB of lOOli, 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, 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
219
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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 &>) , 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 (30 - SE)/(SB - 30)
13.6.23.8.4 This calculation assumes that dilution water
salinity is 30 ± 2&.,
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
2fe and a brine salinity of 66to, 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
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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: Combine brine with reagent water or natural seawater
to achieve a brine of 68-xfe and, unless natural seawater is used
for dilution water, also a brine-based dilution water of 34tb.
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.
(%)
40
Effluent .
X&
800 mL
Brine
(68-x)t-o
800 mL
Dilution
Water* 34fc
400 mL
Step 2. Make serial dilutions from the highest test
concentration.
Effluent Cone. (%)'
20
10
5
2.5
Control
Effluent Source
1000 mL of 40%
1000 mL of 20%
1000 mL of 10%
1000 mL of 5%
none
Dilution Water*
(341-=)
1000 mL
1000 mL
1000 mL ,
1000 mL
1000; mL
INDIVIDUAL PREPARATION: .
Effluent Cone.
(%)
40
20
10
5
2.5
Control
Effluent xlro
400 mL
200 mL
100 mL
50 mL
25 mL
none
Brine (68-x)li
400 mL
200 mL
100 mL :
50 mL
25 mL ;
none
Dilution
Water* 34lb
200 mL
600 mL
800 mL
900 mL
950 mL
1000 mL
*May be natural seawater or brine-reagent water equivalent.
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mL) beaker and stir. Distribute equal volumes into the replicate
test chambers. The remaining test solution can be used for
chemistry.
13.6.23.10 Brine Controls
13.6.23.10.1 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, 13.6.23.8.3) setting SE =
0, and solving for VE.
VE = VB x (SB - 30)/(30 - SE)
If effluent salinity is essentially 03o, 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
13.6.24.1 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.
13.6.24.2 Species Identification
13.6.24.2.1 The three species of mussels included in this method
are presumably native to the west coast. The California mussel
(Mytilus californianus) is distributed along the exposed rocky
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.
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galloprovincialis reported from San Francisco Bay to Baja
California, and M. trossulus reported from Monterey to Alaska.
I
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. galloprovinciallis) may
not be possible without electrophoretic characterization. The
geographic source of the Mytilus spp. broodstock must be
reported. 1
13.6.24.3 Obtaining Broodstock :
13.6.24.3.1 Adult oysters (Crassostrea gigas) and mussels
(Mytilus spp.) can be obtained from commercial suppliers and the
mussels can also be collected from the field. Organisms are best
shipped in damp 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 natural seawater, or salt .water
prepared from 'commercial sea salts is recirculated. The animals
are checked daily and any obviously unhealthy animals 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 (^30Si) 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
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toweling. Upon arrival at the field site, 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 consists of at least 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 provide an average initial embryo density 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).
13.10.1.2 Effluent concentrations are expressed as percent
effluent.
13.10.2 TEST SOLUTIONS
13.10.2.1 Receiving waters
13.10.2.1.1 The sampling point is determined by the objectives
of the test. At estuarine and marine sites, samples are usually
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collected at mid-depth.. Receiving, water toxicity is determined
with samples used directly as collected or with samples passed
through a 60 /im NITEX® filter and compared without dilution,
against a control. Using four replicate chambers per test, each
containing 10 mL, and 400 mL for chemical analysis, 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.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 ins.tream
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 lOOfe MSB is used as a diluent, the maximum concentration of
effluent that can be tested will be 70% at 30tb salinity.
13.10.2.2.2 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%). : ,
13.10.2.2.3 The volume in each test chamber is 10 mL.
i
13.10.2.2.4 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. ' \ '
13.10.2.3 Dilution Water
13.10.2.3.1 Dilution water should be uncontaminated l-/itn-
filtered natural seawater •or hypersaline brine (prepared from
uncontaminated natural seawater) plus reagent water (see Section
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7, Dilution Water). Natural seawater may be uncontaminated
receiving water. This water is used in all dilution steps and as
the control water.
13.10.2.4 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 (CuCl2°H2O). 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 (CuCl2°2H2O) 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. .
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, 9.5, 13.9, 20.4, and 30.0
jug/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
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mussels) and allow time for them to resume pumping (ca. 30.
minutes). 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
Count Control
Brine Control
Dilution Water Control
Effluent cone. 1
Effluent cone. 2
Effluent cone. 3
Effluent cone . 4
Effluent cone. 5
TOTAL Chambers = 41-55
• Test Vials
6
4
4
4-
4-
4
4
4
34
Chemistry
Vials
0
:' 1-3
1-3
1-3
! 1~3
1-3
1-3
1-3
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 mussels) , checking
for spawning. . . - ;
13.10.3.1.3 If no spawning occurs after 30 minutes, 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 once; spawning
begins. Mussels can also be induced to spawn by injection of 0.5
M KC1 into the posterior adductor muscle. Oysters.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 '
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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 with 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 passed through Nytex screen
(ca. 75 fj,m) to separate out clumps of gametes or extraneous
material.
13.10.3.2.6 The pooled eggs are placed into a 1 L 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
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e.g., 100 mL of egg suspension plus 1, 3, and 10 mL of sperm
suspension. This test fertilization should be accomplished
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 microscopic 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 , :
i
13.10*4.1 Prior to Beginning the Test 1
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 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 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 solutions should be adjusted 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 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. Recbrd 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
229
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investigator's name, and safely store it away until after 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 chamber counts on 1 mL subsamples
from a stirred suspension of embryos. Final larval density of
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 randomized
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 the embryos using a 37
(tm screen and backwash with a smaller volume for small counting
chambers. In either procedure, appropriate multiple rinsing1is
needed to achieve quantitative transfer of embryos.
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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-controlled water
bath for 4 8 hours. I
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 to 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 duration).
13.10.5 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE
13.10.5.1 The light quality and intensity should .be at ambient
laboratory conditions. Light intensity should be 10-20. /iE/m2/s,
or 50 to 100 foot candles (ft-c), with a 16 h light and 8 h dark
cycle. i. • • .
•
13.10.5.2 The water temperature in the test chambers should be
maintained at 18 or 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. !
!
13.10.5.3 The test salinity should be in the range of 30 ± 2tr0.
The salinity should vary by no more than ±2t« among the chambers
on a given day. If effluent and receiving water tests are
conducted concurrently, the salinities of these tests should be
similar. I
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 '
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polyethylene plastic may help prevent volatilization 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 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.
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 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.
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
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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 counts.
13.10.8.2 Sample Preservation
13.10.8.2.1 To terminate the test, add 0.25 mL 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,
cafe should be taken to confirm that glutaraldehyde kills,
preserves, and produces no artifacts that would! 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 . i •
13.10.8.3.1 After addition of preservative, observe all the
larvae in each test vial. This can be done by examining the
contents of each test vial with an inverted microscope at about
40X-50X magnification or by quantitative transfer of all larvae
onto a counting chamber and counting using a compound 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 meat 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 considered normal,
provided development has been completed" (ASTM, 1994). Record
the final counts on the data sheet.
233
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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 fo.r 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); 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 N1 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.
2.
3.
4.
Test type:
Salinity:
Temperature :
Light quality:
Static non-renewal
30 + 2lb
20 ± 1°C (oysters)
15 or 18 ± 1°C (mussels)*
Ambient laboratory light
234
-------
5.
6.
7.
8.
9.
10.
11-
12.
13.
14.
15.
16.
Light intensity:
Photoperiod :
Test chamber size :
Test solution volume :
No. larvae per chamber:
No. replicate chambers
per concentration:
Dilution water:
Test concentrations:
Dilution factor:
Test duration:
Endpoint :
Test acceptability
criteria:
10-20 uE/m2/s (Ambient
laboratory levels)
16 h light, 8 h darkness
30 mL
10 mL
150-300
4 (plus 3 chemistry vials)
Uncontaminated l-/im-f iltered
natural seawater or hypersaline
brine prepared from natural
seawater
Effluents: Minimum of 5 and a
control .
Receiving waters: 100%
receiving water and a control
Effluents: 2:0.5
Receiving waters: None or ^0.5
48 hours (or until complete
development up to 54 hours)
Survival and normal shell
development • •:
Control survival must be ^70%
for oyster embryos or ^50% for
mussel embryos in control
vials; ^90% normal shell
development in surviving
controls; and must achieve a
%MSD Of <25% i
235
-------
17. Sampling requirements:
18. Sample volume required:
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 Toxicity Tests)
1 L per test
*Mussel embryo-larval tests were commonly conducted at 156C
(ASTM, 1994). 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 characteristics, or other factors.
13.12 ACCEPTABILITY OF TEST RESULTS
13.12.1 For tests to be considered acceptable, the following
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.
13.13 DATA ANALYSIS
13.13.1 GENERAL
13.13.1.1 Tabulate and summarize the data. Calculate the
proportion of normally developed larvae for each replicate.
sample set of test data is listed in Table 5.
A
236
-------
13.13.1.2 Final calculations are based upon counts 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:
A = 100 (N1 - B')
N'
where:
A = percent abnormal and dead organisms
B1 = the adjusted number of -normal larvae at the end of the
test
N1 = the initial number of embryos in the test chambers
expressed as the mean of the initial counts;
and: if N > N1, where . ;
N = the actual number of larvae at the end of the test
then: B' = B (N1 / N)
where: B = the actual number of normal larvae at the end of the
test but, when N £ N1, then: B1 = 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 = 1QQ (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
(/*g/L)
Control
0.13
0.25
0.50
1.00
2.00
Replicate
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
Initial
Density
25
25
25
30
25
30
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
Number
Surviving
22
25
25
30
23
30
25
24
25
19
21
23
11
14
17
15
8
6
8
11
2
Number
Normal
22
24
25
29
22
29
25
23
23
18
19
22
10
13
15
14
7
5
7 .
9
2
Proportion
Normal
1.00
0.96
1.00
0.97
0.96
0.97
1.00
0.96
0.92
0.95
0.90
0.96
0.91
0.93
0.88
0.93
0.88
0.83
0.88
0.82
1.00
13.13.1.5 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,
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. See the-Appendices for examples
of the manual computations, and examples of data input and
program output.
13.13.2 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 used 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 endpoints and for the
estimation of the IC25 endpoint. Concentrations1at which there
is no normal development in any of the test chambers 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 replicates across all
concentrations and the control, the evaluation of the NOEC and
LOEC endpoints is made via a parametric test, Dunnett's
Procedure, or a nonparametric test, Steel's Many-one Rank Test,
on the arc sine square root transformed data. Underlying
assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality -is the
Shapiro-Wilk's Test, and Bartlett's Test is used!to test for
homogeneity of variance. If either of these tests fails, the
nonparametric test, Steel's Many-one Rank Test, is used to
determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett' s Procedure are met, the endpoints are estimated by the
parametric procedure.
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 Development 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 transformation 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 cpntrol are
listed in Table 5. The data are plotted in Figure 2.
239
-------
.
sTAffitteAi ANwySii
EMBRYOlAKVAL DEVELOPMENT TEST
>^^i
s*
IARVALDEVEU
PROPORTIONS
DATA
LARVAE
'/' -'/*•.:
, '. -: '•'. '••' -1
*
PCHNTESTMATION
i
HYPOTHESIS
! ENDPOfNT ESTIMATE
IC25
ARC SINE SQUARE ROOT
TRANSFORMATION
I
SHAPIRaWILK'S TEST
NORMAL DISTRIBUTION
HOMOGENEOUS VARIANCE
NO
NON-NORMAI DISTRIBUTION
BARTLBTTSTEST
**•«
EQUAL NUMBER OF
REPLICATES?
YES
t TEST WITH
BONFERRONI
ADJUSTK^NT
REWJCMTESt
DUNNETT'S
TEST
NO
YES
STEEL'S MANY-ONE
BONF
Figure 1. Flowchart for statistical analysis of the pacific
oyster, Crassostrea gigas, and mussel, Mytilua 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 lof all
observations within a concentration from each observation in that
concentration. The centered observations are summarized in
Table 6.
TABLE 6. BIVALVE EMBRYO-LARVAL DEVELOPMENT DATA
Copper Concentration (/xg/L)
Replicate
RAW
ARC SINE
SQUARE ROOT
TRANSFORMED
Mean (Yi)
S2
i
i
A
B
C
D
A
B
C
D
-
Control
1.00
0 .96
1.00
0.97
1.571
1.369
1.571
1.397
1.477
0.01191
1
0.13
0.96
0.97
1.00
0.96
1.369
1.397
1.571
1.369
1.427
0.00945
2
0.25
0.92
0.95
0.90
0.96
1.284
1.345
1.249
1.369
1.312 •
0.00303
3
0.50
0.91
0.93
0.88
0.93
1.266
1.303
1.217 ;
1.303
1.272
0.00166
4 .
1.00
0.88
0.83
0.88
0.82
1.217
1.146
1.217
1.133
1.178
0.00203
5
2.00
1.00
0.67
0.75
0.40
1.571
0.959
1.047
0.685
1.066
0.13733
6
13.13.2.5.2 Calculate the denominator, D, of the statistic:
D = E (x,. - x)2
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
X = 1 (-0.002) = 0.000
• 24
D = 0.4963*
241
-------
13.13.2.5.4 Order the centered observations from smallest to
largest
<: X(2> <: . . . * X'n>
where X(i) denotes the ith ordered observation. The ordered
observations for this example are listed in Table 7
TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK1S
EXAMPLE
1
2
3
4
5
6
7
8
9
10
11
12
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
.381
.108
.107
.080
.063
.058
.058
.055
.045
.032
.030
.028
13
14
15
16
17
18
19
20
21
22
23
24
-0
-0
0
0
0
0
0
0
0
0
0
0
.019
.006
.031
.031
.033
.039
.039
.057
.094
.094
.144
.505
13.13.2.5.5 From Table 4, Appendix B, for the number of
observations, n, obtain the coefficients al7 a2, ... ak where k is
n/2 if n is even and (n-1) /2 if n is odd. For the data in this
example, n = 24 and k = 12 . The a.j. values are listed in Table 8.
13.13.2.5.6 Compute the test statistic, W, as follows:
ff = —
D
T k 2
"""1
The differences, x(n-i+1) - X(i>, are listed in Table 8. For the
data in this example:
242
-------
W =
0.4963
(0.6322)2 = 0.805
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK1 S
EXAMPLE
1 0.4493
2 0.3098
3 0.2554
4 0.2154
5 0.1807
6 0.1512
7 0.1245
8 0.0997
9 0.0764
10 0.0539
11 0.0321
12 0.0107
0.886
0.252
0.201
0.174
0.120
0.097
0.097
0,088
0.076
0.063
0.024
0.009
X(24)
' X(23>
X(22)
X(21)
X(20)
X(19)
:, X(18>
X(17)
X(16)
x(as)
: x(14)
X(13)
- X(1>
- X<2)
- x(3)
- X'4'
- x(s>
- x<6>
- x<7>
- X'8'
- x<9>
- X'10'
- x(11)
- 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 value at a significance
level of 0.01 and n = 24 observations is 0.884. Since W = 0.805
is less than the critical value, conclude that the data 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 Rank Test
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, cissign the average
rank to each tied observation.
243
-------
(D
to
•a
M
o
rt
PROPORTION NORMALLY DEVELOPED
1
•8
n
rt
H-
B
o
Mi
g
H
•8
CD
o
b
s s
p
u
o
in
§
p
ta
I i-
8
M
§
-x-
-------
13.13.2.6.2 An example of assigning ranks to the combined data
for the control and .-0.13 /*g/L 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. ; '
TABLE 9. ASSIGNING RANKS TO THE CONTROL AND 0.13
CONCENTRATION LEVEL FOR STEEL'S MANY-ONE RANK TEST
Transformed ;
Proportion ,
Rank Normal Concentration
2 1
2 1
2 1
4.5 1
4.5 1
7 1
7 1
7 1
.369 0.13 ptg/L
.369 ! 0.13 /xg/L
.369 Control
.397
.397
.571
.571
.571
0.13 /Lig/L
Control
0.13 /ig/L
Control
Control
13.13.2.6.3 For this example, determine if the survival in any
of the concentrations is significantly1 lower than the survival in
the control. If this occurs, the rank sum at that concentration
would be significantly lower than the rank sum of the control.
Thus compare the rank sums for the survival at each of the
various concentration levels with some "minimum" or critical rank
sum, at or below which the survival would be considered
significantly lower than the control. At a significance level of
0.05, the minimum rank sum in a test with five concentrations
(excluding the control) and four replicates is 10 (See Table 5,
Appendix E). . i
13.13.2.6.4 Since the rank sums for the 0.50 /ug/L and 1.00 /ig/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
245 .
-------
TABLE 10. TABLE OF RANKS1
Replicate
Replicate
Copper Concentration (pig/L)
Control
0.13
0.25
1
2
3
4
1
1
1
1
.571(7,
.369(2,
.571(7,
.397(4.
7.5,7
4.5,5
7.5,7
5,6,6
.5,7.5,
,5,4}
.5,7.5,
,6,5)
7)
7)
1
1
1
1
.369
.397
.571
.369
(2)
(4.5)
(7)
(2)
1
1
1
1
.284(2)
.345(3)
.249(1)
.369(4.
5)
Copper Concentration (/xg/L) (Continued)
0.50
1.00
2.00
1
2
3
4
1.266(2)
1.303(3.5)
1.217(1)
1.303(3.5)
1.217(3.5)
1.146(2)
1.217(3.5)
1.133(1)
1.571(7)
0.959(2)
1.047(3)
0.685(1)
1Control ranks are given in the order of the concentration with which
they were ranked.
TABLE 11.
RANK SUMS
Concentrat ion
fig/L> Copper)
Rank Sum
0.13
• 0.25
0.50
15.5
10.5
10.0
other concentration has a significantly lower proportion normal
than the control. Because the 0.50 /*g/L concentration shows
significantly lower normal development than the control while the
246
-------
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 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 />tg/L and 0.50 fig/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 not follow a linear trends). ; Therefore, it is
not necessary to smooth the means prior to calculating the 1C.
The observed means, represented by Y.J.. become the corresponding
smoothed means, M^ 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
Copper Means, Yj. Means, M±
Cone. (//g/L) i (proportion) (proportion)
Control
0
0
0
1
2
.13
.25
.50
.00
.00 •
1
2
3
4
5
6
0
0
0
0
0
0
.983
.973
.932
.913
.852
.705
i 0
0
1 0
' 0
i 0
0
.983
.973
.932
.913
.852
.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
-------
*
B
1
9
CO
o
5
rH
5
•H
43
2
4J
Vt
O
Tf
a
4J
O
i
W
•d CM
S H
"l
co (d
(0 'I1
0)
O >
rH (1)
PM -d
S5t_
- 8
ro
a)
Q3d013A3a
•H
fa
248
-------
in a mean proportion of 0.737, where Mid-p/100),. = 0.983(1-
25/100). Examining the means and their associated concentrations
(Table 12), the response, 0.737, is bracketed by C4 = 1.00 jug/L
copper and C5 = 2.00 ptg/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:
(C -C )
ICp = Ct^'
IC25 = 1.00 + [0.983(1 - 25/100) - 0.852] (,2.00 - 1.00)
(0.705 - 0.852)
= 1.78
13.13.2.7.4 When the ICPIN program was used to analyze this set
of data, requesting 80 resamples,. the estimate of the IC25 was
1.7839 /xg/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 Tables 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 pulp mill
effluent. Single -laboratory precision data for the Crassostrea
gigas with the reference toxicant cadmium and lyophilized 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 mill
effluent .
13.14.1.2 Mult i- laboratory Precision ;
249
-------
Cone . ID
Cone. Tested
Response
Response
Response
Response
1
2
3
4
1
0
1.00
0.96
1.00
0.97
0
0
0
1
0
2
.13
.96
.97
.00
.96
0
0
0
0
0
3
.25
.92
.95
.90
.96
0
0
0
0
0
4
.50
.91
.93
.88
.93
1
0
0
0
0
5
.00
.88
.83
.88
.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.
ID
1
2
3
4
5
6
Number
Replicates
4
4
4
4
4
4
Concentration
ug/L
0
0
0
0
1
2
.000
.130
.250
.500
.000
.000
Response
Means
0.
0.
0.
0.
0.
0.
983
973
932
913
852
705 '
Std. Pooled
Dev. Response Means
0
0
0
0
0
0
.021
.019
.028
.024
.032
.247
0.
0.
0.
0.
0.
0.
983
973
932
913
852
705
The Linear Interpolation Estimate:
1.7839 Entered P Value: 25
Number of Resamplings: SOThose resamples not used had estimates
above the highest concentration/ %Effluent.
The Bootstrap Estimates Mean: 1.6188 Standard Deviation:
0.1758
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 provided 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-laboratory precision
data for Crassostrea gigas with the reference toxicant, cadmiumv
and lyophilized pulp mill effluent are provided in Tables 14-15.
The coefficient of variation is 21.3% for cadmium 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 THE MUSSEL,
MYTILUS SPP., DEVELOPMENT TEST PERFORMED WITH CADMIUM
CHLORIDE (CD MG/L) AS A REFERENCE TOXICANT
Month
Oct-92
Nov-92
Dec-92
Jan- 93
Feb-93
Mar- 93
Lab A
2.35
0.86
1.79
3.69
2.81
3.71
Lab B
1.06
3.49
2.51
2.25
2 . 91
2.64
Lab C
2.42
3.89
no data
6.77
5.85
2.62
Lab D
4.20
2.21
2.27
no data
3.75
4.89
.Lab E
4.77
2.39
3.73
1.57
3.05
no data
Mean
SD
CV (%)
2.54
1.11
43.9
2.48
0.81
32.8
4.31
1.94
45.0
3.46 ;
1.19
34.3
3.10
1.23
40.0
# of Labs
5
Statistic
Mean (N=5)
SD
CV(%)
EC25
3.18
0.75
23.6
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.
251
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TABLE 13. SINGLE AND MULTI-LABORATORY PRECISION OF THE MUSSEL,
MYTILUS SPP., DEVELOPMENT TEST PERFORMED WITH
LYOPHILIZED PULP MILL EFFLUENT (%) AS THE TOXICANT
Month
Oct-92
Nov-92
Dec-92
Jan- 93
Feb-93
Mar- 9 3
Lab A
1.78
1.57
1.74
3.17
1.66
1.85
Lab B
1.40
1.94
1.88
2.03
no data
1.66
Lab C
2.02
2.70
3.08
2.46
no data
1.72
Lab D
1.83
1.98
no data
1.07
no data
1.82
Lab E
1.85
no data
1.87
no data
no data
•no data
Mean
SD
CV (%)
1.96
0.60
30.6
1.78
0.25
14.2
2.40
0.54
22.5
1.68
0.41
24.5
1.86
0.28
1.4
# of Labs
5
Statistic
Mean (n=5)
SD
CV(%)
EC25
1.93
0.28
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
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TABLE 14. SINGLE AND MULTI-LABORATORY PRECISION -OF'THE OYSTER,
CRASSOSTREA GIGAS, DEVELOPMENT TEST PERFORMED WITH
CADMIUM CHLORIDE (CD MG/L) AS A REFERENCE TOXICANT ,
Month .
July- 92
Aug-92
Sept-92
Apr- 93
May- 9 3
June - 9 3 .
July- 9 3
Lab A
1.04
0.31
0.68
no data
0.46
0.26
0.28
Lab B
'l.54
1.38
0.20
0.45
0.30
1.55
0.82
Lab C
0.50
0.30
0.49
0.51
1.05
0.93
0.66
Lab D
0.41
0.35
no data
no data
0.52
no data
1.56
Lab E
0.56
no data
no data
0.95
0.83
0.83
•0 . 90
Mean . .
SD
CV (%)
0.51
0.31
60.6
0.89
0.59
66.7
0.63
0.27
42.1 .
0.71
0.57
80.4
0.81
0.15
18.5
# of Labs
5
Statistic
Mean (n=5)
SD
CV(%.) .
EC25
0.71
0.15
21.3'
These data are from: Pastorok, et al. (1994), West Coas't Marine
Species Chronic Protocol Variability Study, PTI Environmental,
Services, Prepared for Washington Department of Ecology,
February, 1994.
253
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TABLE 15. SINGLE AND MULTI-LABORATORY PRECISION OF THE OYSTER,
CRASSOSTREA GIGAS, DEVELOPMENT TEST PERFORMED WITH
LYOPHILIZED PULP MILL EFFLUENT (%) AS THE TOXICANT
Month
July- 9 2
Aug-92
Sept -92
Apr- 9 3
May- 9 3
June -93
July- 9 3
Lab A
no data
1.21
0.76
0.80
1.21
1.09
0.82
Lab B
0.91
1.09
1.66
1.10
0.65
1.32
0.80
Lab C
1.28
0.98
0.83
1.61
1.90
1.72
1.56
Lab D
no data
0.61
no data
1.66
0.93
0.83
1.67
Lab E
1.43
no data
no data
no data
0.93
0.98
1.04
Mean
SD.
CV (%)
0.98
0.21
21.6
1.08
6.34
31.4
1.41
0.40
28.0
1.14
0.49
43.3
1,10
0.23
20.8
# of Labs
5
Statistic
Mean (n=5)
SD
CV(%)
EC25
1.14
0.16
14.2
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.
254
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APPENDIX I. BIVALVE 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 30 ± 2&>. Include brine controls in tests that
use brine. ;
C. Prepare a copper reference toxicant stock solution.
i
D. Prepare a series copper reference toxicant concentrations.
E. Sample effluent and reference toxicant solutions for
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 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 concentration.
PREPARATION AND ANALYSIS OF TEST ORGANISMS
A. Determine test concentrations and appropriate dilution water
based on NPDES permit requirements and guidance from the
appropriate regulatory agency.
255 i
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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 1500-3000/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
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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 required for complete development.
H. Terminate the test by addition of formalin.
I. Count larvae and record the number of normal 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
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Sample data sheet for embryo microscopic examination.
BIVALVE DEVELOPMENT TEST: RESULTS
Bioassay No.
Date
Counter
Number
Sample
Abnormal
Normal
%Normal
Notes
258
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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
i
(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 Scope and Application
14.2 Summary of Method
14.3 Interferences
14.4 Safety , :
14.5 Apparatus and Equipment
14.6 Reagents and Supplies
14.7 Effluents and Receiving Water Collection,
Preservation, and Storage
14.8 Calibration and Standardization
14.9 Quality Control , }
14.10 Test Procedures
14.11 'Summary of Test Conditions and Test
Acceptability Criteria
14.12 Acceptability of Test Results
14.13 Data Analysis ;
14.14 Precision and Accuracy .
Appendix I Step-by Step Summary
259
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SECTION 14
RED ABALONE, HALIOTUS RUFESCENS
LARVAL DEVELOPMENT TEST METHOD
14.1 SCOPE AND APPLICATION
14.1.1 This method estimates the chronic toxicity of effluents
and receiving waters to the larvae of red abalone, Hal lotis
rufescens during a 48-h static 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
chemical 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
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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 may adversely
affect test results (see Section 8, Effluent and Receiving Water
Sampling, and Sample Handling, and Sample Preparation 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 and 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 keeping dilution water
supply, gametes, and embryo stock suspensions at test 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 -- for determining salinity.
14.5.6 Hydrometer(s) -- for calibrating refractometer.
14.5.7 Thermometers, glass or electronic, laboratory grade --
for measuring water temperatures.
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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 -- for determining
DO (optional) and calibrating the DO meter.
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 -- 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).
14.5.16 Volumetric flasks -- Class A, borosilicate glass or non-
toxic 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-liter cubitainers or polycarbonate water cooler jugs
-- for making hypersaline brine.
262
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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 labeled 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 inspecting
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>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. :
,i
14.5.30 Sieve or screened tube, approximately 37 /zm-mesh -- for
retaining larvae at the end of the test. j
14.5.31 60 /zm NITEX® filter -- for filtering receiving water.
14.6 REAGENTS AND SUPPLIES -. - !
I
I
14.6.1 Sample containers -- for sample shipment and storage (see
Section 8, Effluent and Receiving Water Sampling, and Sample
Handling, and Sample Preparation for Toxicity Tests).
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. .
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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 -- for personal protection from
contamination.
14.6.7 Pipets, serological -- 1-10 mL, graduated.
14.6.8 Pipet tips -- for automatic pipets.
14.6.9 Coverslips -- 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 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 mm thick).
14.6.17 Formaldehyde, 37% (Concentrated Formalin) -- for
preserving larvae. Note: formaldehyde has been identified as a
264
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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 (hydroxymethyl) aminomethane and hydrogen peroxide
(for H2O2 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 and Section 14.6.24, Hypersaline Brines. The
dilution water should be uncontaminated l-/im-filtered natural
seawater. Hypersaline brine should be prepared from dilution
water. ;
i • -
14.6.23 HYPERSALINE BRINES ' \
14.6.23.1 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.
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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 (lOOli) is used as a
diluent, the maximum concentration, of effluent (0&>) 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 /zm 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 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
lOOio. It is advisable not to exceed about 7Oli 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 jtxm 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
266
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4°C (even room temperature has been acceptable). HSB is usually
of acceptable quality even after several months in storage.
14.6.23.5 Heat Preparation of Brine
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
Salinity
%>
0
1
2
3
4
5
10
15
20
25
Brine
60
&
43.33
44.07
44.83
45.61
.46.43
47.27
52.00
57.78
65.00
74.29
Brine
70
&
51.43
52.17
52.94
53.73
54.55
55.38
60.00
65.45
72.00
80.00
Brine
80
Si
57.50
58.23
58.97
59.74
60.53
61.33
65.71
70.77
76.67
83 .64
Brine
90
fc
62.22
62.92
63.64
. 64.37
65.12
65.88
70.00
74.67
80.00
86.15
Brine
100
fc
66.00
66.67
67.35
68.04
68.75
69'.47
73.33
77.65
82.50
88.00
14.6.23.5.1 The ideal container for making brine using heat-
assisted 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
267
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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 /zm 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 lOOfe 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 /urn 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 Artificial Sea Salts
14.6.23.6.1 No data from red. abalone tests using sea salts or
artificial seawater (e.g., GP2) are available for evaluation at
this time, and their use must be considered provisional.
14.6.23.7 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 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
268
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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.,
14.6.'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 brine is lOOib and the test
is to be conducted at 34tr0, lOOti divided by 34tb ==2.94. The
proportion of brine is 1 part plus 1.94 parts reagent water. To
make 1 L of dilution water at 34lb salinity from a HSB of lOOib,
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 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 68to/ 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, 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).
14.6.23.8.3 To calculate the amount of brine to add to each
effluent dilution, determine the following quantities: salinity
of the brine (SB, in tb) , the salinity of the effluent OSE, in
to), 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
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VB « VE X (34 - SE)/(SB - 34)
14.6.23.8.4 This calculation assumes that dilution water
salinity is 34 ± 2fe.
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 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-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
2fe and a brine salinity of 66lo, 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 (SB - 34)/(34 - SE)
If effluent salinity is essentially•0&, 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.
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14.6.24 TEST ORGANISMS
14.6.24.1 The test organisms used for this test are red abalone,
Haliotis rufescens. This large gastropod mollusc is harvested
commercially in southern California and supports a popular
recreational fishery throughout the state. It, consumes a 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
equivalent.ejecting large numbers of gametes into the water
column, where fertilization takes place externally. Free-
swimming 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 stored in the yolk sack, supplemented perhaps by the
uptake of dissolved amino acids. Once larvae come into contact
with suitable substrate, they metamorphose and begin 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 life history
and culture to Martin et al. (1977), Morse et al (1979) and Hunt
and Anderson (1989 and 1993) for previous toxicity 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 smooth,
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).
i
14 . 6 . 24 . 3 Obtaining Broodstock j
14.6.24.3.1 Mature 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
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TABLE 2. EXAMPLES OF EFFLUENT DILUTION SHOWING VOLUMES OF
EFFLUENT (3*), 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-xfe and, unless natural seawater is used for dilution water, also
a brine-based dilution water of 34fc.
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.
40
Effluent
x&
800 mL
Brine
800 mL
Dilution Water*
34&
400 mL
Step 2 , Use either serially prepared dilutions of the hiahest test
concentration or individual dilutions of 100% effluent.
Effluent Cone. (%)
20
10
5
2.5
Control
Effluent Source
1000 mL of 40%
1000 mL of 20%
1000 mL of 10%
1000 mL of 5%
none
Dilution Water*
(34&)
1000 mL
1000 mL
1000 mL
1000 mL
1000 mL
INDIVIDUAL PREPARATION
Effluent Cone.
(%)
40
20
10
5
2.5
Control
Effluent x&
400 mL
200 mL
100 mL
50 mL
25 mL
none
Brine (68-x)&>
400 mL
200 mL
100 mL
50 -mL
25 mL
none
Dilution Water*
34&
200 mL
600 mL
800 mL
900 mL
950 mL
1000 mL
*May be natural seawater or brine-reagent water.
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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 the wild can be induced to
spawn, those grown or conditioned in the laboratory have been
more dependable. Commercial mariculture facilities in California
produce large.numbers of abalone, and distribution systems exist
to supply live spawners to a number of market areas. In any
case, broodstock should be obtained from sources free of
contamination by toxic substances to avoid genetic 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 clean covered
plastic buckets filled with seawater. Use compressed air, or
battery powered pumps to supply aeration. Compressed oxygen is
not recommended because bubbled oxygen may induce unintended
spawning (Morse et al., 1977). Maintain water temperatures
within 3°C of the temperature at the collecting site. Four
abalone in a 15-liter bucket should remain healthy for up to four
hours, under these conditions.
14.6.24.3.4 Abalone can be transported for up to 30 hours in
sealed, oxygen-filled plastic bags containing moist (seawater)
polyfoam sponges (Hahn, 1989). Cut the polyfoam into sections
(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. ' It is important
that there 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 ice 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 above 30°C.
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14.6.24.4 Broodstock Culture and Handling
14.6.24.4.1 At the testing facility, place 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 than 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, diill-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.
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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 trying 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. When the
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 (recrudescent) spawners have a
distinct color difference between the-'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).- Though 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, depending on test 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 of suitable materials to be used in laboratory culture
of abalone. 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.
14.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION, AND
STORAGE
14.7.1 See Section 8, Effluent and Receiving Water Sampling,
Sample Handling, and Sample Preparation for Toxicity Tests.
14.8 CALIBRATION AND STANDARDIZATION
14.8.1 See Section 4, Quality Assurance. ';
14.9 QUALITY CONTROL
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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 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 /mi 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 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 (IWC). At least two of the effluent
treatments must be of lesser effluent concentration than the IWC,
276
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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%b salinity.
14.10.2.2.2 If the effluent is known or suspected to be highly
toxic, a lower range of effluent concentrations ishould 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 '200 mL. .
14.10.2.2.4 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.
14.10.2.3 Dilution Water
14.10.2.3.1 Dilution water should be uncontamiriated l-//m-
filtered 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 water1-is 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 for red abalone is
zinc sulfate (ZnSO4oH2O). 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°H2O) to one
liter of reagent water in a polyethylene volumetric flask.
Alternatively, certified standard solutions can be ordered from .
commercial companies. •• -..,-
14.10.2.4.3 Reference toxicant solutions should be five
replicates each of 0 (control), 10, 18, 32, and 56, and 100 /xg/L
total zinc. Prepare one liter of each concentration by adding 0,
277
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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 ± 2tb.
14.10.3 COLLECTION OF GAMETES FOR THE TEST
14.10.3,.! 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 one-
cell 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 stimulating
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 to the laboratory by a
supplier, it is important to allow two days or more for
laboratory acclimation before spawning induction; this should
278
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increase the probability of achieving a successful spawn of
viable gametes. Always bring brood stock up to acclimation
temperature slowly to avoid premature spawning.;
14.10.3.2 Hydrogen Peroxide Method i-
!
14.10.3.2.1 Select four ripe male abalone and Ifour 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-/mi-filtered
seawater (15°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 without food to eliminate
wastes. Three hours prior to the desired spawning time, drain
the buckets, wipe and rinse out mucus and debris, and refill with
6 liters of 1 /zm-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 aerated until
spawning begins.
14.10.3.2.1 Dissolve 12.1 g of Tris into 50 ml/ of reagent water.
When the Tris has dissolved completely, mix the hydrogen peroxide
(H2O2) solution in a separate flask by pouring 10 mL of fresh*
refrigerated H2O2 (30%) into 40 mL of refrigerated reagent water
(1:5 dilution). Pour 25 mL of Tris solution and 25 mL of H2O2
solution into each of the spawning buckets (male and female).
Stir well to mix; the final concentration in the spawning buckets
will be approximately 6 mM Tris (pH = 9.1) and 5 m H2O2. 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 H2O2 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 male spawning 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 H202 in small containers (100
279 ;
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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 ' (1 liter/minute) 20-/mi-
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 ±
16C to counter the temperature increase caused by the slow
280
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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), abalone should begin
spawning by ejecting 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 respiratory 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 continue spawning over a longer period.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 risking 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 cases this
will provide time for more than one of each sex to spawn. More
important than multiple spawning, however, is avoiding delay of
fertilization. Eggs should be fertilized within one hour of
release (Uki and Kikuchi 1974). All sperm should be pooled, and
all eggs should be pooled prior to fertilization. This can be
accomplished by gentle swirling within the spawning buckets.
Note: Take care to avoid contaminating eggs with sperm prior to
the intended fertilization time. It is important that
development is synchronous among all test embryos.
14.10.3.5 Fertilization . - ,
14.10.3.5.1 As the females spawn, allow the eggs to settle to
the bottom. If necessary, gently stir to evenly distribute 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 passed since the first
individual of the second sex has spanned (2) multiple individuals
of each sex have spawned, or 3) there are too many eggs on the
" , *
281 i
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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 ± l°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 10s 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. Using 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 mix 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.
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14.10.3.5.4 Note: Once fertilized eggs have settled to the
bottom of the bucket (15 minutes after addition of sperm), the
following steps {rinsing, concentrating, and counting the
embryos) must proceed 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 take's place in the toxicant
solution).
14.10.3.5.5 After embryos have settled, carefully pour or siphon
off the water from above the settled 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 ± 1°C) . 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 sample 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 that the embryos appear
fuzzy (»100 sperm/egg), the abalone may develop abnormally and
should not be used.
14.10.4 START OF THE TEST '
\
14.10.4.1 Prior to Beginning the Test
14.10.4.1.1 The test should begin as soon as possible,
preferably within 24 h of sample collection. Thei 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 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
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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 ±.1°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 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
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 stirring. 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
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embryos after counting. Density of embryos in the beaker should
be between 200 and 300 embryos/mL. Dilute if the concentration
is too high, let embryos settle and pour "off excess water if
concentration is too low. Take the mean of 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 before'taking aliquots. The
temperature of the embryo suspension must 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, j
14.10.4.4 Incubation !
14.10,.4.4.1 Incubate test organisms for 48 hours in the test
chambers at 15 ± 1°C under low lighting (approximately 10
/iE/m2/s) with 16L:8D photoperiod. Fertilized embryos become
trochophore larvae, hatch, and develop into veliger larvae in the
test solutions during the exposure period..
14.10.5 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE
14.10.5.1 The light quality and intensity should be at ambient
laboratory conditions. Light intensity should be 10-20 /xE/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 s'hould be
.maintained at 15 ± 1°C. If a water bath is used to maintain the
285 !
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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 + 2I».
The salinity should vary by no more than ±2&> among the chambers
on a given day. If effluent and receiving water tests are
conducted concurrently, the salinities of these tests should be
similar.
14.10.5.4 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
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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. i"
14.10.7.1.3 Record all the measurements on the;data sheet.
14.10.8 TERMINATION OF THE TEST i
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. j
14.10.8.2 Sample Preservation i
14.10.8.2.1 After 48 hours 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. i
• i
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 /im-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
and less accurate. Add enough buffered formalin to preserve
larvae in a 5% solution (some laboratories have successfully
preserved larvae with lower formalin concentrations. Under-
preserved larvae disintegrate quickly, however, and whole tests
may have to be rejected if larvae have not been adequately
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 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.
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 10OX 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 across 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 typeis 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 with broken shells,
(4) larval shells separated from the rest of the animal, and (5)
larvae found remaining in the egg membrane (however, 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 ACCEPTABILITY CRITERIA
14.14.1 A summary of test conditions and test acceptability
criteria is listed in Table 3. ;
14.12 ACCEPTABILITY OF TEST RESULTS
14.12.1 Test results are acceptable only if all the following
requirements are met: I
-
(1) the mean larval normality must be at least 80% in the
controls.
(2) the response from 56 /Kj/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 reference toxicant.
14.13 DATA ANALYSIS j •
14.13.1 GENERAL
14.13.1.1 Tabulate and summarize the data. Calculate the
proportion of larvae with normally developed shells for each
replicate. A sample set of test data is listed in Table 4.
14.13.1.2 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. ;
289
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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 (i.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.
B. Obviously abnormal larva with transparent
shell and numerous shell deformities.
C. Normal larva with some shell thinning
and mild flattening of shell curvature near
the leading edge (left side of photograph).
>-, "^itis,""^;-
D. Abnormal larva with multiple slight
indentations and transparency near the
leading edge (left side of photograph)
291
-------
it
E. Normal larva with well calcified
(striated) shell but uneven shell outline.
G. Normal larva, anterior (rather than
lateral) view. Well striated, smooth
rounded shell outline.
F. Abnormal larva with transparent shell and
large indentation.
H. Abnormal larva, anterior (rather than
lateral) view. Transparent irregular shell with
indentations.
292
-------
J. Abnormal larva with shell
transparencies, indentations, and
irregular shape.
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.
L. Abnormal Istrva with arrested development
at an early stage. Any larva found within the
egg membrane, no matter how ell developed, is
counted as abnormal.
•293
-------
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY
CRITERIA FOR, HALIOTIS RUFESCENS, LARVAL DEVELOPMENT
TEST WITH EFFLUENTS AND RECEIVING WATERS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Test type :
Salinity:
Temperature :
Light quality:
Light intensity:
Photoperiod :
Test chamber size:
Test solution volume:
Larvae density per
chamber :
No. replicate chambers
per concentration:
Dilution water:
Test concentrations:
Dilution factor:
Test duration:
Endpoint :
Static non- renewal
34 ±21-0 . .
15 ± 1°C
Ambient laboratory light
10 piE/mVs
(Ambient laboratory levels)
16 h light, 8 h darkness
600 mL
200 mL/re'plicate
5-10 per 'mL
5
Uncontami-nated l-/xm-f iltered
natural seawater or hyper saline
brine plus reagent water
Effluents: Minimum of 5 and a
control
Receiving waters: 100%
receiving water and a control
Effluents: s:0.5
Receiving waters: None or kO.5
48 h
Normal shell development
294
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16. Test acceptability
criteria:
17. Sampling requirements:
18. Sample volume required:
5:80% normal shell 'development in
the controls; must have
statistical significant effect
at 56 /ig/L zinc; must achieve a,
%MSD of <20% |
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 Toxicity
Tests)
2 L per test
14.13.1.3 The endpoints of toxicity tests using the red abalone
are based on the reduction in proportion of normal 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 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.
14.13.2' EXAMPLE OF ANALYSIS OF RED ABALONE, HALIOTUS 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 shells 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 'excluded from
statistical,analysis of the NOEC and LOEC, but included in the
estimation of the IC25.
295
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TABLE 4. DATA FROM RED ABALONE, HALIOTUS RUFESCENS,
DEVELOPMENT TEST
Effluent
Concentration
(%) Replicate
Brine A
Control B
C
D
E
Dilution A
Control B
C
D
0.56 A
B
C
D
E
1.00 A
B
C
D
E
1.80 A
B
C
D
E
3.20 A
B
C
D
E
5.60 A
B
C
D
E
10.00 A
B
C
D
E
No . Larvae
Counted
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Number
Normal
100
98
100
99
99
99
99
99
100
99
99
98
100
100
99
100
99
99
100
99
99
99
98
97
39
57
61
65
80
0
0
0
0
0
0
0
0
0
0
Proportion
Normal
1.00
0.98
1.00
0.99
0.99
0.99
0.99
0.99
1.00
0.99
0.99
0.98
1.00
1.00
0.99
1.00
0.99
0.99
1.00
0.99
0.99
0.99
0.98
0.97
0.39
0.57
0.61
0.65
0.80
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
296
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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 endpoihts is made via a parametric test, Dunnett's
Procedure, or a nonparametric test, Steel's Many-one Rank Test,
on the arc sine square root transformed data. Underlying
assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality is the
Shapiro-Wilk1s Test, and Bartlett's Test is used, to test for
homogeneity of variance. If either of these tests fails, the
nonparametric test, Steel's Many-one Rank Test, is used to
determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the
parametric procedure.
14.13.2.3 If unequal numbers of replicates occur among the'
concentration levels tested, there are parametric and
nonparametric alternative analyses. The parametric analysis Is a
t test with the Bonferroni adjustment (see Appendix D). The
Wilcoxon Rank Sum Test with the Bonferroni adjustment is the
nonparametric alternative.
14.13.2.4 Comparison of Brine and Dilution Controls
14.13.2.4.1 This example uses toxicity data from a red abalone,
Haliotus rufescens, larval development test performed with
effluent. The response of interest is the proportion of larvae
with normally developed shells, -thus each replicate 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 normal shell
development proportions. The raw and transformed data, means and
variances of the transformed observations for the two controls
are listed in Table 5.
i
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 normally distributed
population. Thus in comparing the brine and dilution controls,
the data for each concentration must be separately checked for
normality. When the two response groups are tested separately,
it is not necessary to center the data. \
I
i
297
-------
, ;-;;V'p'|L*|^f,|-,,p /ilf*!!!.:
;-J'-' ';• ' .-'^it,,fMfkXf^"^ffL,-\""^fl "
', ••'-;• ':^^l^i^^^--':^/^^m
: - - 1^il»ft'^^s'-1^;^J^|=
_t__j " '. • ' 3s ' S« -!« -« .,
a^ni1"
k^
PROPORTION OF NORMAL LftRVAE
^T^
^ •KK'^i
, *,".,
m
,,:-:;,-,-:
POINT ESTIMATION
ENDPOINT ESTIMATE
IC25
HOMOGENEOUS VARIANCE
NO
HYPOTHESIS"TfeNG
NORMAL DlSTRIBUTfON
EQUAL NUMBER OF
REPLICATES?
YES
tTESTWrTH
BONFERRONI
ADJUSTMENT
EQUAL NUMBER OF
DUNNETTS
TEST
YES
STEEL'S MANY-ONE
RANKTEST
ENDPOINT ESTIMATES
NOEC,LOEC
Figure 2. Flowchart for statistical analysis of red abalone,
Ha.ld.otus rufescens, development data.
298
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TABLE 5
RED ABALONE, HALIOTUS RUFESCENS, LARVAL
DEVELOPMENT DATA FROM BRINE AND DILUTION
CONTROLS
Replicate
RAW A
B
C .
D
E
ARC SINE A
SQUARE ROOT B
TRANSFORMED C
D
E
Mean (y"±)
s?
i
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
1.521
; 1.484 .
0.000625
2
14.13.2.4.2.2 Calculate the denominator, D, of the statistic
for each control group : • ' ' :
i-l
Where: Xt = the ith centered observation
X = the overall mean of the centered observations
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) = 1.484
4
D = 0.00191
14.13.2.4.2.4 Order the observations for each control group from
smallest to largest
x
-------
14.13.2.4.2.5 From Table 4, Appendix B, for the number of
observations; n, obtain the coefficients a.lt a2, . * . ak where k is
n/2 if n is even and (n-'l) /2 if n is odd. For the datasets in
this example/ n = 5 and k = 2 for the brine control group, and n
= 4 and k = 2 for the dilution control group. The a± values are
listed in Table 7.
i
TABLE 7. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S
EXAMPLE
Brine Control Group
1
2
0.6646
0.2413
0.092
0.050
X<5)
X<4)
- X'1'
- 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, for each group as
follows: ,
W = -[Ea.(X{n-ifl)-XU))J :
D i,i * .
The differences, x(n-i+1) - X(i), are listed'in Table 7. For the
data in the brine example:
W = 1 (0.07321)2 = 6.880 '' '
0.00609
For the data in the dilution example:
W = 1 (0.03436)2 = 0.618 ;
0.00191
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'normally 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
Trans formed
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
302
-------
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 the brine control
group is 25 and the sum of the ranks in the dilution control
group is 20.
14.13.2.4.3.3 For this situation, we wish to determine if the
proportions of normally developed larvae in the two control
groups are significantly different. To do this,,compare 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 replicates in the> other
is 11 (See Snedecor and Cochran, 1980). ; • .
i , '
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 that 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 control 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 replicate must first
be transformed by the arc sine square root transformation
procedure described in Appendix B. The raw and transformed data,
means and variances of the transformed observations at each
effluent concentration and 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 10,. 0%
concentrations, they are not included in the statistical 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:
D = £ (X.-~x)2
.i-i
Where: Xi = the ith centered observation
X~ = the overall mean of the centered observations
n = the total number of centered observations
14.13.2.6.3 For this set of data, n = 24
iT = 1 (-0.004) = 0.000
24
D = 0.1127
14.13.2.6.4 Order the centered observations from smallest to
largest
X(1) <. X(2) * ... z X(n)
where X(1> 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 ax, a2, ... ak where k is
n/2 if n is even and (n-l)/2 if n is odd. For the data in this
example, n = 24 and k = 12 . The a£ values are listed in
Table 12.
14.13.2.5.6 Compute the test statistic, W, as follows:
a = -
D
304
-------
soe
PROPORTION NORMAL .
000000
(D
cr M
fa o
M ft
o
0 O
n> HI
Hi (D
Su fo
M.
O *O
£3
C ft
H, H-
| S
(D O
£3 H,
CQ
(D
81
(D
O,
(D
-------
TABLE 9. RED ABALONE, HALIOTUS RUFESCENS, SHELL DEVELOPMENT
DATA
Replicate Control
RAW
ARC SINE
SQUARE ROOT
TRANSFORMED
Mean (YI)
si
i
A
B
C
D
E
A
8
C
D
E
0
0
0
1
1
1
1
1
1.
0.
1
.99
.99
.99
.00
.471
.471
.471
.521
484
000625
0
0
0
0
1
1
1
1
1
1
1
1.
0.
,2
Effluent Concentration (%)
.56
.99
.99
.98
.00
.00
.471
.471
.429
.521
.521
483
001523
1
0
1
0
0
1
1
1
1
1
1
1.
0.
3
.00
.99
.00
.99
.99
.00
.471
.521
.471
.471
.521
491
000750
1
0
0
0
0
0
1
1
1
1
1
1.
0.
4
.80
.99
.99
.99
.98
.97
.471
.471
.471
.429
.397
448
001137
3.20
0.39
0.57
0.61
0.65
0.80
0.674
0.856
0.896
0.938
1.107
0.894
5.6 10.0
0 0
0 0
0 0
0 0
0 0
-
-
-
-
-
-
0.024288 -
5
6 7
TABLE 10. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S
EXAMPLE
Effluent Concentration (%)
Replicate
A
B
C
D
E
Control
-0
-0
-0
0
.013
.013
.013
.037
0
-0
-0
-0
0
0
.56
.012
.012
.054
.038
.038
1
-0
0
-0
-0
0
.00
.020
.030
.020
.020
.030
1
0
0
0
-0
-0
.80
.023
.023
.023
.019
.051
3
-0
-0
0
0
0
.20
.220
.038
.002
.044
.213
306
-------
TABLE. 11. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-
WILK'S EXAMPLE
1
2
3
4
5
6
7
8
-0.220
-0.054
-0.051
-0.038
-0.020
-0.020
-0.020
'-0.019
13
14
15
16
17
18
19
20
-0.
0.
0.
0.
0.
0.
0.
0.
012
002
023
023
023
030 -
030
037
TABLE 12. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK'S
EXAMPLE
1
2
3
4
5
6
7
8
0.4493
0.3098
0.2554
0.2145
0.1807
0.1512
0.1245
0.0997
0.433
0.098
0.089
0.076
0.057
0.050
0.050
0.042
x<24'
X<23>
X(22)
1 x(211
• i X'20'
x(19)
XU8>
x<17)
- x<"
- x(2>
- x<3)
- X'4'
- x(5)
- X'6'
- x(7)
- 'X(8)
The differences, xln-i+1) - X(i> , are listed in Table 12. For the
data in this example :
W =
(0.2974)2 = 0.7848
0.1127
307
-------
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 significance 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 are not
normally distributed.
14.13.2.5.8 Since the data do not meet the assumption of
normality, the Wilcoxon Rank Sum Test with the Bonferroni
Adjustment will be used to analyze the shell development data.
14.13.2.6 Wilcoxon Rank Sum Test with the Bonferroni Adjustment ,
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 (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.
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 each 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 critical rank
sum, at or below' which the survival would be considered
significantly lower than the control. At a significance level of
0.05, the minimum rank sum in a test with four concentrations
(excluding the control), four control replicates and five
concentration replicates is 15 (See Table 5, Appendix F).
308
-------
TABLE 13. ASSIGNING RANKS TO THE CONTROL AND 0.56%
CONCENTRATION LEVEL FOR THE WILCOXQN RANK SUM
TEST WITH THE BONFERRONI ADJUSTMENT
Rank
i
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
Concentration
1 ,.0.56 %
0.56 %
0.56 %.J
! Control
Control
Control
0.56 %
0.56 .%
! Control
TABLE 14. TABLE OF RANKS1
Repli-
cate
1
2
3
4
5 '
1
1
1
1
Control
.471(4,3.
.471(4,3.
.471(4,3.
.521-(8,8,
5,5.5,
5,5.5,
5,5.5,
9,9)
7)
7)
7)
Effluent
0.56
1.471(4)
1.471(4)
1.429(1)
1.521(8)
1.521(8)
1
1
1
1
1
1.00
.471(3.
.521(8)
.471(3.
.471(3.
.521(8)
Concentration (%)
5)
5)
5)
1.. 80
1.471(5.5)
1.471(5.5)
1.471(5.5)
1.429(2)
1.397(1)
0
0
0
0
1
3.20
.674(1)
.856(2)
.896(3)
.938(4)
.107(5)
'•Control ranks are given in the order of the concentration with which
they were ranked.
309
-------
TABLE 15. RANK SUMS
Concentration
(% Effluent) Rank Sum
0.56
1.00
1.80
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 1C. In the following discussion, the observed
means are represented by Y ± and the smoothed means by MI .
14.13.2.7.2 Starting with the control mean, Yx = 0.993 and Y 2 =
0.992, we see that Y^ > Y~2. Set Mj = Y^. Comparing Y~2 to Y~3/Y~2< Y~3.
14.13.2.7.3 Calculate.the smoothed means:
M2 = M3 = (Y~2 + Y~3)/2 = 0.993
14.13.2.7.4 Since Y~7 = 0 < Y~6 = 0 < Y~5 = 0.604 < Y~4 = 0.984 < Y~3
» 0.993, set M3 = 0.993, M4 = 0.984, Ms = 0.604, M6 = 0, and set M7
- 0.
310
-------
8
1VW80N
^ VO
O H
»M
•d
-a a)
0)
CD O
M
•O «H
c
OS IK
4J
- (0
CD T3
C
flj 4J
0) C
CD O
4) >
CO Q>
JQ TJ
O
* 10
S
flj O
•o w
4)
^ «M
« P
M M
«4-4 CO
4J O
O -H
- ^
5
01
9) e
fe
311
-------
TABLE 16. RED ABALONE, HALIOTUS RUFESCENS, MEAN SHELL
DEVELOPMENT RESPONSE AFTER SMOOTHING
Effluent
Cone. (%)
Control
0.56
1.00
1.80
3.20
5.60
10.00
i
1
2
3
4
5
6
7
Response
Means, Y"A
(proportion)
0.993
0.992
0.994
0.984
0.604
0.000
0.000
Smoothed
Means , Mi
(proportion)
0.993
0.993
0.993
0.984
0.604
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 Mid-p/lOO) = 0.993(1-25/100).
Examining the means and their associated concentrations
(Table 16), the response, 0.745, is bracketed by C4 = 1.80%
effluent and C5 = 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:
ICp
IC25 = 1.8 -i- [0.993(1 - 25/100) - 0.984] (3.2 - 1.8)
(0.604 - 0.984)
= 2.68%.
14.13.2.7.8 When the ICPIN progreim 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 precision of the
Haliotis rufescens larval development method using zinc sulfate
are shown in Table 17. Zinc concentrations were 18, 32, 56, and
100 pg/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
Cone. Tested
Response
Response
Response
Response
Response
1
2
3
4
5
1
0
.99
.99
.99
1.00
2
.56
.99
.99
.98
1.00
1.00
3
1
.99
1.00
.99
.99
1.00
4
1.8
.99
.99
.99
.98
.97
5
3.2
,39
.57
61
.65
.80
6
5.6
0
0
0
0
0
7
10
0
0
0
0
0
Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Effluent
Test Start Date: Test Ending Dat2:
Test Species: Red Abalone
Test Duration: 48 hours
DATA FILE: abalone.icp
OUTPUT FILE: aoalone.125
Cone.
ID
1
2
3
4
5
6
7
Number Concentre . ion
Replicates
4
5
5
S
5
5
5
Response
% Means
0.
0.
1.
1.
3.
5.
10.
000
560
OOD
?TD
200
SOO
000
0
0
0
0
0
0
0
.993
.992
.994
.984
.604
.000
.000
Std.
Dev.
0
0
0
0
0
0
0
.005
.008
.005
.009
.148
.000
.000
Pooled
Response Means
0.
0.
0.
0.
0.
0.
0.
993
993
993
984
604
000
000
The Linear Interpolation Esti.tiate:
2.6818 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Expanded Confidence Limits:
Resampling time in Seconds:
2.7085 Standard Deviation: 0.1510
lower: 2.5000 Upper: 3.1262
Lower: 2.4091 Upper: 3.3484
0.27 Random Seed: -770872716
Figure 5. ICPU? program output for the IC25.
314
-------
TABLE 17. SINGLE LABORATORY PRECISION DATA FOR THE RED ABALONE,
HALIOTIS RUFESCENS LARVAL DEVELOPMENT TEST WITH ZINC
(ZN //G/L) SULFATE AS A REFERENCE TOXICANT
Test Date
March 1990
May 1990
January 1991
February 1991
Mean
SD
CV (•%)
NOEC (/xg/L)
32
32
18
18
EC50 (/.ig/L)
421 !
391
341
402
38.4
3.0
7.8
1 Source: Hunt et al., 1991
2 Source: Anderson et al., 1994
315
-------
TABLE 18. MULTI-LABORATORY PRECISION OF THE RED ABALONE, HALIOTIS
RUFESCENS LARVAL DEVELOPMENT TEST PERFORMED WITH ZINC
(ZN /zG/L) SULFATE AND EFFLUENT (%) AS THE TOXICANTS
Test Date
March 1990
March 1990
March 1990
March 1990
March 1990
March 1990
May 1990
May 1990
May 1990
May 1990
May 1990
May 1990
January
1991
January
1991
January
1991
January
1991
January
1991
January
1991
Toxicant
Effluent
Effluent
Effluent
Zinc
Zinc
Zinc
Effluent
Effluent
Effluent
Zinc
Zinc
Zinc
Effluent
Effluent
Zinc
Zinc
Effluent
Effluent
Lab
A
B
C
A
B
C
A
D
C
A
D
C
A
C
A
C
A
C
NOEC
>3.2%
>3 .2%
0.32%
32
18
18
3.2%
1.8%
3.2%
32
32
32
<0.56%
1.25%
18
32
1.0%
1.8%
EC50
nc
nc
1.83
41
28
31
4.7
3.5
3.8
39
46
37
1.5
1.8
34
48
2.7
2.8
CV
nc
20% '
16%
f
12%
13%
24%
3.0%
Mean Interlaboratory CV = 15% Interlaboratory CV based on 6 tests for which
CV values could be calculated. Source: Hunt et al., 1991.
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 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
/xg/L) ,by adding 0.0440 g of zinc sulfate (ZhSO4o7H2O) to 1
liter of reagent water.
D. Prepare zinc reference toxicant solution of 0 (control) 10,
18, 32, 56. and 100 /Kj/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 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 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, pH, 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 H2O2 and Tris or UV irradiated seawater (300 mL/min
flow rate through the UV unit) . All solutions should be .
maintained at 15 ± 1°C.
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 1°C 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 beaker and the solutions
are at 15 ± 1°C. 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 jiim-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 of Brine
VDW = VBL - VBS
VDW = Volume of Dilution Water (Adjusted to 34+2%)
VBL = Largest Volume of Brine added to adjust salinity
VBS = Volume of Brine added to each Sample
Total Volume = VB added + VDW added
(Total volume should be the same for all samples)
Site Code (ID OrgTT
or concentration
Initial
, Salinity
TS
34±2
34±2
• 34±2
34±2
34±2
34±2
34±2 ,
34±2 .
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
34±2
Vol. of
Brine
Vol.Dil.
Water
Total
Volume
Final
Salinity
Precision and
Accuracy for
Refractometer
Initials:
Double Checked:
319
-------
Data Sheet for Mollusc Larval Development Toxicity Test
Mollusc Species:
Collection/Arrival Date:
Broodstock Source:
Test Start Date: Start Time:
Test End Date: End Time:
Reference
Toxicant:Reference
Toxicant:
Sample Source:
Sample Type: Effluent RefTox Solid Elutriate Pore Water WaterSample Type: Effluent RefTox
Solid Elutriate Pore Water Water
Test
Cont.
H
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Station
Code or
Concentration
Sample
ID#
After 48 hours
formal
Larvae
Abnormal
Larvae
Notes
0
Computer Data StorageComputer Notes
Dala Storage
Disk:
File:
320 .
-------
SECTION 15
PURPLE URCHIN, Strongylocentrotus purpuratus
AND SAND DOLLAR, Dendraster 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
15,1 Scope and Application
15.2 Summary of Method
15.3 Interferences
15.4 Safety
15.5 Apparatus and Equipment
15.6 Reagents and Supplies
15.7 Effluents and Receiving Water Collection,
Preservation, and Storage
15.8 Calibration and Standardization !
15.9 Quality Control
15.10 Test Procedures ,
1,5.11 Summary of Test Conditions and Test
Acceptability Criteria
15.12 Acceptability of Test Results
15.13 Data Analysis
15.14 Precision and Accuracy
Appendix- I 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 toxicity 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 and
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 volatile and highly
degradable toxicants in the source may not be detected in the
test.
15.1.4 This test is commonly used in one of two forms: (1) a
definitive test, consisting of a minimum -of five effluent
concentrations and a control, and (2) a receiving water test(s),
consisting of one or more receiving water concentrations and a
control.
15.1.5 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
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performing a 72-h static non-renewal test using the early
development of the purple sea urchin, Strongylocentrotus
purpuratus, and the sand dollar, Dendraster excentricus, to
determine the toxicity of substances in marine and estuarine
waters. The test endpoint is normal larval development and may
include mortality if modified for total counts at test initiation
and termination. •
15.3 INTERFERENCES
15.3.1 Toxic substances may be introduced by contaminants in
dilution water, glassware, sample hardware, and testing equipment
(see Section 5, Facilities, Equipment, and Supplies). '
15.3.2 Improper effluent sampling and handling may adversely
affect test results (see Section 8, Effluent and Receiving Water
Sampling 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 test 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.
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15.5.4 Water purification system -- Millipore Super-Q, Deionized
water (DI) or equivalent.
15.5.5 Refractometer -- for determining'salinity.
15.5.6 Hydrometer(s) -- 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 non-
toxic 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
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15.5.19 Wash bottles -- for reagent water, for topping off
graduated cylinders, for rinsing small glassware and instrument
electrodes and probes.
/'" . , ' ;
15.5.20 Wash bottles -- for dilution water.
15.5.21 20-liter cubitainers or polycarbonate water cooler jugs
-- for making hypersaline brine.
15.5.22 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.
15.5.23 Beakers, 5-10 mL borosilicate glass -- for collecting
sperm from sand dollars.
15.5.24 Beakers, 250 mL borosilicate glass -- for preparation of
test solutions. • . • . :
15.5.25 Beakers, 100 mL borosilicate glass -- for spawning; to
support sea urchins and to collect sea urchin and sand dollar
eggs.
15.5.26 Beakers, 1,000 mL borosilicate glass -- 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 (10Ox), 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 of an
inverted scope is not required, but recommended. Its use reduces
the exposure of workers to hazardous fumes (formalin Or
glutaraldehyde) during the counting of larvae and reduces sample
examination time. Alternatively, a Sedgewick-Rafter cell may.be
used on a. regular compound scope.
15.5.29 Counter, two unit, 0-999 -- for recording sperm, egg,
embryo, and larval counts. '.
325 • ! '
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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 counting sperm.
15.5.33 Siphon hose (3 mm i.d.) -- for removing wash water from
settled eggs.
*
15.5.34 Perforated plunger -- for maintaining homogeneous
distribution of 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 (0.5mm mesh) -- cleaning egg solutions.
15.5.37 60 [j.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 _-- for labelling test chambers and
containers.
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.
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15.6.7 Pipets, serological -- 1-10 mL, graduated.
15.6.8 Pipet tips -- for automatic pipets. Note; 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".
'1
15.6.9 Coverslips -- for microscope slides. i
i
i
15.6.10 Lens paper -- for cleaning microscope optics.
15.6.11 Laboratory tissue wipes -- for cleaning and drying
electrodes, microscope slides, etc.
i
15.6.12 Disposable countertop covering -- for protection of work
surfaces and minimizing spills and contamination.j
15.6.13 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).
15.6.14 Membranes and filling solutions -- for dj
probe (see USEPA Method 360.-1, USEPA, 1979), or reagents for
modified Winkler analysis.
15.6.15 Laboratory quality assurance samples and
for the above methods.
ssolved oxygen
standards --
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 under,conditions of
maximal ventilation and minimal opportunity for volatilization
into room air. i
15.6.18 Glutaraldehyde, 1% in seawater -- for preserving larvae.
327- i
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Figure 1. Sample experiment set-up sheet.
SEA URCHIN EMBRYO DEVELOPMENT TEST
EXPERIMENT DESCRIPTION
Bioassay No.
Invest igator_
Start Date_
End Date
Start time_
End Time
Sample
Numbers
Sample
Concentration
Other Information
328
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Figure 2. Sample worksheet for urchin spawning information.
Bioassay no.
Spawning
SEA URCHIN DEVELOPMENT TEST
SPAWNING WORKSHEET
Date
No.
1
2
3
4
5
6
7
8
9
10
11
12
Injection
time
Sex
Accepted?
(Comments)
i
1 • •
Sperm density
#sperm counted=_
(mean)
x _ (5 x 10s).
mean=
sperm/mL
Egg dilution
# eggs counted=
mean=
(mean)
x 100=
eggs/mL in stock -i- 1,000=
in spock
i
Egg dilution factor
329
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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
s eggs in dilution
_eggs in dilution x 500 sperm/egg
sperm needed
_sperm needed -3- ^_sperm/mL in sperm
dilution= mL sperm dilution needed
Percent fertilized after 10 min
Time of inoculation
330
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Figure 4. Sample data sheet for embryo microscopic examination,
SEA URCHIN DEVELOPMENT TEST: RESULTS
Bioassay No.
Date
Counter
Number
Sample
-
Abnormal
Normal
%Normal
Notes
331
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15.6.19 Acetic acid, 10%, reagent grade,' in filtered
seawater -- for preparing killed sperm dilutions for sperm
counts.
15.6.20 Haemo-Sol or equivalent cleaner -- for cleaning
hemacytometer and cover slips.
15.6.21 0.5 M KC1 solution -- for inducing spawning.
15.6.22 Syringe, disposable, 3 or 5 mL -- for injecting KC1 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.
15.6.26 Microscope well-slides -- for pre-test assessment of
sperm activity and egg condition.
15.6.27 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).
15.6.29 Effluent and receiving water -- see Section 8, Effluent
and Surface Water Sampling, and Sample Handling, and Sample
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-/«n-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
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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.
15.6.31.2 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 (Oti) that can be
tested is 66% effluent at 34ii salinity (see Table 1).
15.6.31.3 High quality (and preferably high salinity) seawater
should be filtered to at least 10 ptm before placing into the
freezer or the brine generator. Water should be collected on an
incoming tide to minimize the possibility of contamination.
15.6.31.4 Freeze Preparation of Brine :
15.6.31.4.1 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).
333
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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
Salinity
So
0
1
2
3
4
5
10
15
20
25
Brine
60
So
43.33
44.07
44.83
45.61
46.43
47.27
52.00
57.78
65.00
74.29
Brine
70
So
51.43
52.17
52.94
53.73
54.55
55.38
60.00
65.45
72.00
80.00
Brine
80
So
57.50
58.23
58.97
59.74
60.53
61.33
65.71
70.77
76.67
83.64
Brine
90
So
62.22
62.92
63.64
64.37
65.12
65.88
70.00
74.67
, 80.00
86.15
Brine
100
Si
66.00
66.67
67.35
68.04
68.75
69.4.7
73.33
77.65
82.50
88.00
15.6.31.4.2 It is preferable to monitor the water until the
target salinity is achieved rather than allowing total freezing
followed by partial thawing'. Brine salinity should never exceed
1002o. It is advisable not to exceed about 70§b 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 jan 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
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15.6.31.5.1 The ideal container for making brine using heat-
assisted evaporation of natural seawater is one that (1) has a
high surface to volume ratio, (2) is made of a rion-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. :
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. . . . •
i
15.6.31.5.3 Seawater should be filtered to at least 10 /zm 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 lOOtb 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 /im 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). i HSB is usually
of acceptable quality even after several months in storage.
I
15.6.31.6 Artificial Sea Salts J
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 ;
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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 individually 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 lOOli and "the test is to be
conducted at 34&, 100& divided by 341s, = 2.94. The proportion
is 1 part brine 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.
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 34tb 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.
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
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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 tb) , 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: j
VB = VE x (34 - SE)/(SB - 34)
15.6.31.8.4 This calculation assumes that dilution water
salinity is 34 ± 2%b.
15.6.31.9 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 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
2tb and a brine salinity of 661-0, 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 (100-250
mL) beaker and stir. Distribute equal volumes into the replicate
test chambers. The remaining test, solution can be used for
chemistry.
337
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TABLE 2. EXAMPLES OF EFFLUENT DILUTION SHOWING VOLUMES OF
EFFLUENT (AT Xfe), 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-xfe 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.
(%)
40
Effluent x&>
800 mil
Brine (68-
x)&
800 mL
Dilution Water*
34&
400 mL .
Step 2. Make serial dilutions from the highest test concentration.
Effluent Cone. {%)
20
10
5
2.5
Control
Effluent Source
1000 mL of 40%
1000 mL of 20%
1000 mL of 10%
1000 mL of 5%
none
Dilution Water*
(34fe)
1000 mL
1000 mL
1000 mL
1000 mL
1000 mL
INDIVIDUAL PREPARATION:
Effluent Cone.
(%)
40
20
10
5
2.5
Control
Effluent xio
400 mL
200 mL
100 mL
50 mL
25 mL
none
Brine (68-x)&>
400 mL
200 mL .
100 mL
50 mL
25 mL
none
Dilution Water*
34&
200 mL
600 mL
800 mL
900 mL
950 mL
1000 mL
*May be natural seawater or brine-reagent water equivalent.
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15.6.31.10 Brine Controls
15.6.31.10.1 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 dilution 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. I
VE = VB x (SB - 34)/(34 - SE)
If effluent salinity is essentially Olb, 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.
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 matter of
confirming general body color, size, and spine appearance, those
unfamiliar with the species should seek confirmation from local
experts.
15.6.32.3 Obtaining Broodstock
15.6.32.3.1 Adult sea urchins (Strongylocentrotus purpuratus)
can be obtained from commercial.suppliers or collected .from
uncontaminated intertidal 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 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.
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15.6.32.4 Broodstock Culture and Handling
15.6.32.4.1 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.
15.6.32.4.2 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.
15.6.32.4.3 Food for sea urchins -- kelp, recommended, but not
necessarily limited to, Laminaria sp., Hedophyllum sp.,
Nereocystis sp., Macrocystis sp., Egregia 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.
15.6.32.4.4 Natural seawater (>30§-o) is used to maintain the
adult animals and (;>32!i) as a control water in the tests.
15.6.32.4.5 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.
15.6.32.4.6 To successfully maintain about 25 adult animals for
seven days at a field site, 40-L glass aquaria using aerated,
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recirculating, clean saline water (32&) and a gravel bed
filtration system, are housed within a water bath, such as an
INSTANT OCEANR Aquarium. The sexes should be held 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 subtidal
zones (most areas) or from inter-tidal 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 besst 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 seiawater, or
saltwater prepared from commercial sea salts is !recirculated.
The animals are checked daily and any obviously unhealthy animals
are discarded. For longer periods than a few daiys, several
centimeters or more of a sand substrate may be desirable.
15.6.33.4.2 Although ambient temperature seawater is usually
acceptable, maintaining sand dollars in spawning condition
usually requires holding at a relatively constant temperature.
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The culture unit should be capable of maintaining a constant
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 phy top lank ton, 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 (>30t<>) is used to maintain the
adult animals and (^32fe) 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 site, trays or 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.
15.6.33.4.6 To successfully maintain about 25 adult animals for
seven days at a field site, 40-L glass aquaria using aerated,
recirculating, clean saline water (>30ti) 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
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15.10.1 TEST DESIGN
I
15.10.1.1 The test consists of at least 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. . , . !
I
15.10.1.2 Effluent concentrations are expressed as percent
effluent. I
i
•
15.10.2 TEST SOLUTIONS I
15.10.2.1 Receiving waters '
l
15.10.2.1.1 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 /xm NITEX® filter and compared without dilution,
against a control. Using four replicate chambers per test, each
containing 10 mL, and 400 mL for chemical analysis, would require
approximately 440 mL of sample per test.
•
15.10.2.2 Effluents |
15.10.2.2.1 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 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 instreaxn
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.
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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-/im-
faltered 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 (CuCl2oH2O). 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 (CuCl2°2H2O) 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.
15.10.2.4.3 Prepare a control (0 A*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
ng/li, 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
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and filling to 100-mL with dilution water). Alternatively,
certified standard solutions can be ordered from'commercial
companies. Start with control solutions and progress to the
highest concentration to minimize contamination.
15.10.2.4.4 If the effluent and reference toxicjmt 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 ± 2ti.
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
number of sea urchins or sand dollars (based upoii 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. '
I
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 onto a clean
tray covered with several layers of seawater moistened paper
towels. !
I
15.10.3.1.4 Handle sexes separately once known; this minimizes
the chance of accidental egg fertilization. Throughout 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.
15.10.3.1.5 Fill a 3 or 5 mL syringe with 0.5 M KC1 and inject
345 '
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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 KCl.
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 will 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 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.
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 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.
15.10.3.1.11 Sections 15.10.4.2 and,, 15 .10 . 6 .4 describe
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.
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Figure 5. Showing the location and orientation used in the
injection of KC1 into sea urchins to stimulate spawning.
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
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 semen from each
male into separate 1-15 mL conical test tubes, stored in an ice
water bath. ,. • ! !
15.10.3.3 Viability of Sperm I
15.10.3.3.1 Early in the spawning process, place a very small
amount of sperm from each male sea urchin or sand dollar into
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dilution water on a microscope slide (well slides work -nicely).
Examine the sperm for motility; 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 2xl09 and 2xl010 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 mL of 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
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onto each 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 refill 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)(hemaevtometer conversion factor)(mm3/mL)
number of squares counted
dilution=100 j
conversion factor=4000 :
mm3/mL=1000
number of squares=80
Therefore:
#sperm/mL= (count) (5 x 106) ; (Equation 2A)
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 microscope. . Look for
the presence of significant quantities of immature 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 females whose eggs did
not fertilize during the test in Section-15.10.5.1.
15.10.5.2 Pooling of Eggs j
15.10.5.2.1 Allow eggs to settle in the collection beakers.
Decant some of the water from the collection beakers taking care
not to pour of£ many eggs. Pour the remaining sea urchin eggs
349 ' ~ '
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through the Nitex® screen (to remove fecal material and other
debris) into a 1 liter beaker. Repeat with each of the "good"
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 parafilm 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 (Equation 3)
15.10.5.4 Dilution of Eggs
15.10.5.4.1 When using scintillation vials as the test chamber,
the final concentration of eggs in the diluted stock must 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/mL in stock * 1,000= Dilution factor (Equation 4)
15.10.5.4.2 The dilution factor must be greater than one. If
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not, concentrate the eggs and recount (starting at Section
15.4.5.3 The dilution factor minus 1 equals the 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.
15.10.5.4.3 Make a dilution of the egg stock so that there is .
more than "enough volume to perform the bioassay.
15.10.5.5 Fertilization of Eggs i
15.10.5.5.1 'The recommended initial sperm to egg ratio for
fertilization of the eggs is 500:1. Calculate the volume of
sperm dilution (Section 15.10.5.1) 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
5A) . . ' ' !
total # of eggs in dilution x 500 sperm/egg= # of sperm needed
(Equation 5B) •• '•• • - . '
# of sperm needed * # sp'erm/mL in sperm dilutidn= mL of sperm solution
(Equation 5C) , '
• ' i
15.10.5.5.2 Add this volume of the sperm dilution to the egg
. dilution and mix gently with a plunger-. Wait 10 min, then check .
for fertilization. If fertilization is not at least 90%, add a
second volume of the sperm dilution. Wait 10 min and r'echeck.
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 .j
15.10.6.1 Prior to Beginning the Test .
15.10.6.1.1 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
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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
± 1°C) 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 + 1°C) .
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 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 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.
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15.10.6.2 Delivery of Fertilized Eggs
15.10.6.2.1 Gently mix the solution of fertilized 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 mm opening.
Deliver the embryos into the test chambers directly from the
pipette, taking care not to touch the pipette to the test
solution. The egg solution temperature'must be within 1°C of the
test solutions. Keep the eggs well mixed during 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 after test
initiation. If they are sampled and counted in a 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, PHOTQPERIOD, SALINITY AND TEMPERATURE
15.10.7.1 The light quality and intensity should be at ambient
laboratory conditions. Light intensity should be 10-20 /xE/m2/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 the test cups
should be as deep as possible without floating the chambers.
15.10.7.3 The test salinity should be in the range of 34 ± 2li.
The salinity should vary by no more than ±2ti 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
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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.
15.10.8 DISSOLVED OXYGEN (DO) CONCENTRATION
15.10.8.1 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 0). The DO should not fall below 4.0
tng/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.
15.10.9 OBSERVATIONS DURING THE TEST
15.10.9.1 Routine Chemical and Physical Observations
15.10.9.1.1 The DO should be measured in each test solution at
the beginning of the exposure period.
15.10.9.1.2 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 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.
15.10.9.1.3 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 circumstances. '
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15.10.10 TERMINATION OF THE TEST |
i
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) . i •. .
i
15.10.10.2 Sample Preservation
!
15.10.10.2.1 To terminate the test, add 1.0 mL 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 chamber.
Tightly cap and gently mix each chamber and store for later
evaluation. (If the test is performed in larger chambers, a 10
mL subsample of well mixed test solution is to be taken from each
chamber and preserved). :
• i
15.10.10.3 Counting ! .
15.10.10.3.1 It is recommended that the embryos be examined
within one week of preservation. Longer storag'e times may also
be used, but run the risk of sample degradation due to improper
preservation. Larvae can be counted directly in 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 lOOx 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
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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 length 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. ;
(3) Inhibited: larvae at the blastula origastrula stage
that have no gut differentiation or have no or
underdeveloped skeleton. These larvae appear to be
developing regularly, but are at a stage earlier than
attained by control organisms (e.g., Figure 6A-C).
!
(4) Gut abnormalities: larvae whose overall appearance is
normal, but have guts that are lacking,
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 exhibit a
separation of the rods at the apex. This may be caused
by preservation and should not" be termed abnormal.
Since the test is started with already fertilized eggs,
any unfertilized eggs that are encountered should not
be counted as either'normal or abnormal, but should be
ignored. ' • -
15.11 SUMMARY OF TEST CONDITIONS
15.11.1 A summary of test conditions and test.acceptability
criteria is listed in Table 3.
15.12 ACCEPTABILITY OF TEST RESULTS
15.12.1 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 I
-------
o/.arm std
Figure 6. Stages of sea urchin embryo development
(modified from Kume and Dan 1957). A. blastula; B,
gastrula; C. prism; D. pluteus (frontal view); E.
pluteus (lateral view). al.am?: 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 !
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13 .
14.
15.
Test type :
Salinity:
Temperature :
Light quality:
Light intensity:
Photoperiod:
Test chamber size:
Test solution volume:
No. replicate chambers
per concentration:
Dilution water:
Test concentrations:
Dilution factor:
Test duration:
Endpoint :
Test acceptability
criteria :
Static non- renewal
34 ± 2t» . •
15 ± 1°C :
Ambient laboratory !
illumination
10-20 /iE/m2/s (Ambient
laboratory levels)
16 h light-, 8 h darkness
30 mL ;
10 mL :
4 - . ' ;
Uncontaminated l-/iiri-f iltered
natural seawater or,
hypersaline brine prepared
from natural seawater
Effluents : Minimum of 5 and a
control ,
Receiving waters: 100%
receiving water and a control
Effluents : . ^0 .5 '
Receiving waters: 100%
receiving water and a control
72 ± 2 hr ;
Normal development;; ..'••. .
mortality can be included
^80% normal shell development
in the controls; must, achieve
a %MSD of <25%
359
-------
16 . Sampling requirements :
17. Sample volume
required :
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 Toxicity
Tests)
1 L per test
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 jusing 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 IC25. 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 the 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,
TEST
DEVELOPMENT
Copper
Concentration
(|ig/L)
Control
3.2
5.6
10.0
18.0
Replicate
A
B
C
D
E
A
B
C
D
E
A
B
C
D
E
A
B
C
D
E
A
B
C
D
E
No . Larvae
Exposed
100
100
100
101
74
110
100
100
100
100
102
100
100
107
100
100
100
100
100
100
100
100
100
100
100
No . Larvae
Normally
Developed
87
89
81
89
62
98
82
91
83
89
86
89
85
90
85
70
71 ;
77
74
87
7 ', ,
12
14 !
16
10
Proportion
Normal
0.87
0.89
0.81
0.88
0.84
0.89
0.82
0.91
0.83
0.89
0.84
0.89
0.85
0.84
0.85
0.70
0.71
0.77
0.74
0.87
0.07
0.12
0.14
0.16
0 . 10
361
-------
—- -^y'•_;:.• - ^"i-f^"_'ir'77*?
f STATlSf!cAL>M^g41| |EA:
':. r'pE^dl^lf^pST'
I
ENDPO1NT ESTIMATE
IC23
i>:.
£• • " - ' I BARUETTSTEST
•HOMOGENEOUS VARIANCE ' '•—-
* • * .. . • <
' _ • V ' ,.-.',,
LARVAL DEVELOPMENT 8ATA
PROPORTION NORMAL LARVAE
. L . f*'1 , V,,,"-,, '•;; ''"'•^fjf^'-
T •-• '" •-'"'•' r" '"I'S1';'"'""^!??^*
j-' ;'V..^;'•.^i'jjJMi^
T . "^i^yaaaj
, I ii ill i i "! 'H't'l'SllPlj'ifl-ii!
*v -- "•» if,. ««m>"»"»..*«t''. iL ti u t« I' A , ^"UnM
SHAPIRO-WILKSTESTj ; }< .,•.••,'. ^"'^
J I •••r". ??; SSS
3UTTON I ' .•'•-./L-;iL>--;y«p>if
W ,- - "' .-• ,- ; -".'3-'*
POINT ESTIMATION
_.~~*
SHAt%
NORMAL DISTRIBUTION
1 " ' i »» : -.
__ . , „ _ , I -'-;•';• .•••,•! ;,•' ',- ;• ••-'^ ;^g,,,
EQUAL NUMBER OP EQUAL NUMBER OF
REPLICATES? | / j ' , RgPdE^Ai^f ^'
VETO "' ! VCO ' ' ''''"'''"''f^S^.'Ui
• "CO t , , TCO _ ^as^rn
". - -j. -"•'•li^X^ '"", "'"' *--',''!'„'• i 'f-'F.: '. **"''• ^"^
lUMBER^F;^ _ : •,,•',. •> rf,, a^ v, -,/1 J;'
DUNNETTS STEEL'S MANY-ONE
TEST I - ' • RASlK-,TEST. ,",'
Figure 7. Flowchart for statistical analysis of sea urchin,
Strongylocentrotus purpuratus, 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. Underlying
assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality is the
Shapiro-Wilk1s Test, and Bartlett's Test is used to test for
homogeneity of variance. If either of these tests fails, the
nonparametric test, Steel's Many-one Rank Test, is used to
determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the
parametric procedure.
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 proportion of
normally developed larvae, thus each replicate must first be
transformed by the arc sine square root transformation procedure
described in Appendix B. The raw and transformed data, means and
variances of the transformed observations at each copper
concentration and control are listed in Table 5» The data are
plotted in Figure 8. Because there is zero normal development in
all five replicates of the 32.0 ptg/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 . j
15.13.2.5.1 The first step of the test for normality is to
center the observations by subtracting the mean of all
observations within a concentration from each observation in that
concentration. The centered observations are summarized in
Table 6.
15.13.2.5.2 Calculate the denominator, D, of the statistic:
x, - x)2 ;
363
-------
Where: X± = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
15.13.2.5.3 For this set of data, n = 25
D = 0.0680
TABLE 5.
SEA URCHIN, STRONGYLOCENTROTUS PURPURATUS,
DEVELOPMENT DATA
Copper Concentration (/xg/L)
Replicate Control
3.2
5.6
10.0
18. 0
RAW
ARC SINE
SQUARE ROOT
TRANSFORMED
Mean (Yi)
SJ
i
A
B
C
D
E
A
B
C
D
E
0.87
0.89
0.81
0.88
0.84
1.202
1.234
1.120
1.217
1.159
1.186
0.00215
1
0.89
0.82
0.91
0.83
0.89
1.234
1.133
1.266
1.146
1.234
1.203
0.00351
2
0.84
0.89
0.85
0.84
0.85
1.159
1.234
1.173
1.159
1.173
1.180
0.00097
3
0.70
0.71
0.77
0.74
0.87
0.991
1.002
1.071
1.036
1.202
1.060
0.00725
4
0.07
0.12
0.14
0.16
0.10
0.268
. 0.354
0.383
0.412
0.322
0.348
0.00311
5
364
-------
S9£
PROPORTION NORMALLY DEVELOPED
rt H-
I rf.
l—i OP
0 •
O
(D *d
a M
n- o
rt
rt O
C rt
C H
i^ ft
81 H-
l-h
pj p
5 H
fD
O
•o
(D
10
(D
§
io
-------
15.13.2.5.4 Order the centered observations from smallest to
largest
<: X(2> <; . . . * X(n)
where X(i) denotes the ith ordered observation. The ordered
observations for this example are listed in Table 7.
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WILK1S
EXAMPLE '
Copper Concentration (/xg/L)
Replicate
Control
3.2
5.6
10.0
18.0
A
B
C
D
E
0.016
0.048
-0.066
0.031
-0.027
0.031
-0.070
0.063
-0.057
0.031
-0.021
0.054
-0.007
-0.021
-0.007
-0,069
-0.058
0.011
-0.024
6 . 142
-0.080
0.006
0.035
0.064
-0.026
15.13.2.5.5 From Table 4, Appendix B, for the number of
observations, n, obtain the coefficients ax/ a2, ... ak where k is
n/2 if n is even and (n-l)/2 if n is odd.. For the data in this
example, n = 25 and k = 12 . The a± values are listed in Table 8.
15.13.2.5.6 Compute the test statistic, W, as follows:
W = -•[
D
The differences, x(n-i+1) - X(i) , are listed in Table 8
data in this example:
For the
W =
(0.2545)2 = 0.953
0.0680
366
-------
TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK1S
EXAMPLE ;
i
1
2
3
4
5
6
7
8
9 '
10
11
12
13
XU>
-0.080
-0.070
-0.069
-0.066
. -0.058
-0.057
-0.027
-0.026
-0.024
-0.021
-0.021
-0.007
-0.007
i
14
15
16
17
18
19
20
21
22
23
24
25
X(i)
0.006
0.011
0.016
0.031
0.031
0.031
0.035
0.048
0.054
C'.063
0.064
0.142
< •
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK1S
EXAMPLE
i . a, X— - x«»
1 0.4450
2 0.3069
3 0.2543
4 0.2148
5 0.1822
6 0.1539
7 0.1283
8 0.1046
9 0.0823
10 0.0610
11 0.0403
12 0.0200
0.222
0,134
0.132
0.120
0.106
0.092
0.058
0.057
0.055
0.037
0.032
0.013
X(25)
X<24>
X(23)
X(22>
X<21'
X(20)
X(19)
X(18)
X(17>
X(16)
X(1S)
X(14)
- X<»
- X(2>
- X13'
- x(4>
- x<5>
- x(6>
- X'7'
- x(e>
- x(9)
- x(10)
- x(11)
- 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 than the critical
value, conclude that the data are not normally distributed. For
j
367 i
-------
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
[ (Ev.) In S2 - Ev. In S?]
B £1 £1
Where: Vi « degrees of freedom for each concentration and
control,
Vi = (Hi - 1)
p = number of concentration levels including the control
nt = the number of replicates for-concentration i.
In = loge
i = 1,2, ..., p where p is the number of concentrations
including the control
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, V± = 4 for all i.
368
-------
15.13.2.6.3 Bartlett's statistic is, therefore:
p
B =[ (20) In {0.00340 )- 4 Eln(S?) ]/ 1.100
1.1
[20(-5.6840) - 4 (-29.4325)]/1.10.0
= 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 i.n
Table 9.
Where: p = number of concentration levels including the
control
\
TABLE 9. ANOVA TABLE
Source df Sum of Squares Mean Square(MS)
(SS) (SS/df)
Between
Within
P - 1
N - p
SSB
SSW
2 '
SB = SSB/ (p-1)
2 i
Sw = SSW/(N-p)
Total N - 1 SST
N = total number of observations
. 369
n2 ... + np
-------
n.i = number of observations in concentration i
P
SSB - Er2/n.-G2/w Between Sum of Squares
SST - EEr2-G2/w Total Sum of Squares
i-ij-l lj
ssw = SST-SSB Within Sum of Squares
G = the ^rand total of all sample observations,
G = ET.
i-l Z
Ti = the total of the replicate measurements for
concentration i
Yij = 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 example:
n± = n2 = n3 = n4 = ns = 5
N = 25
T! = YU + Y12 + Y13 + Y14 + Y15 = 5.932
T2 = Y21 + Y22 + Y23 + Y24 + Y25 = 6.013
T3 = Y31 + Y32 + Y33 + Y34 + Y35 = 5.898
T4 = Y41 + Y42 + Y43 + Y44 + Y45 = 5.302
T5 = Y51 + Y52 + Y53 + Y54 + Y5S = 1.739
G = Tj_ + T2 + T3 + T4 + T5 = 24.884
p
SSB = Er?/ni-G2/W
a (137.267)/5 - (24.884)2/25 = 2.685
p ni
SST = EEr2.-G2/w
J^.j-ZT.521 - (24.884)2/25 = 2.752
SSff = SST-SSB = 2.752 - 2.685 = 0.067
Si = SSB/(p-l) = 2.685/(5-l) = 0.6713
Sg = 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 df Sum of Squares Mean Square(MS)
(SS) (SS/df)
Between
Within
4
20
2.685
0.067
0.6713
0.0034
Total 24 2.752
15.13.2.7.4 To perform the individual comparisons, calculate the
t statistic for each concentration, and control combination as
follows:
Where: Y± = mean proportion normal larvae for concentration i
Y! = mean proportion normal larvae for the control
Sw = square root of the within mean square
nx = number of replicates for ,the control
n± = number of replicates for concentration i.
Since we are looking for a decreased response from the control in
the proportion of normally developed larvae, the ;concentration .
mean is subtracted from the control mean.
371
-------
15.13.2.7.5 Table 11 includes the calculated t values for each
concentration and control combination. In this example,
comparing the 3.2 /xg/L copper concentration with the control the
calculation is as.follows:
(1.186 - 1.203 )
t2 « —=
0.0583 ^(1/5)+{l/5)
TABLE 11. CALCULATED t VALUES
Copper Concentration (jtg/L)
3.2
5.6
10.0
18.0
2
3
4
5
-0.461
0.163
3.417
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 value 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 ti is
greater than the critical value. Therefore, the 10.0 j^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 £ig/L copper and the LOEC is 10.0 /ig/L copper.
15.13.2.7.7 To quantify the sensitivity of the test, the minimum
significant difference (MSD) that can be statistically detected
may be calculated:
MSD = d S^CL/nJ *(l/n)
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)
•! ' '
HI = the number of replicates in the control.
- i
15.13.2.7.8 In this example,
MSD = 2.30 (0.0583) ^(1/5)'+ (1/5) '
I
= 2.30 (0.0583) (0.6325)
= 0.085 i
!
i
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
2. Obtain the untransformed values for the control mean and..
the difference calculated, in step-1 of 13.2.7.9. .
[ Sine (1.186) ]2 = 0.859
[ Sine (1.101) ]2 =0.795
3. The untransformed MSD (MSDU) is determined by subtracting
the untransformed values from step 2 in 14.2.7.9.
MSDU = 0.859 - 0.795 = 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 concentration 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.
i .
15.13.2.8 Calculation of the ICp I
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 respect to concentration.
,
373
-------
H-
1
(D
vo
PROPORTION NORMALLY DEVELOPED
g 2
-------
Therefore, the means must be smoothed prior to palculating the
1C. |
l
15.13.2.8.2 Starting with the observed controU mean, Yx = 0.858,
and the observed mean for the lowest copper concentration, Y2 =
0.868, we see that Yx is less than Y2. ;
15.13.2.8.3 Calculate the smoothed means: :
j
M! = M2 = (Y! + Y2)/2 = 0.863 ;
15.13.2.8.4 Since Y3 = 0.854 > Y4 = 0.758 > Y5 = 0.118 > Y6 =
0.0, set M3 = 0.854, M4 = 0.758, M5 = 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 N^ (l-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 /Kj/L
copper and C5 = 18.0 /xg/L copper. ;
TABLE 12. SEA URCHIN, STRONYLOCENTROTUS PURPURATUS,
MEAN PROPORTION OF NORMALLY DEVELOPED LARVAE
Copper
Cone.
(/zg/L)
Control
0. 05
0.10
0.15
0.20
0.40
i
1
2
3
4
5
6
Response
, Means , Yi
(proportion)
0.858
0.868
0.854
0.758
0.118
0.000
Smoothed
Means , M±
(proportion)
0.863
0.863
0.854
0.758
0.118
p. 000
15.13.2.8.6 Using the equation from Section 4.2 in Appendix L,
the estimate of the IC25 is calculated as follows:
fc(j.i>-cj>
J'+ 1 J (M,.,.-M.)
375
-------
Cone . ID
Cone. Tested
Response
Response
Response
Response
Response
1
2
3
4
5
1
0
.87
.89
.81
.88
.84
2
3.2
.89
.82
.91
.83
.89
3
5.6
.84
. .89
.85
.84
.85
4
10
.70
.71
.77
.74
.87
5
18
.07
.12
.14
.16
.10
6
32
0
, 0
0
0
0
*** Inhibition Concentration Percentage Estimate ***
Toxicant/Effluent: Copper Chloride
Test Start Date: Test Ending Date:
Test Species: Purple Sea Urchin, Strongylocentrotus purpuratus
Test Duration: 72 hours
DATA FILE: urch_dev.icp
OUTPUT FILE: urch dev.i25
Cone.
ID
1
2
3
4
5
6
Number
Replicates
5
5
5
5
5
5
Concentration
ug/L
0.000
3.200
5.600
10.000
18.000
32.000
Response
Means
0.858
0.868
0.854
0.758
0.118
0.000 •
Std. Pooled
Dev. Response Means
0.033
0.040
0.021
0.068
0.035
0.000
0.863
0.863
. 0.854
0.758
0.118
0.000
The Linear Interpolation Estimate:
11.3844 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Expanded Confidence Limits:
Resampling time in Seconds:
11.3702 Standard Deviation: 0.2898
Lower: . 10.7785 Upper: 11.9375
Lower: 10.4756 Upper: 12.2141
0.16 Random Seed: 83761380
Figure 10. 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 /xg/L.
15.13.2.8.7 When the ICPIN program was used to analyze this set
of data, requesting 80 resamples, the estimate of the IC25 was
11.3844 /xg/L. The empirical 95.0% confidence interval for the
true mean was 10.7785 /xg/L to 11.9375 ptg/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 precision of the
development test using copper as a reference toxicant is provided
in Table 13. The NOEC varied by only one concentration interval
indicating good precision. The coefficient of variation for the
EC50 and EC25 were 22% and 21% indicating acceptable 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 toxicant is provided
in Table 14. The NOEC for laboratory's A and B were identical.
The difference in NOEC observed for lab C was probably due the
wide range of concentrations used (See Footnote 4). The
coefficient of variation for the EC50 was 39%, indicating
acceptable interlaboratory precision.
15.14.2 ACCURACY
15.14.2.1 The accuracy of toxlicity tests cannot be determined.
377
-------
Table 13. SINGLE-LABORATORY PRECISION OF THE PURPLE SEA URCHIN,
STRONGYLOCENTROTUS PURPURATUS, DEVELOPMENT TEST WITH
COPPER (CU /iG/L) SULFATE AS A REFERENCE TOXICANT1.
Test Number
1
2
3
4
5
Mean
CV(%)
NOEC Ug/L)
10.0-
10.0
5.6
5.6
5.6
EC50 (/xg/L)
19.4
18.3
10.8
14.3
16.8
15.9
22.0
EC25 (ftg/L)
15.1
15.4
9.0
11.0
12.9
12.7
21.0
1 Tests performed by Marine Pollution Studies Laboratory,
Granite Canyon, Monterey California.
TABLE 14. MULTI-LABORATORY.PRECISION OF THE PURPLE SEA URCHIN,
STRONGYLOCENTROTUS PURPURATUS, DEVELOPMENT TEST WITH
COPPER (CU /iG/L) SULFATE AS A REFERENCE TOXICANT.1
Lab
A2
B3
C4
Mean
CV(%)
NOEC .(/ig/L)
10.0
10.0
1.8
EC50 (jig/L)
22.5 -
15.2
10.1
15.9 '
39.0
xData 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
sTest 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 MEG 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
378
-------
APPENDIX I. SEA URCHIN DEVELOPMENT: STEP-BY-STEP SUMMARY
•••.'•:' • | ••.
PREPARATION OF TEST SOLUTIONS
- • f • . •.-.',
A. Determine test concentrations and appropriate dilution water
based on NPDES permit conditions and guidance from the
appropriate regulatory agency. i .
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 ± 2t<>. Include brine controls in tests that"
use brine.
C. Prepare a copper reference toxicant stock solution. '
D. Prepare a copper reference toxicant series.; Add 1-0 mL of
test solution each vial. '
E. Sample effluent and reference toxicant solutions for
physical/chemical analysis. Measure salinity, pH and
dissolved oxygen of 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 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
I. At the end of the test, measure salinity, pH, and dissolved
oxygen concentration from each test concentration.
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 place on tray
covered with seawater moistened paper towels. Induce
379 ':'
-------
spawning by injecting each sea urchin with 0.5 mL of 0.5 M
KC1. Place animals back onto 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 1-5 min after spawning begins,
D. Collect semen using either a Pasteur pipette or a 100 //L
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.
P. 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 0.05 'mL 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
15°C dilution water. Stir thoroughly with a Pasteur
pipette. Test eggs from each female to .determine if they
can be fertilized. , •
I. Take 0.5 mL subsample of sperm dilution and add to 5 mL of
10% 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 lOOx 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
determine the density 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 fertilize 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 mtn
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 water quality measurements as at the start.
S. After 72 hours, add 1.0 mL of 37% buffered formalin or 0.5
mL of 1.0% glutaraldehyde to each:test chamber. Tightly cap
and gently mix each vial. :
T. Examine each sample with a microscope and determine the
percentage of normally developed embryos.
U. Analyze the data.
V. Include standard reference toxicant point estimate values in
the standard quality control charts. i
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
i r*Ar»iMr* M/YTVIJ
LUAUINui NU 1 t»n
1
i
r
i:
P
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i
i
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1
i
m
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,
^
s
n
L
H.
i
!
i
*
!
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!!
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t
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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 coverslip (0.1 mm), provide
space for a specific microvolume of aqueous sample (0.1 mm3).
SIDE VIEW:
Counting
Area / Coverslip
.-*-> tr
I
t
Overflow Well
Loading
Notch
END VIEW THROUGH MID-CROSS SECTION:
Coverslip
Counting | Counting
Loading
Notch— *
Are a 4
1
1 Area
!-*•%
Overflow Well
Loading
•*— Notch
383
-------
This volume of liquid and the cells suspended therein (e.g.,
blood cells or sperm cells) represent 1/10,000th of the liquid
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 counted sample represents a
dilution of a more concentrated original sample, the above 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/mli = (dilution) (4.000 squares/mm3) (1,000 mm3/cm3) (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 100, the
calculation becomes:
cells/mL = (4.000) (4.000) (1,000) (loo)
80
- 20,000,000,000 cells/mL
384
-------
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 I Counting
Area 4 | 4 Area
Overflow Well
Loading
-Notch
385
-------
The- loaded heraacytometer 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 lOOx. Once the grid is properly positioned, the
microscope is adjusted to 20Ox or 40Ox, 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 or
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 uppe:r left hand corner
(square 1) and preceding right to square 4, down to square 5,
left to square 8, etc. until all 16 squares are counted.
„
' 0
1 *
a.
f
8* „
0
»
9 "
(C ,
'16°
f>
2 o "
^.
0
p
7
•o
0
10
0
15 »
3 & ' ' f
0
6
11
^
&
14 °
0
p
4
CJ
^
•b
0 **
12
•o
13
386
-------
Because sperm that appear on lines might be counted as being, in
either square, it is important to avoid double counting or non-
counting. 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 fall on the upper and left
lines, but not on the lower or right lines: •, ,-, •:
27.
28
*
54
=> 2
•1 03
4a '
^25
24
~o26 yt
C.*F
O *
029
30*
•f 31
52
<*" <
, »S3
•o
j>8
•5 ,
60 '*>
20
22
••*
^
v ^19 - t^
->*• JJ
«34
35
' ' ' 0
•51 49 jjg
50b
all
9
^
ib .n>
18°
19
^
36a,37
380
46 -»
3- 44
47
°45
"
o 13
12
p14t
. '
is**
^16
**>17 v
<>39 * • /^
' V.
^ &+
>>43
i
@-i—
-— 1
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, SI and 54
touch lines and are counted as being in the square below them or
to their right. The circled sperm are not; 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).
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 following procedure is recommended:
Count the number of sperm in the first large square.
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, 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
8
4
7
5
3
6
2
a
1
10
11
20
21
2
3
12
19
22
3
8
13
18
23
4
7
14
17
24
5
6
15
16
25
Pattern no. 1
Pattern no. 2
Pattern no. 3
The final consideration in achieving good replicate counts is
keeping the hemacytometers and coverslips 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
PURPLE URCHIN/ Strongylocentrotus purpuratus
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 CONTENTS
16.1 Scope and Application
16.2 Summary of Method
16.3 Interferences
16.4 Safety
16.5 Apparatus and Equipment
16.6 Reagents and Supplies
16.7 Effluents"and Receiving Water Collection,
Preservation, and Storage
16.8 Calibration and Standardization ,
16.9 Quality Control
16.10 Test Procedures .•
16.11 Summary of Test Conditions and Test
Acceptability Criteria
16.12 Acceptability of Test Results
16.13 Data Analysis
16.14 Precision and Accuracy
Appendix I 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 adversely 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 results become routine.
16.2 SUMMARY OF METHOD
16.2.1 The method provides the step-by-step instructions for
exposing sperm suspensions (appropriate sperm deilsity may first
be determined in a trial fertilization test) to effluents or
receiving waters for 20 minutes. Eggs are then added to the
sperm suspensions and, twenty minutes after the eggs 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. ;
I
16.3 INTERFERENCES '
i
i
16.3.1 Toxic substances may be introduced by contaminants in
dilution water, glassware, sample hardware, and testing equipment
(see Section 5, Facilities, Equipment, and Supplies).
16.3.2 Improper effluent sampling and handling may adversely
affect test results (see Section 8, Effluent and Receiving Water
Sampling and Sample Handling, and Sample Preparation for Toxicity
Tests).
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 maintaining
seawater at 10-20°C), with appropriate filtration and aeration
system.
391
-------
16.5.2 Air pump, air lines, and air stones -- for aerating water
containing broods'tock or for supplying air to test solutions with
low dissolved oxygen.
16.5.3 Constant temperature chambers or water baths -- for
maintaining test solution temperature and keeping dilution water
supply, gametes, and embryo stock suspensions at test temperature
(12°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.)
16.5.4 Water purification system -- Millipore Super-Q, Deionized
water (DI) or equivalent.
16.5.5 Refractometer -- for determining salinity.
16.5.6 Hydrometer(s) -- for calibrating refractometer.
16.5.7 Thermometers, glass or electronic, laboratory grade --
for measuring water temperatures.
16.5.8 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 solutions.
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 gametes or embryos and
test solutions).
16.5.16 Volumetric flasks -- Class A, borosilicate glass or non-
toxic plastic labware, 10-1000 mL for making test solutions.
16.5.17 Pipets, automatic -- adjustable, 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. i
16.5.20 Wash bottles -- for dilution water.
16.5.21 20-liter cubitainers or polycarbonate water cooler jugs
-- for making hypersaline brine.
16.5.22 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.
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; formaldehyde
similarly stored has induced abberant partial membrane 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 arid sand dollar
eggs.
16.5.25 Beakers, 1,000 mL borosilicate glass --; for rinsing and
settling sea urchin eggs. . :
393
-------
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 t'o test
tubes.
16.5.34 60 /urn NITEX® filter -- for filtering receiving water.
16.6 REAGENTS AND SUPPLIES
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) -- for data recording (see
Figures 1 and 2) .
16.6.3 Tape, colored -- for labelling test chambers and
containers.
16.6.4 Markers, water-proof -- for marking containers, etc.
394
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16.6.5 Parafilm --to cover graduated cylinders and vessels
containing gametes. ;
16.6.6 Gloves, disposable. -- for personal protection f.rom
contamination.
, * . * * ' , ' • ' " '
16.6.7 Pipets, serological -- 1-10 mL, graduated.
16.6.8 Pipet tips;,-- for automatic pipets. Not e: 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.
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.minimizing spills and contamination. : •.
16.6.13 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).
16.6.14 Membranes and filling solutions'-- for dissolved oxygen
probe .(see USEPA Method 360.. 1.,- USEPA, 1979), or reagents for
modified Winkler analysis. :
16.6.15 Laboratory quality assurance samples and standards --
for the above methods. - ,
16.6.16 Test chambers -- test tubes, borosilicate glass, 16 x
100 mm or 16 x 125 mm, with .-caps for conducting the test, four
chambers per concentration. •.
395
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Figure 1. Sample data sheet for spawning record.
Animal Time
No. Sex Injected Spawn Comments
2
8
10
11
12
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 - i
Sample • Dilution Count Eacrs /mL
For 100 mL egg suspension at 2,240 eggs/mL use:
100 mL x 2,240 eggs/mL / (counted eggs/mL) ;=.mL of egg stock
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:
( eggs/mL / 2,240 eggs/mL) x 100 = _J % volume
SPERM COUNTS -
Sample Dilution Count Square's Sperm/mL
SPERM/tnL = (DIL.FACT.) (COUNT) (4000) (1000)
(NO. SQUARES COUNTED)
397
-------
16.6.17 Formaldehyde, 10%, in seawater -- for preserving eggs.
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 under conditions of
maximal ventilation and minimal opportunity for volatilization
into room air.
16.6.18 Glutaraldehyde, 1% in seawater -- for preserving eggs.
16.6.13 pH buffers 4, 7, and 10 (or as per instructions of
16.6.19 Acetic acid, 10%, reagent grade, in filtered (10//)
seawater -- for preparing killed sperm dilutions for sperm
counts. .
16.6.20 Haemo-Sol or equivalent cleaner -- for cleaning
hemacytometer and cover slips.
16.6.21 0.5 M KC1 solution -- for inducing spawning.
16.6.22 Syringe, disposable, 3 or 5 mL -- for injecting KC1 into
sea urchins and sand dollars to .induce spawning.
16.6.23 Needles, 25 gauge -- for injecting KC1.
16.6.24 Pasteur pipets and bulbs .-- for .sampling eggs from
spawning beakers.
16.6.25 Hematocrit capillary tubes -- for sampling sperm for
examination and for loading hemacytometers.
16.6.26 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 uiicontaminated l-/zm-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 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.
16.6.31.2 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 (lOOli) > is used as a
diluent, the maximum concentration of effluent (6ts>) that can be
tested is 66% effluent at 34& salinity (see Table 1).
16.6.31.3 High quality (and preferably high salinity) seawater
should be filtered to at least 10 /xm before placing into the
freezer or the brine generator. Water should be collected on an
incoming tide to minimize the possibility of contamination.
399 !
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16.6.31.4 Freeze Preparation of Brine
16.6.31.4.1 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).
16.6.31.4.2 It is preferable to monitor the water until the
target salinity is achieved rather than allowing total freezing
followed by partial thawing. Brine salinity should never exceed
lOOIo. • It is advisable not to exceed about 70& brine salinity
unless it is necessary to test effluent concentrations greater
than 50%.
16.6.31.4.3 After the required salinity is attained, the HSB
should be filtered through a 1 /mi 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.
16.6.31.5 Heat Preparation of Brine
16.6.31.5.1 The ideal container for making brine using heat-
assisted 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 applied, use only
oil-free air compressors to prevent contamination. ,
16.6.31*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
400
-------
brine. A good quality biodegradable detergent should be used,
followed by several (at least three) thorough reaigent water .
rinses. . i
16.6.31.5.3 Seawater should be filtered to at least- 10 /zm 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 lOOti and that the temperature does not exceed 4Q°C.
Additional seawater may be added to the brine to obtain the
volume of brine required, i
16.6.31.5.4 After the required salinity is attained, the HSB
should be filtered through a 1 /zm 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.
16.6.31.6 Artificial Sea Salts
16.6.31.6.1 No data from sea urchin or sand dollar 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 seawater.
16.6.31.6.2 The GP2 reagent grade chemicals (Table 2) should.be
mixed with deionized (DI) water or its equivalent 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 be autoclaved,
sodium bicarbonate is added after the solution has cooled. A
stock solution of sodium bicarbonate is made up by'dissolving
33.6 g NaHCO3 in 500 mL of reagent water. ,Add 2.15 mL of this
stock solution for each liter of the>GP2 artificial seawater.
16.6.31.7 Dilution Water Preparation from Brine
401
-------
16.6.31.7.1 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 affluent
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.
16.6.31.7.2 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 lOOii arid the test is to be
conducted at 34&, 100& divided by 34tc.= 2.94. Thus, the
proportion is one part brine plus"1.94 parts reagent water). To
make 1 L of dilution water at 34&> salinity from a HSB of lOOli,
340 mL of brine and 660 mL of reagent water are required. Verify
the salinity of the resulting mixture using a refractometer.
16.6.31.8 Test Solution Salinity Adjustment
16.6.31.8.1 Table 3 illustrates the preparation of test
solutions (up to 50% effluent) at 34t<> 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 3.
16.6.31.8.2 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
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TABLE 1. MAXIMUM EFFLUENT CONCENTRATION (%) THAT CAN BE TESTED
AT. 34to WITHOUT THE ADDITION OF DRY SALTS GIVEN THE
INDICATED EFFLUENT AND BRINE SALINITIES.
Effluent
Salinity
&
0
1
2
3
4
5
10
15
20
25
Brine
60
to
43.33
44.07
44.83
45.61
46.43
47.27 '
52.00
57.78
65.00
74.29
Brine
70
&>
51.43
52.17
52.94
53.73
5.4.55
55.38 ,
60.00 '
, 65.45
72.00
80.00
Brine
80
&
57.50.
58.23
58.97
59.74
60.53
61.33
65.71
70.77
76.67
83.64
Brine
90
to
62.22
62.92
63.64
64.37
65.12 !
65.88
70.00
74.67
. 80.00
86.15
. Brine
100 '••
ti
66.00
66.67
67.35
68.04
68.75
. 69.4.7
73.33 '
77.65
82.50
88.00
16.6.31.8.3 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
%b) , 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)
16.6.31.8.4 This calculation assumes that dilution water
salinity is 34 ± 2%>. • " • - •
16.6.31.9 Preparing Test Solutions
403
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TABLE 2. ' REAGENT GRADE CHEMICALS USED IN THE PREPARATION OF GP2
ARTIFICIAL SEAWATER FOR THE PURPLE URCHIN
STRONGYLOCENTROTUS PURPURATUS, AND SAND DOLLAR
DENDRASTER EXCENTRICUS TOXICITY TEST1-2
Compound Concentration Amount (g)
(g/L) , Required for
20 L
NaCl
Na2SO4
KC1
KBr
Na2B4O7 .10 H2O
MgCl2 . 6 H20
CaCl2 • 2 H2O
SrCl2 . 6 H2O
NaHCO3
23.
4.
0.
0.
0.
10.
1.
0.
0.
90
00
698
100
039
80
50
025
193
478
80
13
2
0
216
30
0
3
,0
.0
.96
.00
.78
.0
.0
.490
.86
Modified GP2 from Spotte et al. (1984)
2The constituent salts and concentrations were taken from USEPA
(1990b). The salinity is 34.0 g/L.
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&> and a brine salinity of 66ib, 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 into the replicate
test chambers. The remaining test solution can.be used for
chemistry.
16.6.31.10 Brine Controls j .,,.....
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 dilution water.
Calculate the amount of reagent water to add to brine controls by
rearranging the above equation, (See, 16.6.33.8.3) setting SE =
0, and solving for VE. ''<.'..
i
VE = VB x (SB - 34)/(34 - SE) .
i
If effluent salinity is essentially Oto, 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. • . • ! •.•''••
> . ' * •
16.6.32 TEST ORGANISMS, PURPLE URCHINS j
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 collected from
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 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.
405
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TABLE 3. EXAMPLES OF EFFLUENT DILUTION SHOWING' VOLUMES OF •
EFFLUENT (AT Xfe), 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-xts> and, unless natural seawater is used
for dilution water, also a brine-based dilution water of 34le.
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.
(%)
40
Effluent x&
800 mL
Brine (68-
x}&
800 mL
Dilution Water*
34&
400 mL
Step 2. Use either serially prepared dilutions of the highest test'
concentration or individual dilutions of 100% effluent.
Effluent Cone. (%) ,
20
10
5
2.5
Control
Effluent Source
1000 mL of 40%
1000 mL of 20%
1000 mL of 10%
1000 mL of 5%
none
Dilution. Water*
(34&)
1000 mL
1000 mL
1000 ,mL « ' '
1000 mL
1000 mL
INDIVIDUAL PREPARATION:
Effluent Cone.
(V)
40
20
10
5
2.5
Control
Effluent x&
400 mL
200 mL
100 mL
50 mL
25 mL
none
Brine (68-x)&>
400 mL
200 mL
100 mL
50 mL • ,
• 25 mL
none
Dilution Water*
34&
200 mL
6,00 mL ,
800 mL
, 900 mL
950 mL
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
animals 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 capable of maintaining a constant
temperature between 10 and 14°C with a water temperature control
device.
16.6.32.5 Food for sea urchins --- kelp, 'recommended, but not
necessarily limited,to, Laminaria sp., Hedophyllum sp.,
Nerepcystis sp., Macrocystis sp. , Egregia sp., Alaria sp. or
romaine lettuce. The kelp should be gathered frbm 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. i
i
16.6.32.6 Natural seawater (>30&) is used to maintain the adult
animals and (^32li) as a control, 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 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 hour£.
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 (32tb) and a gravel bed
filtration system, are housed within a water bath, such as an
407 i
-------
INSTANT OCEAN* Aquarium. 'The sexes should be held separately if
possible.
16.6.33 TEST ORGANISMS, SAND DOLLARS
16.6.33.1 Sand Dollars, Dendraster excentricus, (approximately 6
of each sex per test).
16.6.33.2 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," surrounded 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.
16.6.33.3 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 salts 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.
16.6.33.4 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 control
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.
16.6.33.6 Natural seawater (>30&>) is used to maintain the adult
animals and (i323o) 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 control water,
loosely covered with a styrofoam sheet and allowed to equilibrate
to the holding temperature before animals are added.
16.7 EFFLUENT AND RECEIVING WATER COLLECTION, PRESERVATION AND
STORAGE
16.7.1 Section 8, Effluent and Receiving Water Sampling and
Sample Handling, and Sampling Preparation for Toxicity Tests.
16.8 CALIBRATION AND STANDARDIZATION
16.8.1 See Section 4, Quality Assurance.'
16.9 QUALITY CONTROL
16.9.1 See Section 4, Quality Assurance.
16.10 TEST PROCEDURES
16.10.1 TEST 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 Receiving waters
16.10.2.1.1 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
409
-------
through a 60 /zm 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.
16.10.2.2 Effluents
16.10.2.2.1 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 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 (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 34li 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%).
16.10.2.2.3 The volume in each test chamber is 5 mL.
16.10.2.2.4 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.
16.10.2.3 Dilution Water
16.10.2.3.1 Dilution water should be uncontaminated l-/xm-
filtered 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
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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 (CuCl2oH20). Reference toxicant
tests provide an indication o'f 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 (GuCl2°2H2O) 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 A«J/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
/Kj/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
and filling to 100-mL with dilution water). Start with control
solutions and progress to the highest concentration to minimize
contamination. " i
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. ± 2l"o.
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 beakers and place in 12°C
bath or room. Allow to come to temperature. Select a sufficient
number of sea urchins or sand dollars (based upoii 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
411'
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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.
16.10.3.1.2 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. ,
16.10.3.1.3 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.
16.10.3.1.4 Handle sexes separately once known; this minimizes
the chance of accidental egg fertilization. Throughout 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.
16.10.3.1.5 Fill a 3 or 5 mL syringe with 0.5 M KCl 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).
16.10.3.1.6 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 KCl.
16.10.3.1.7 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 will release cream-colored semen.
16.10.3.1.8 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
412
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(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 minutes, a
second 0.5 mL injection may be 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, glass centrifuge
tube, covered with a cap or parafilm, on ice. Air 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 describe
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. j
i
1
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 semen from each
male into separate 1-15 mL conical test tubes, stored 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
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dilution water on a microscope slide (well slides work nicely).
Examine the sperm for motility; use sperm from males with 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 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 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 (<5°C) . The sperm should be
used in a toxicity test within 4'h of collection.
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, small, 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.
16.10.4.2 Pooling of Eggs
16.10.4.2.1 Allow eggs to settle in the collection beakers.
Decant some of the water from the collection beakers taking.care
not to pour off many eggs. The sea urchin eggs are pooled into a
1 L beaker, and the volume brought to 600 mL with 12°C dilution
water. The eggs are suspended by swirling and the eggs allowed
414
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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 • i
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 into vial 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 Sedgewick-
Rafter 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 dilution water 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 eggs7mL = ; 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 10 or (Count B) x 100,
If the egg stock is <2,240 eggs/mL (A <224 eggs/mL) , 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
«:5.6xl06/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 sets 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
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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 density 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 required to obtain
a sperm stock (3.36 x 107 sperm/mL) for the maximum sperm density
(6 x 105 sperm/200 eggs/mL--3,000:1) needed for the trial. A
sperm density of about 1 x 107 is convenient to count. If the
approximate sperm density is known, the dilution procedures
outlined in Table 4 can be followed without initial 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 mL. Table 4 is
provided for guidance as a quick reference for dilution volumes
if sperm density of pooled semen is can be reasonably estimated,
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 with
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 dilution waiter (e.g., 10 mL
of 10% glacial acetic acid plus 90 mL of dilution water).
417
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**** ' **
Figure 3. Showing the location and orientation used in the
injection of KCl 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.
16.10.5.8 Mix the chilled suspension of pooled sand dollar sperm
(16.10.5.6) 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
1% glacial acetic acid in dilution water (e.g., 10 mL of 10%
glacial acetic acid plus 90 mL of dilution water).
16.10.5.9 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
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least 80% of the higher count use the mean count to estimate
sperm density in semen and the required dilution volume for the
test stock. If the two counts do not agree within 20%, count the
two fields on the other hemacytometer. Calculate 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 water necessary to
dilute the sea urchin semen or the sand dollar sperm suspension
to the sperm density (sperm/mL) required for the; 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 IxlO8 to 9xl09 are for volume
(mL) of sperm stock for total volume of 100 mL; Stable values for
sperm densities ilxlO10 are for dilution water volumes for.0.025
mL of semen. Table 5 is used as follows:- given ;a sperm density
in the semen stock (e.g., 4.7xl09) find the row containing the
integer (characteristic) and the exponent (4xl09) in the left
hand column, then read across to the column corespondirig to the
mantissa (0.7). The value at the intersection of the row and
column (0.71 mL) is the volume of semen per lOOmL needed for
sperm stock to achieve a 3000:1 sperm:egg ratio in the trial.
16.10.5.12 For the approximate sperm:egg ratios dilute the
3000:1 stock as follows:
1288:1 5 mL 3000:1 stock with 6.6 mL dilution water
550:1 2 mL 3000:1 stock with 9.9 m'L dilution water
234:1 1 mL 3000:1 stock with 11.8 mL 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 should include
3,000:1 and several lower ratios. The ratios 100:1, 234:1,
550:1, 1288:1 and 3,000:1 are recommended because they evenly
divide the log sperm:egg ratio. Fertilization appears to be a
linear function of the log of sperm density (Figure 4).
Recommended sperm dilution procedures are given in Table 5.
419
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16.10.6.4 Quantitive evaluation of the sperm density trial
should be obtained by counting 100 eggs from each tube until a
suitable sperm density can be determined 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 loss 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.
16.10.6.5 Prepare killed sperm preparations of the trial sperm
stock suspensions to provide confirmation of the nominal
sperm:egg ratios. It saves timea if these can be prepared and
loaded onto hemacytometers while the trial is being conducted.
Alternatively, once the trial has been evaluated, the selected
nominal sperm density can be confirmed by direct hemacytometer
count.
16.10.6.6 Record all the counts made, select a target sperm .-egg
ratio for the test, and calculate tjae dilution of the stored
sperm stock needed to provide the necessary sperm density for the
definitive test.
16.10.6.7 Table 5 can be used for deriving the volumes needed
for preparing the final sperm stock. For a pooled sperm
suspension density of 4xl09 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
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TABLE 4. Dilution volume guide for initial count of sperm
density to achieve recommended counting density of 1 x
107/mL.
Initial
Sperm/mL
1 x 10s,
2 X 10"
3 x 10"
' 4 x 10s
5 X 108
6 X 10"
7 X 10"
8 X 108
9 X 10s
1 X 109
2 X 109
3 X 10s
4 X 10s
5 X 109
6 X 109
7 x 10'
8 X 10s
9 X 109
1 x 10l°
• 2 x 10l°
3 x 1010
4 x 1010
5 x 1010
6 X 1010
7 x 1010 •
8 x 1010
9 X 1010
mli/lOmL
1.000
0.500
0.333
0.250
0.200
0.167
0.143
0.125
0.111
0.100
0.050
0.033
0.025
mL/100mL
1.000
0.500
0.333
0.250
0.200-
0.167
0.143
0.125
0.111
0.100
0.050-
0.033
0.025
mL/200m!j
, 1.000
0.667
0.500
0.400
0.333
6.286
0.250
0.222
0.200
0.100
0.067
0.050
0.040
0.033
0.029
0.025
, 0.022
Note; 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 Xwhich usually settle to the test tube bottom upon vortexing); and (4)
wiping semen from the pipet tip with tissue be done with care to avoid wicking semen from
within the pipet tip. ;
421
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Bring the indicated volume of sperm stock to 100 mL
Density
l.OOe+08
2.006+08
3.006+08
4.006+08
S.OOe+08
6.006+08
7.006+08
S.OOe+08
9.006+08
1. OOe+09
2.000+09
3.006+09
4.000+09
S.OOe+09
£.00e+09
7.006+09
8.00e+09
9.006+09
0.0
33.60
16.80
11.20
8.40
6.72
5.60
4.80
4.20
3.73
3.36
1.68
1.12
0.84
0.67
0.56
0.48
0.42
0.37
0.1
30.55
16.00
10.84
8.20
6.59
5.51
4.73
4.15
3.69
3.05
1.60
1.08
0.82
0.66
0.55
0.47
0.41
0.37
0.2
28.00
15.27
10.50
8.00
6.46
5.42
4.67
4.10
3.65
2.80
1.53
1.05
0.80
0.65
0.54
0.47
0.41
0.37
0.3
25.85
14.61
10.18
7.81
6.34
5.33.
4.60
4.05
3.61
2.58
1.46
1.02
0.78
0.63
0.53
0.46
0.40
0.36
0.4
24.00
14.00
9.88
7.64
6.22
5.25
4.54
4.00
3.57
2.40
1.40
0.99
0.76
0.62
0.53
0.45
0.40
0.36
0.5
22.40
13.44
9.60
7.47
6.11
5.17
4.48
3.95
3'. 54
2.24
1.34
0.96
0.75
. 0..61
0.52
0.45
0.40
0.35
0.6
21.00
12^92
9:33
7.30
6.00
5.09
4.42
3.91
3.50
2.10
1.29
0.93
0.73
0.60
0.51
0.44
' 0.39
0.35
0.7
19.76
12.44
9.08
7.15
5.89
5.01
4.36
3.86
3.46
1.98
1.24
0.91
0.71
0.59
0.50
0.44 •
0.39
0.35
0.8
18.67
12.00
8.84
7.00
5.79
4.94
4.31
3.82
3.43
1.87
1.20
0.88 .
0.70
0.58
0.49
0.43
0.38
0.34
0.9
17.68
11.59
8.62
6.86
5.69
4.87
4.25
3.78
3.39
1.77
1.16
0.86
0.69
0.57
0.49
0.43
0.38
0.34
To dilute dense semen: add 0.025 mL of semen into these volumes (mL) of dilution water
Density
l.OOe+10
2.006+10
3 . OOe+io
4.006+10
S.OOe+10
6.006+10
7.006+10
8.006+10
9.006+10
0.0
7.44
14.88
22.32
29.76
37.20
44.64
52.08
59.52
66.96
0.1
8.44
15.88
23.32
30.76
38.20
45.64
53.08
60.52
67.96
0.2
9.44
16.88
24.32
31.76
39.20
46.64
54.08
• 61.52
68.96
0.3
10.44
17.88
25.32
32. 76
40.20
47.64
55.08
62.52
69.96
0.4
11.44
18.88
26.32
33.76
41.20
48.64
56.08
63.52
70.96
0.5
12.44
19.88
27.32
34.76
42.. 20
49.64
57.08
64.52
71.96
0.6
13.44
20.88
28.32
35.76
43.20
50.64
58.08
65.52
72.96
0.7
14.44
21.88
29.32
36.76
44.20
51.64
59.08
66.52
73.96
0.8
15.44
22.88
30.32
37.76
45.20
52.64
60.08
67.52
74.96
0.9
16.44
23.88
31.32
38.76
46.20
53 .64
61.08
68.52
75.96
TABIiB 5. DILUTION VOLUMES OF SPERM STOCK
ACHIEVE THE SPERM STOCK DENSITY (3.36X107)
OF INDICATED DENSITY (1.0X108 TO
FOR A 3000:1 SPERM:EGG RATIO.
9.9X1010) TO
422
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100
90
1 70
S 60
1 50
£
15 40
| 30
£ 20
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i!
1
a
i
i-
-------
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
100:1
234:1
550:1
1288:1
3000:1
case 1
100*
100
100
100
100
case 2
95*
98
100
100
100
case 3
85
95*
98
100
100
case 4
70
80
98*
100
100
case 5
40
64
82
84
88*
case 6
70
85*
89
90
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
spentuegg 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.
(11,200 = (1,120 eggs/tube)(0.1 mL of sperm stock/tube)).
(stock sperm/mL)/(target sperm/mL) = dilution; e.g., if stock
sperm has 4xl09 sperm/mL, then dilution = 4xl09 / 5.6xl06 = 714
16.10.8.1.1 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
424
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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).
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
± 1°C) and maintained at that temperature during the addition of
dilution water.
16.10.8.1.3 Increase the temperature of the water 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 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 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 the end of the
test.
i
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 copy of the randomization 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
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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 0.100 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 .the 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 1 mL 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 -
spermregg 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
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>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 thermometer.
I
16.10.9.3 Adding Eggs to the Test - . |
16.10.9.3.1 Exactly 20 minutes after the sperm addition to the
test was begun, begin to add the eggs, 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 used with the sperm
so that each test tube has a sperm incubation period of 20
minutes. Note the time of start and finish of egg addition.
This duration should be within one minute of that used for the
sperm.
16.10.9.3.2 In order to maintain the same sperm:egg- ratio in
each test tube,, the eggs 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 perforated plunger
appears to be the best method of achieving a uniform
distribution. Frequent inversion and mixing of egg stock in
either a graduated cylinder or a multiple injection pipet may be
acceptable.
i
16.10.9.3.3 The eggs should be injected 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
contains 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 masking 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 ±2li 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
-------
I
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. j
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. j .
16.10.12.1.2 Temperature, pH, and salinity are measured at the
beginning of the exposure period in one test chaimber 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.
16.10.12.1.3 Record all the measurements on the data sheet.
16.10.13 TERMINATION OF THE TEST !
j
16.10.13.1 Ending the Test i :
16.10.13.1.1 Record the time the test is terminated.
16.10.13.1.2 Because of the short test duration water quality
measurements are not necessary at the end. j
16.10.13.2 Sample Preservation i
16.10.13.2.1 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 [zygotes]) and to
preserve the eggs for examination. Again, the time allotted to
fixative addition should be about the same as that for sperm and
egg addition and the sequence of addition the same as for the
introduction of the gametes.
429
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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 adding 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 presence, or absence of an elevated
fertilization membrane. 'Newly fertilized 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 membrane are counted as
fertilized.
430
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Normal Fertilized
Egg
a.
Unfertilized
Not Mature Eggs
(do not count]
Figure 5 . Examples of typical fertilized and unfertilized 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);
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 hyaline
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 iri 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 preservative), 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.
•
d.
•
e.
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 spawned 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
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16.10.13.3.3 It is convenient to concentrate the eggs prior to
counting. If the eggs are allowed to completely settle (ca 30
minutes after termination and mixing), most of the overlying
solution can be removed with a pipet, leaving the eggs
concentrated in a much smaller volume. The eggs are then
resuspended by filling and emptying a 1 mL pipet 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 fertilization due 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 the preservative,
and the period between preservation and counting. However, other
sampling procedures may be used once demonstrated not to 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 or with an inverted
microscope... / , .
i -
16.10.13.4 Endpoint j
!
16.10.13.4.1 In a count of at least 100 eggs, record the number
of eggs with fertilization membranes and the number 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 acceptability
criteria is listed in Table 7. • '
TABLE 7. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY'
CRITERIA FOR,, STRONGYLOCENTROTUS PURPURATUS AND
DENDRASTER EXCENTRICUS, FERTILIZATION TEST WITH
EFFLUENTS AND RECEIVING WATERS
1.
2.
3.
4.
Test type :
Salinity:
Temperature :
Light quality:
Static non- renewal ,' .
34 ± 2&
12 ,± 1°C ' '.
Ambient, laboratory light
during test preparation
433
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5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Light intensity:
Test chamber size :
Test solution volume : •
Number of spawners:
No. egg and sperm cells
per chamber:
No. replicate chambers
per concentration:
Dilution water:
Test concentrations :
Dilution factor:
Test duration:
Endpoint :
Test acceptability
criteria:
Sampling requirements :
Sample volume required:
10-20 uE/m2/s (Ambient
laboratory levels)
16 x 1QO or 16 x 125 mm
5 mL
Pooled sperm from up to four
males and pooled eggs from up
to four females are used per
test
About 1,120 eggs and not more
than 3,360,000 sperm per test
tube
4
Uncontaminated l-/xm- filtered
natural seawater or
hypersaline brine prepared
from natural seawater or
artificial sea salts
Effluents: Minimum of 5 and a
control
Receiving waters: 100% a
control
Effluents: ^0.5
Receiving waters: None or :>0.5
40 min (20 min plus 20 min)
Fertilization of eggs
;>70% egg fertilization in
controls; %MSD of <25%; and
appropriate sperm counts
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, and Sample
Preparation for Toxicity
Tests)
1 L
434
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16.12 ACCEPTABILITY OF TEST RESULTS
16;12.1 Test results are acceptable only if all the fallowing
requirements are met: ;
(1) Egg .fertilization at the NOEC must be greater than 80%
of that in the controls.
(2) The minimum significant difference (%MSD) 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 the 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. Calculate 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 statistics.
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
interpolation Method (see Section 9, Chronic Toxicity Test
Endpoints and Data Analysis). LOEC and NOEC values for fecundity
are obtained using a hypothesis testing approach such as
Dunnett's Procedure (Dunnett, 1955) or Steel's Many-one Rank Test
(Steel, 1959; Miller, 1981) (see Section 9). Separate 'analyses ,
are performed for the estimation of the LOEC and NOEC endpoints
435
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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
Concentrat ion
(%)
Control
0.05
0.10
0.15
0.20
0.40
0.60
0.80
Replicate
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
No . of Eggs
Counted
100
100
100
100
100
100
100
100
100
100
100
100
10.0
100
100
100
100
100
100
100
100
100
100
100
No. of Eggs
Fertilized
97
90
100
100
100
98
100
97
99
98
96
97
94
88
97
43
63
46
2
1
9
0
0
0
Proportion
Fertilized
0.97
0.90
1.00
1.00
1.00
0.98
1.00
0.97
0.99
0.98
0.96
0.97
• 0.94
0.88
0.97
0.43
0.63
0.46
0.02
'0.01
0.09
0.00
0.00
0.00
436
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16.13.2 EXAMPLE.OF ANALYSIS OF SEA URCHIN, STRONGYLOCENTROTUS
PURPURATUS, AND SAND DOLLAR, DENDRASTER EXCENTRICUS,
FERTILIZATION DATA
I
16.13.2.1 Formal statistical analysis of the fertilization data
is outlined in Figure 7.
The response used in the analysis is the proportion of fertilized
eggs in each test or control chamber. Separate analyses are
performed for the estimation of the NOEC and LOEC endpoints and
for the estimation of the IC25 endpoint. Concentrations at which
there are no eggs fertilized in any of the test chambers are
excluded from statistical analysis of the NOEC and LOEC, but •
included in the estimation of the IC25.
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, Durinett' s
Procedure, or a nonparametric .test, Steel's Manyfone 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-Wilk1s Test, and Bartlett's Test is used to test for
homogeneity of variance. If either of these tests fails, the
nonparametric test, Steel's Many-one Rank Test, is used to
determine the NOEC and LOEC endpoints. . If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the
parametric procedure.
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 adjustment is the
nonparametric alternative. •
16.13.2.4 Example of Analysis of Fecundity Data
.
16.13.2.4.1 This example uses toxicity data from a sea. urchin,
Strongylocentrotus purpuratus, fertilization test performed with
effluent. The response of interest is the proportion of
fertilized eggs, thus each replicate must first .be transformed by
the arc sine square root transformation procedure described in
Appendix B. The raw and transformed data, means and variances of
the transformed observations at each effluent concentration and
control are listed in Table 9. The data are plotted in Figure 8.
Because there is zero fertilization in all threeireplicates for
the 0.80% effluent concentration, it was not included in the
statistical analysis and is considered a qualitative fecundity
effect.
437
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STATISTICAL ANALYSIS OF SEA URCHIN AND
SAND DOLLAR FERTILIZATION TEST
FERTILIZATION DATA
PROPORTION OF FERTILIZED EGGS
POINT ESTIMATION
t
'HVPOTHE^TESTINO
ENDP01NT ESTIMATE
IC25
•••
ARC SINE SQUARE ROOT
TRANSFORMATION
I
SHAPIRO-WILlCS ,TEST
NORMAL DISTRIBUTION
HOMOGENEOUS VARIANCE
NO
EQUAL NUMBER OF
REPLICATES?
1
VARIANCE
NO
EQUAL NUMBER OF
REPLICATES?
YES
t TEST WITH
BONFERRONI
YES
'Hlh
K
A: ,
WILCOXON RANK SUM
itESTWITH '
BONFERRONI ADJUSTMENT
Hit |tt !) !! tt88g! lVt H
NOEC,LOEO
Figure 7. Flowchart for statistical analysis of sea urchin,
Strongylocentrptus purpuratus, and sand dollar,
Dendraster excentricus, test.
438
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CQ
3
4J
S
o
o
f
4J
CO
m
(U
-------
16.13.2.5 Test for Normality
16.13.2.5.1 The first step of the test for normality is to
center the observations by subtracting the mean of all
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 {%)
Rep. Control 0.05
0.10
0.15
0.20
0.40
0.60
RAW
ARC SINE
SQUARE ROOT
TRANSFORMED
Mean (YI>
s?
i
A
B
C
A
B
C
0.97
0.90
1.00
1.397
1.249
1.521
1.389
0.01854
1
1.00
1.00
0.98
1.521
1.521
1.429
1.490
0.00282
2
1.00
0.97
0.99
1.521
1.397
1.471
1.463
0.00389
3
0.98
0.9.6
0.97
1.429
1.369
1.397
1.398
0.00090
4
0.94
0.88
0.97
1.323
1.217
1.397
1.312
0.00819
5
0.43
0.63
0.46
0 . 7.15
0.917
0.745
0.792
0.01188 0
6
0.02
0.01
0.09
0.142
0.100
0.305
0.182
.01173
7
TABLE 10. CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S
EXAMPLE
Effluent "Concentration (%)
Replicate Control
0.05
0.10
0.15
0.20
0.40
0.60
A
B
C
0.008
-0.140
0.132
0.031
0.031
-0.061
0.058
-0.066
0.008
0.031
-0.029
-O.OOl'
0.011
-0.095
0.085
-0.077
0.125
-0.047
-0.040
-0.082
0.123
440
-------
16.13.2.5.2 Calculate the denominator, D, of the statistic:
D
- X)
i-l
Where: XL = the ith centered observation
X = the overall mean of the centered observations
n = the total number of centered observations
16.13.2.5.3 For this set of data,, n = 21
X =
(0.005) = 0.000
21
D = 0.1159
16.13.2.5.4 Order the centered observations from smallest to
largest
]£(D ^ X'2' £ £ X'n'
where X(i) denotes 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 number of
observations, n, obtain the coefficients a17 a2, ... ak where k is
n/2 if n is even and (n-1)/2 if n is odd. For the data in this
example, n = 21 and k = 10. The a± values are listed in
Table 12. '
16.13.2.5.6 Compute the test statistic, W, as follows:
1 * (nil) (i) 2
w = — [Ea .(Xln"il)-xu>) ]
D y.i 1 .
The differences, x(n-i+1) - X(i), are listed in Table 12. For the
data in this example:
W =
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
1
2
3
4
5
6
7
8
9
10
11
-0.140
-0.095
-0.082
-0.077
-0.066
-0.061
-0.047
-0.040
-0.029
-0.001
0.008
12
13
14
15
16
17
18
19
20
21
0.008
0.011
0.031
0.031
0.031
0.058
0.085
0.123
0.125
0.132
TABLE 12. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK1S EXAMPLE
1 0.4643
2 0.3185
3 0.2578
4 0.2119
5 0.1735
6 0.1399
7 0.1092
8 0.0804
9 0.0530
10 0.0263
0.272
0.220
0.205
0.162
0.124
0.092
0.078
0.071
0.040
0.009
X<21)
X(20)
X(19)
X(18)
X<»>
X(16)
XUS>
X<14>
X'13'
Xd2)
- X'1'
- x<2)
- X(3)
- X<4>
- x<5>
- X'6'
- X'7'
- x<8'
- x(9>
- X'10'
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's Test
(Snedecor and Cochran, 1980). The test statistic is as follows:
p _ p ', •. •
[ (Ev.) In S2 - T,V. In S?J
i
Where: V± = degrees of freedom for each concentration and
control, :
V± = {ni - 1)
p = number of concentration levels including the control
n£ = the number of replicates for concentration i.
In = loge . .
i = 1,2, ..., p where p is the number of concentrations
including the control
16.13.2.6.2 For the data in this example (see Table 8), all
effluent concentrations including the control have the same
number of replicates (n± = J3 for all i) . Thus, V^ = 2 for all i
16.13.2.6.3 Bartlett's statistic is, therefore:
p : •
B = I (14) In {0.008279 ) -2Eln(S*) ] / 1.1905
i-l
[14C-4.7940) - 2 (-36.1047)'] /1.1905
= 5.0934/1.1905
= 4.2784 '.'.-••
16.13.2.6.4 B is approximately distributed as chi-square with
p-1 degrees of freedom, when the variances are in; fact the same.
Therefore, the appropriate critical value for this test, at a
significance level of 0.01 with 6 degrees of freedom, is 16.81.
Since B = 4.2784 is less than the critical value of 16.81,
conclude that the variances are not different.
i
"
i
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
Between p - 1
Within N - p
Total N - 1
Sum of Squares
(SS)
SSB
SSW
SST
Mean Square (MS)
(SS/df)
2
SB = SSB/(p-l)
2
Sw = SSW/(N-p)
Where: p
number of concentration levels including the
control
N = total number of observations nx + n2
+ n,,
= number of observations in concentration i
SSB - ET?/n.-G2/N Between Sum of Squares
1.1
SST
.-
J
1JJ-1
SSff * SST-SSB
Total Sum of Squares
Within Sum of Squares
G = the g,rand total of all sample observations,
G - Er.
1.1
Ti = the total of the replicate measurements for
concentration i
ij = 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: ;
nx = n2 = n3 = n4 = ns - = n6 = • n7. = 3
N = 21
T! = Ylx + Y12 + Y13 = 4.167 \
.T2 = Y21 + Y22 + Y23 = 4.471
T3 = Y31 + Y32 + Y33 = 4.389
T4 = Y41 + Y42 + Y43 = 4.194
T5 = Y51 + Y52 + Y53 = 3.937
T — Y -u V -i-V — 9 777
J-6 — 161 + X62 + X63 — ^ • -3 ' '
T7 = Y71 + Y72 + Y73 = 0.547
T! + T2 + T3 + T4 + T5 + T6 + T7 = 24.082
SSB = Er2/n .-G2/W
i=a i95z.656)/3 - (24..082)2/21 = 4.269
p n±
SST = EEy2-G2/w
«j4J^Q01 - (24.082)2/21 = 4.385 .
SStf = SST-SSB = 4.385 - 4.269 = 0 M16 .
SB = SSB/(p-l) = 4.269/(7-l) = 0.7115
sl = 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 combination as
follows:
(v^v - !
445
-------
TABLE 14. ANOVA TABLE FOR DUNNETT'S PROCEDURE
EXAMPLE
Source df Sum of Squares Mean Square(MS)
(SS) (SS/df)
Between 6 4.269 0.7115
Within 14 0.116 0.0083
Total 20 4.385
Where: YA = mean proportion fertilized eggs for concentration i
Yx = mean proportion fertilized eggs for the control
SH = square root of the within mean square
n^ = number of replicates for the control
n± = number of replicates for concentration i.
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 :
(1.389 - 1.490)
-J..JOO
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 control if t± is greater than the
446
-------
critical value. Therefore, the 0.40% 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 Concentration (%)
0
0
0
0
0
- o
.05
-10
.15
.20
.40
.60
i
2
3
4
5
6
7
ti
-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 (MSB) that can be statistically detected
may be calculated:
MSD = d
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-L =. the number • of replicates in, the control.
16.13.2.7.8 In this example, ' .
MSD =2.53 (0.0911 ) W
= 2.53 (0.0911)(0.81&5)
= 0.188 !
16.13.2.7.9 The MSD (0.188) is in transformed units. To-
determine the MSD in terms of proportion of fertilized eggs,
carry out the following.conversion.
1. Subtract the .MSD from the transformed control mean.
1.389 - 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) ]2 = 0.967
[ Sine (1.201) ]2 = 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 ICp
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 1C.
16.13.3.8.2 Starting with the observed control mean, Yx = 0.957,
and the observed mean for the lowest effluent concentration, Y2
- 0.993, we see that Yx is less than Y2.
16.13.3.8.3 Calculate the smoothed means:
M! = M2 = (Y! + Y2)/2 = 0.975
16.13.3.8.4 Since Y3 = 0.987 is larger than M2, average Y3'with
the previous concentrations:
Mj. = M2 = M3 = (Mi + M2 + Y3)/3 = 0.979.
16.13.3.8.5 Since M3 > Y4 = 0.970 > Y5 = 0.930 > Y6 = 0.507 > Y7 =
0.040 > Y8 = 0.0, set M4 = 0.970, M5 = 0.930, H6 = 0.507, M7 =
0.040, and M8 = 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 M^l-p/lOO) = 0.979(1-25/100).
Examining the means and their associated concentrations
448
-------
(Table 16), the response, 0.734, is bracketed by C5 = 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 follows:
ICp
IC25 = 0.20 + [0.979(1 - 25/100) - 0.930] (0.40 - 0.20)
,(0.507 - 0.930)
= 0.29%.
TABLE 16. SEA URCHIN, STRONYLOCENTROTUS PURPURATUS,
MEAN PROPORTION OP FERTILIZED EGGS
Effluent
Cone.
(%)
Control
0.05
0.10
0.15
0.20
0.40
0.60
0.80
i
1
2
' 3
4
5
6
7
8
Response
Means , Y±
(proportion)
0.957
0.993
• 0.987
0.970
0.930
0.507
0.040
0.000
Smoothed
Means? , M±
(proportion)
0.979
0.979
0.979
0.970
0.930
O.S07
0 . 040
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
-------
16.14.1.1.1 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 19. 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
16.14.1.2.1 Multi-laboratory precision data for
Strongylocentrotus purpuratus, with the reference toxicant
copper, tested in natural seawater, are provided in Table 20.
The coefficient of variation for the EG25 was 52%, based on data
from five laboratories.
16.14.2 ACCURACY
16.14.2.1 The accuracy of toxicity tests cannot be determined.
450
-------
Q)
CQ
CO
fl
co
i
•d
.5 .
4J ra
o tn
o o
e a)
GO
S-B
a
•8 §
S 4J
" P
0) »H
ra -u
*§
- o
rt
5 §
•H
4J ^J
O O
iH M
CM 3
Q3Z 1 1
NO
&
•H
451
-------
Cone. ID 1 2 3 4 5 678
Cone. Tested 0 .05 .10. .15 ..20 .40 .60 .80
Response
Response
Response
l
2
3
.97
.90
1.00
1
1
.00
.00
.98
1.00
.97
.99
.98
.96
.97
.94
.88
.97
.43
.63
. .46
.02
.01
.09
0
0
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
ID Replicates
1
2
3
4
5
6
7
8
3
3
3
3
3
3
3
3
Concentration
%
0
0
0
0
0
0
0
0
.000
.050
.100
.150
.200
.400
.600
.800
Response
Means
0.
0.
0.
0.
0.
0.
0.
0.
957
993
987
970
930
507 .
040
000
Std. Pooled
Dev. Response Means
0.
0.
0.
0.
0.
0.
0.
0.
051
012
015
010
046
108
044
000
0
0
0
0
0
0
0
0
.979
.979
.979
.970
.930
.507
.040
.000
The Linear Interpolation Estimate:
0.2925 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Expanded Confidence Limits:
Resampling time in Seconds:
0.2917 Standard Deviation: ' 0.0141
Lower: 0.2739 Upper: 0.3241
Lower: 0 . 2533 "Upper: 0.3589
0.22 Random Seed: -25579058
Figure 10. ICPIN program output for the IC25.
452
-------
TABLE 17. SINGLE LABORATORY PRECISION OF THE SEA URCHIN,
STRONGYLOCENTROTUS PURPURATUS FERTILIZATION TEST
PERFORMED IN SEAWATER USING GAMETES FROM ADULTS
MAINTAINED IN SEAWATER AFTER BEING COLLECTED FROM
NATURAL POPULATIONS WITH COPPER (CU jLtG/L) SULFATE .AS
THE REFERENCE TOXICANT
Test Number
1
2
3
4
5
6
Mean
CV{%)
NOEC (/ig/L)
6.9
23.0
11.2
16.0
15.3
10.8
EC25 (Atg/L)
9.7
26.2
19.6
16.4 :
17.8
18.6
18.1
29.0 ;
EC50 (Aig/D
14.3 ",'.' .
30.9
25.8 .
31.1
24.6
28.3
25.8
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 concentrations were 5, 8, 12, 17, 25, 35, and 50
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 AlG/L) SULFATE AS THE REFERENCE TOXICANT
Test
Date
7/11/94
7/14/94
7/17/94
7/19/94
Mean
SD
CV(%)
Test
Number
1*
2**
3***
1*
2**
3***
1*
2**
3***
1*
2**
3***
1
2
3
overall
1
2
3
overall
1
2
3
overall
NOEC (/ig/L)
5.0
5.0
12.0
<5.0
17.0
8.0
5.0
12.0
12.0
17.0
12.0
EC25 (pig/D
9.4
14.6
16.0
16.7
19.6
23.0
15.3
13.5
13.4
12.8
18.6
13.3
13.5
16.6
16.4
3.2
3.0
4.6
24%
18%
28%
EC50 (/ig/L)
12.6
17.5
18.6
20.9
25.8
30.5
17.7
16.4
17.0
15.6
22.1
16.0
16.7
20.5
20.5
3.5
4:. 3
6.7
21%
21%
33%
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 /iG/L) SULFATE AS THE REFERENCE
TOXICANT. i
Test Number
1
2
3
4
5
Mean
CV(%)
NOEC (Atg/L)
17.0
25.0
12.0
8.0
25.0
EC25 (jig/L)
25.8
34.3
31.1
14.2
27.2
26.5
29.0
EC50 (Mg/D
31.0
41.8
43.7
19.8
30.5 .
33.4
29.0
Tests performed by Gary Chapman and Debra Denton at EPA's Pacific
Ecosystems Branch of ERL-Narragansett, Newport, OR.
Copper concentrations were nominal; nominal concentrations were
5, 8, 12, 17, 25, 35, and 50
455
-------
TABLE 20. MULTIPLE LABORATORY PRECISION OF THE SEA URCHIN,
STONGYLOCENTROTUS PURPURATUS, FERTILIZATION TEST
PERFORMED WITH COPPER (CU /-iG/L) SULFATE AS A REFERENCE
TOXICANT
Lab
A
B
C
D
E
# of Tests
3
2
6
2
6
Statistic
Mean
SD
CV(%)
Mean
SD
CV(%)
Mean
SD
CV(%)
Mean
NA
CV(%)
Mean
SD
CV(%)
EC25 (Mg/D
7.8
3.0
38%
4.0
18.0
5.4
30%
14.9
19.3
10.5
54%
# of Lab
Means
5
Statistic
Mean
SD
CV(%)
EC25
12.8
6.6
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 i
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 ± 2to. Include brine controls in tests that
use brine.
/
C. Prepare a copper reference toxicant stock solution (2,000
mg/L) by adding 5.366 g of copper chloride (CuCl2o2H2O) 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 /ug/L) plus 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 A*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 and
filling to 100-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 until
after the test samples have been analyzed.
F. Sample effluent and reference toxicant solutions for
physical/chemical analysis. Measure salinity, 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 test (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 toxicant point estimate values in
the standard quality control charts.
458
-------
APPENDIX II. USING THE NEUBAUER HEMACYTOMETER ,TO ENUMERATE SEA t"
URCHIN SPERM ,
:our
TOP
itirig grids and a coverslip. .
VIEW:
COVERSLIP
SUPPORT
LOADING NOTCH
1
i
i
1
:{
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> COUNTING GRIDS
(size exaggerated]
(see detail next page)
i
459
-------
Together, the total area of each grid (1 mm2) and the vertical
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
Area y Coverslip
Overflow Well
Loading
Notch
END VIEW THROUGH MID-CROSS SECTION:
Coverslip
Counting I Counting
Loading
Notch— *•
Area; 1
•(•Area
Overflow Well
Loading
«— Notch
460
-------
This volume of liquid and the cells suspended therein'(e.g.,
blood cells or sperm cells) represent 1/10,000th of the liquid
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 tnm3/cm3, the answer is the number of sperm in one
railliliter 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 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/mm3) (1.000 mn^/cm3) (cell count)
(number of squares counted)
Thus, with a dilution of 4000 (0.025 mL of semen: in 100 inL of
dilution water), 80 squares counted, and a count of 100, the
calculation becomes:
cells/mL = (4.000) (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 I Counting
Area-L I •!• Area
_«-. + .-IK
Overflow Well
Loading
-Notch
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 lOOx. Once the grid is properly positioned, the
microscope is adjusted to 20Ox or 40Ox, 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 preceding right to square 4, down to square 5,
left to square 8, etc. until all 16 squares are bounted.
0
* 0
1 *
0.
f
•o
8
0
•o
9 "
0" ,
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•"
2 o °>
o
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7
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0
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0
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15 ^>
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0
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4
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5
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12
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13
463
-------
Because sperm that appear on lines might be counted as being in
either square, it is important to avoid double counting or non-
counting. 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 fall on the upper and left
lines, but not on the lower or right lines:
27
54
9,2
'1 03
4tt '
o.25
24
~<>26 23
o
o29
30*
'31
52
' <
' °53
p8
5 -,
60 1 °>
20
22 021
p
^
^32 33
<>34
35
0
•51 49 48
50b .
9
0,
10 11*
18°
19
o, 13
3G*37
3«o
46 ^
& 44
47
"45
o 13
12
.014
15s"
^
t>17 V^
1° ^«
o39 " (\
^.
•o
41 42tf
•e43
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 are not 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 square.
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, 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
8
4
7
5
3
6
2
9
,1
10
11
20
21
2
9
12
19
22
3
8
13
18
23
4
7
H
17
24
5
6
15
16
25
Pattern no. 1
Pattern no. 2
Pattern no. 3
The final consideration in achieving good replicate counts is
keeping the hemacytometers and coverslips 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.
465
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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
17.1 Scope and Application
17.2 Summary of Method
17.3 Interferences
17.4 Safety
17.5 Apparatus and Equipment
17.6 Reagents and Supplies
17.7 Effluents and Receiving Water Collection,
Preservation, and Storage
17.8 Calibration and Standardization
17.9 Quality Control
17.10 Test Procedures
17.11 Summary of Test Conditions and Test
Acceptability Criteria
17.12 Acceptability of Test Results
17.13 Data Analysis
17.14 Precision and Accuracy
Appendix I Step-by Step Summary
466
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SECTION 17
GIANT KELP, 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 because 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 receiving 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
needed before acceptable results become routine.
17.2 SUMMARY OF METHOD
17.2.1 This method provides step-by-step instructions for
performing a 48-h day static non-renewal 'toxicity test using
giant kelp to determine the toxicity of substances in marine and
467
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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 contaminants 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 -- for determining salinity. ,
17.5.6 Hydrometer(s) -- for calibrating .refractometer.
468
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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 -- Analytical, capable of accurately weighing to
0.0001 g. , .
17.5.13 Fume hood -- to protect the analyst from effluent or
formaldehyde fumes. r
17.5.14 Glass stirring rods -- for mixing test solutions.
17.5.15 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). . ; ,
17.5.16 Volumetric flasks —Class A, borosilicate 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 -- PROPIPET® or equivalent.
17.5.19 Wash bottles -- for reagent water, for topping off
graduated cylinders, for rinsing small glassware and instrument
electrodes and probes. . . i
17.5.20 Wash bottles -- for dilution water.
469
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17.5.21 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.
17.5.23 Beakers, 250 borosilicate glass -- for mixing test
solutions.
17.5.24 Beakers, 1,000 mL borosilicate glass --' for holding
sporophyll blades.
17.5.25 Inverted or compound microscope -- for inspecting
zoopspores and embryonic gametophytes.
17.5.26 Hemacytometer (bright-line rbc) -- for measuring
zoospore density.
17.5.27 Counter, two unit, 0-999 -- for recording counts of
zoopspores.
17.5.28 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.
17.5.30 60 /zm NITEX® filter -- for filtering receiving water.
17.6 REAGENTS AND SUPPLIES
17.6.1 Sample containers -- for sample shipment and storage (see
Section 8, Effluent and Receiving Water Sampling, and Sample
Handling, and Sample Preparation for Toxicity Tests).
17.6.2 Data sheets (one set per test) -- for data recording
(Figures 1 and 2).
17.6.3 Tape, colored -- for labelling test chambers and
containers.
470
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17.6.4 Markers, water-proof -- for marking containers, etc.
17.6.5 Parafilm -- to cover graduated cylinders and vessels.
17.6.6 Gloves, disposable --for personal protection from
contamination. .
17.6.7 Pipets, serological -- 1-10 mL, graduated.
17.6.8 Pipet tips -- for automatic pipets.
17.6.9 Coverslips -- for microscope slides.
17.6.10 Lens paper -- for cleaning microscope optics.
17.6.11 Laboratory tissue wipes -- for cleaning and drying
electrodes, microscope slides, etc. \
17.6.12 Disposable countertop covering -- for protection of work
surfaces and minimizing spills and contamination.
17.6.13 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).
17.6.14 Membranes and filling solutions -- for dissolved oxygen
probe (see USEPA Method 360.1, USEPA, 1979), or, 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, the 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
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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-/nm-filtered natural
seawater. Hypersaline brine should be prepared from dilution
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 derived from natural seawater contains the
necessary trace metals, biogenic colloids, and some of the
472
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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 (IQOii); is used as a
diluent, the maximum concentration of effluent (;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
Salinity
t-o
0
1
2
3 ,
4
5
10
15
20
25
Brine
60
%i
43.33
44.07
44.83
45.61
46.43
47.27
52.00
57.78
65.00
74.29
Brine
70
l-c
51.43
52.17
52.94
53.73
54.55
55.38
60.00
65.45
72.00
80.00
Brine
80
&
57.50
58.23
58.97
59.74
60.53-
61.33
65.71
70 .77
76.67
83.64
Brine
90
&
62.22 :
' 62.92
63.64
64.37
j
65.12
65.88 i
70.00
74.67 ;
80.00 ;
86.15
Brine
100
&
66.00
66.67
67.35
68.04
68.75
69.47
73.33
77 . 6.5
82.50
88.00
17.6.23.3 High quality (and preferably high salinity) .seawater
should be filtered to at least 10 /mi before placing into the
freezer or the brine generator. Water should be collected on an
incoming tide to minimize the possibility of contamination.
17.6.23.4 Freeze Preparation of Brine • ;
17.6.23.4.1 A convenient container .for making HSB by freezing is
one that has a bottom drain. One liter of brine can be made from
473
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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
lOOti. It is advisable not to exceed about 701*, 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 /im 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 heat-
assisted 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.
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 detergent should be used,
followed by several (at least three) thorough reagent water
rinses.
474
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17.6.23.5.3 Seawater should be filtered to at least 10 fim before
being put into the brine generator. The temperature of the
seawater is increased slowly t.o 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 lOOSi and that the temperature does not exceed 40°C.
Additional 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 pirn 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.
17.6.23.6 Artificial Sea Salts
17.6.23.6.1 No data from giant kelp tests using,sea salts, or
artificial seawater (e.g., GP2) are available for evaluation at
this time, and their use must be considered 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 associated with discernible adverse
effects on test organisms. For this reason, it is recommended
that only the minimum necessary volume of brine a,nd 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.
17.6.23.7.2 When HSB and reagent water are used, thoroughly
mix together the reagent water and HSB before mixing in the
475
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effluent. Divide the salinity of the MSB 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&>, lOOtb 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 lOOfe,
340 mL of brine and 660 mL of reagent water are required. Verify
the salinity of the resulting mixture using a refractometer.
p ,
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 -the effluent (SE, in
fe), 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
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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 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 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
2to 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.
17.6.23.10 Brine Controls
17.6.23.10.1 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, 17.6.23.8.3) setting SE -
0, and solving for VE.
VE = VB x (SB - 34.)/(34 - SE)
If effluent salinity is essentially Ofe, 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.
17.6.24 TEST ORGANISMS
17.6.24.1 The test organisms for this method are the zoospores
of the giant kelp, Macrocystis pyrifera. ' Macrbeyjstis is the
dominant canopy forming Laminarian alga in southern and central ,
California and forms extensive subtidal forests along the coast.
Giant kelp forests support a rich diversity of marine life and
provide habitat and food for hundreds of invertebrate and
vertebrate species (North, 1971; Foster and Schiel, 1985). It
I
477
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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 reagent 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.
(%)
40
Effluent x&
800 mL
Brine (68- '
x)Sb
800 tnL
Dilution Water*
341:,
400 mL
2 . Use either serially prepared dilutions of the highest test
concentration or individual dilutions of 100% effluent.
Effluent Cone. (%)
20
10
5
2.5
Control
Effluent Source
1000 mL of 40%
1000 mL of 20%
1000 mL of 10%
1000 mL of 5%
none
Dilution Water*
(34&)
1000 mL
1000 mL
1000 mL
1000 mL
1000 mL
INDIVIDUAL PREPARATION
Effluent Cone .
(%)
40
20
10
5
2.5
Control
Effluent x&
400 mL
200 mL
100 mL
50 mL
25 mL
none
Brine (68-x)&
400 mL
200 mL
100 mL
50 mL •
25 mL
none
Dilution Water*
34&
200 mL
600 mL
800 mL
900 mL
950 mL
1000 mL
*May be natural seawater or brine-reagent water equivalent.
478
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is an appropriate toxicity test species because of its , .
availability, economic and ecological importance, history of
successful laboratory culture (North, 1976; Luning, 1980;
Kuwabara, 1981; Deysher and Dean, 1984; Linfield, 1985), and
previous use in toxicity,testing (Smith and Harrison, 1978; James
et al., 1987; Anderson and Hunt, 1988; Hunt et al., 1989;
Anderson et al., 1990). Other Laminarian alga species have
proven to be useful for laboratory toxicity testing (Chung and
Brinkhuis, 1986; Thompson and Burrows, 1984; Hopkin and Kain,
1978; see Thursby et al., 1993 for review).
17.6.24.2 Like all kelps, Macro cystis has a life cycle that
alternates between a microscopic gametophyte stage and a
macroscopic sporophyte stage. It is the sporophyte stage that
forms, kelp forests., ,. These plants produce, reproductive blades
'(sporophylls) at their base. The sporophylls develop patches
(sdri) in which biflagellate, haploid zoospores are produced.
The zoospores are released into the water column where they swim
and eventually settle onto the., bottom and germinate. The
'dioecious spores develop into either male or female gametophytes.
The male gametophytes produce flagellated gameteis which may
'fertilize eggs produqed,by the female gametophytes. Fertilized '
eggs develop into sporophytes within,12- 15 days, completing the
lifecycle.
: {'
17.6.24.3 The method described here focuses on'germination of
the zoospores and the initial growth of the developing
gametophytes. It involves the controlled release of.zoospores-.:
from the sporophyll blades, followed by the introduction of a •
spore suspension of known 'density into the test containers'. The
zoospores swim through the test solution'and eventually settle
onto glass microscope slides. The settled spores germinate by :
extruding the cytoplasm of the spore through the germ-tube into
the first gametophytic cell. This stage is often referred to as,
the '"dumbell" stage. The two endpoints measured after 48 hours
are germination/success and growth of the. embryonic gametophytes'•
, (germ-tube length) ...
17.6.24.4. Species Identification .. . ...
17.6.24.4.1 Although there is some debate over the taxonomy of-
the genus Macrocystis, Abbott and Hollenberg (1976) consider only
two species in California: M. pyrifera, and M. integrifolia. The
479
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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, creeping
holdfast. Consult Abbott and Hollenberg (1976) for a more
detailed taxonomic discussion of the two species.
17.6.24.5 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 collected from areas free
of point and non-point source pollution to minimize the
possibility of genetic or physiological adaptation to pollutants.
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 algae 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 jj-m. filtered
seawater to remove diatoms and other epiphytic organisms. The
individual blades can be gently rubbed between fingers under
480
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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 zoospores 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, PRESERVATION, AND
STORAGE
17.7.1 See Section 8, Effluent and Receiving Water Sampling,
Sample Handling, and Sample Preparation for Toxicity Tests.
17.8 CALIBRATION AND STANDARDIZATION j
I
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 1
17.10.1.1 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.
]
17.10.1.2 Effluent concentrations are expressed as percent
effluent. • ;
i
17.10.2 TEST SOLUTIONS - !
• 481 !
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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 £im 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-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 (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& MSB is used as a diluent, the maximum concentration of
effluent that can be tested will be 66% at 34lb 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 each 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 replicates. Dispense into the appropriate
effluent test chambers.
17.10.2.3 Dilution Water
17.10.2.3.1 Dilution water should be uncontaminated 1-^m-
482
-------
filtered 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. . : ••.....
17.10.2.4 Reference Toxicant Test
17.10.2.4.1 Reference toxicant tests should be conducted as
described in Quality Assurance (see Section 4.7).
17.10.2.4.2 The preferred reference toxicant for giant kelp is
copper chloride (CuCl2°2H2O). 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 /zg/L copper stock solution
by adding 0.0268 g of copper chloride (CuCl2°2H2Q) 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, 10, 18, 32, 100, and 180
/ig/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. 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.
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 isame way and test
solutions delivered to the test chambers at the same time.
Reference toxicant tests must be conducted at 34 ± 2&>.
17.10.3 RELEASE OF ZOOSPORES FOR THE TEST •
17.10.3.1 Zoospores are released by slightly desiccating 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
-------
17.10.3.2 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 /zm-filtered seawater, then placed in a one
L glass or plastic beaker filled with 0.2 /inn filtered seawater at
15-16°C. The release water should never exceed 18°C.
17.10.3.3 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 (10Ox).
17.10.3.4 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-than two 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.
17.10.3.5 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 (lOOx). 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 1-mL of 37% buffered
formalin (or acetic acid) in a test tube. Shake the sample well
before placing it on the hemacytometer.
17.10.3.6 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. To prevent
over-dilution of the test solution, this volume should not exceed
1% of the test solution volume. If this volume exceeds 1% of the
test solution volume, it should be noted in the results.
17.10.3.7 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
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the swimming zoospores as soon as they are counted. Zoospore
release and counting should be done in a room separate from that
used for toxicant preparation, and care should be taken to .avoid
contaminating the zoospores prior to testing.
i
17.10.4 START OF THE TEST •
i
17.10.4.1 Prior to Beginning the Test , ,
17.10.4.1.1 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 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.Test).
17.10.4.1.2 Just prior to test initiation (approximately 1 h),
the temperature of a sufficient quantity of the sample to make
the test solutions should be adjusted to the test temperature (15
+ 1°C) and maintained at that temperature during the addition of
dilution water. . ' |
17.10.4.1.3 Increase the temperature of the water bath, room, or
incubator to the required test temperature (15 ± 1°C) . .
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. 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
gametophyte spores have been examined at the end of the test.
17.10.4.1.5 Note: Loss of the randomization shelet would
invalidate the test by making it impossible to analyze the data
afterwards. Make a copy of the randomization she'et and store
'
485 ' i
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separately. Take care to follow the numbering system exactly
while filling chambers with the test solutions.
17.10.4.1.6 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.
17.10.4.2 Estimation of Zoospore Density
17.10.4.2.1 After determining the zoospore density and
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.
17.10.4.2.2 Incubate the developing gametophytes for 48 hours in
the test chambers at 15°C under 50 /xE/m2/s. The zoospores
germinate and develop to the "dumbell" gametophyte stage during
the exposure period.
17.10/5 LIGHT, PHOTOPERIOD, SALINITY AND TEMPERATURE
17.10.5.1 The lights used in this method are cool white
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 /iE/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.
17.10.5.2 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 the test cups
should be as deep as possible without floating the chambers.
15.10.5.3 The test salinity should be in the range of 34 ± 2tb.
The salinity should vary by no more than ±2lo 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
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15.10.5.4 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. ,
17.10.6 DISSOLVED OXYGEN (DO) CONCENTRATION j '
17.10.6.1 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 Water1 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.
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 the exposure
period in one test chamber at each test 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 test chamber at each •
concentration and in the control. Temperature should also be
monitored continuously or observed and recorded daiily 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.
i
i
17.10.7.1.3 Record all the measurements 'on the da'ta sheet.
17.10.8 TERMINATION OF THE TEST !
487
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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 replicates 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 material as a preservative,
study the material data safety sheets from the supplier and
488
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follow strict safety precautions. Make sure test chambers and
solutions contaminated with this material are disposed 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 the1test, reading can
begin after 45 hours and must be completed within six hours.
Remove the slide without decanting the test solution. The test
slide can be lifted from the bottom of the test chamber with a
separate clean microscope slide. Blot the bottom on a paper
towel and place an 18-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, l
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 determine whether
they are circular (non-germinated) or have a protuberance that
extends at least one spore diameter (about 3.0 /j;m) from the edge
of the spore (germinated). Spores with a germination tubes less
than one spore diameter are considered non-germinated. •
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
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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 the 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 102OOE, Standard Methods 17th
edition, for micrometer/microscope calibration procedures). In
situations where germination is significantly 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 germ-
tube 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% in the
controls.
(2) Mean germination-tube length in the.controls must be at
least 10 /im in the controls.
490
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(3) The germination-tube growth NOEC must be below 35 £tg/liter
in the reference toxicant test.
(4) The minimum significant difference (%MSD) is <20% relative
to the control for both germination and germ-tube length in
the reference toxicant test.
TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY
CRITERIA FOR GIANT KELP, MACROCYSTIS PYRIFERA,
GERMINATION AND GERM-TUBE LENGTH TEST WITH EFFLUENTS
AND RECEIVING WATERS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12. .
13.
14.
Test type:
Salinity:
Temperature :
Light quality,.- ,
Light intensity:
Photoperiod:
Test chamber size:
Test solution volume:
Spore density per test
chamber :
No. replicate chambers
per concentration:
Dilution water:
Test concentrations:
Dilution factor:
Test duration:
Static non-renewal.
34 ± 2&. ' :
15 ± 1°C • .
Ambient laboratory illumination ,
50 ± 10 /LiE/m2/s
16 h light, 8 h darkness
600 mL '
200 mL/replicate
7500/mL of test solution
5 ' ';
Uncontaminated l-/itn- filtered natural
seawater or hyper saline brine
prepared from natural seawater
Effluents: Minimum of 5 and a
control 1
Receiving waters: ;100% receiving
water and a control
Effluents : *0 . 5 ;
Receiving waters: None or ^0.5
48 h
491
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15. Endpoints:
Germination and germ-tube length
16. Test acceptability
criteria:
^70% germination in the controls;
^10 jLtm germ-tube length in the
controls and the NOEC must be below
35 /xg/L in the reference toxicant
test; must achieve a %MSD of <20 for
both germination and germ-tube
length in the reference toxicant.
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
Toxicity Tests)
18. Sample volume
required:
2 L per test
17.13 DATA ANALYSIS
17.13.1 GENERAL
17.13.1.1 Tabulate and summarize the data.
sample set of germination and growth data.
Table 4 presents a
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 arfe
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 and program output.
17.13.1.3 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.
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 germination in any of the
test chambers are excluded from statistical analysis of the NOEC
and LOEC, but included in the estimation of the 1C endpoints.
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, Dunnett's
Procedure, or a nonparametric test. Steel's Many-one Rank Test,
on the arc sine square root transformed data. Underlying
assumptions of Dunnett's Procedure, normality and homogeneity of
variance, are formally tested. The test for normality .is the
Shapiro-Wilk's Test, and Bartlett's Test is used to test for
homogeneity of variance. If either of these tests fails, the
nonparametric test, Steel's Many-one Rank Test, is used to
determine the NOEC and LOEC endpoints. If the assumptions of
Dunnett's Procedure are met, the endpoints are estimated by the
parametric procedure.
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 adjustment is the
nonparametric alternative.
493
-------
TABLE 4. DATA FROM GIANT KELP, MACTOCYSTIS PYRIFERA GERMINATION AND
GROWTH TEST
Copper Cone.
(fig/D
Control
5.6
10.0
18.0
32.0
56.0
100.0
180.0
Replicate
Chamber
1
2
3
4
5
1
2
3
4
5
1
2
'3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
, 4
5
1
2
3
4
5
Number
Counted
100
100
100
100
100
100
100
100 ,
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Number
Germinated
89
88
85
89
91
82
55
84
96
85
90
90
70
83
87
88
52
83
54
49
71
82
86
81
82
84
68
62
80
83
66
72
63
72
71
37
69
0
32
48
Proportion
Germinated
0.89
0.88
0.85
0.89
0.91
0.82
0.55
0.84
0.96
0.85
0.90
0.90
0.70
0.83
- 0.87
0.88
0.52
0.83
0.54
0.49
0.71
0.82
0.86
0.81
0.82
0.84
0.68
0.62
0.80
0.83
0.66
0.72
0.63
0.72
0.71
0.37
0.69
0.00
0.32
0.48
Mean
Length
19.58
18.75
19.14
16.50
17.93
18.26
16.25
16.39
18.70
15.62
13.31
18.92
15.62
14.30
15.29
18.59
12.88
16.28
15.38
19.75
12.54
10.67
15.95
12.54
11.66
11.44
11.88
11.88
11.00
11.55
7.92
7.59
8.25
9.13
8.80
6.49
7.25
--
7.63
8.13
494
-------
GERMINATK3N PROPORTION
ENDPO1NT ESTIMATE
,l"C25 1
EQUAL NUMBER OF
t TESTwrm.
BONFERRONI
ADJUSTlMENf
BONFtiRRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOE&tOEG
Figure 1. Flowchart for statistical analysis of giant kelp,
Macrocystis pyrifera, germination data.
495
-------
17.13.2.4 Example of Analysis 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:
n _
D = £ (X.-X)2
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 :
<; X(2) <: . . . <: X(n)
496
-------
TABLE 5. GIANT KELP, MACTOCYSTIS PYRIFERA GERMINATION DATA
COPPER
CONCENTRATION
(jug/L)
Control
5.6
10.0
18.0
32.0
56.0
100.0
180.0
REPLICATE
CHAMBER
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
RAW .
DATA
0.89
0.88
0.85
0.89
0.91
0.82
0.55
0.84
0.96
0.85
0.90
0.90
0.70
0.83
0.87
0.88
0.52
0.83
0.54
0.49
0.71
. 0.82
0.86
0.81
0.82
0.84
0.68
0.62
0.80
0.83
0.66
0.72
0.63
0.72
0.71
0.37
0.69
0.00
0.32
0.48
ARC SINE
SQUARE ROOT
TRANSFORMED
1.233
1.217
1.173
1.233
1.266
1.133
0.835
1.159
1.369
1.173
1.249
1.249
0.991
1.146-
1.202
1.217
0.805
1.146
0.825
0.775
1.002
1.133
1.187
1.120
1.133
1.159
0.970
0.907
1.107
1.146
0.948
1.013-
0.917
1.013
1.002
0.654
0.980
0.050
0.601
0.765
MEAN
i Y S?
1 1.224 0.00114
1
21 1.134 0.03670
|
3! 1.167 0.01152
1
4 0.954 0.04423
5i 1.115 0.00466
|
6! 1.058. 0.01272
j
7, 0.979 0.00191
'
:
;
8 0.610 0.11914
1
497
-------
CD
-W_V_
AA~~A~
•X-
O
8
to
in
. P
(0
-H
tn
4-1 (U
O >
Q)
CQ rH
a
O 4J
•H a
tJ I
O J->
a nj
O 0)
o u
•H m
Q) [«
Cn M
, co K
T- o o d
O
O4 CQ
•H
. -U
CN JO
(U O
M O
iq v. co eg
o o o o
NOIlHOdOMd
498
-------
TABLE 6. CENTERED OBSERVATIONS FOR SHAPIRO-WALK'S EXAMPLE
Conner Concentration (ucr/L)
Rep
1
2
3
4
5
Control
0
-0
-0
0
0
.009
.007
.051
.009
.042
-0
-0
0
0
0
5.6
.001
.299
.025
.235
.039
10.0
0.
0.
-0.
-0.
0.
082
082
176
021
035
0
-0
0
-0
-0
18.0
.263
.149
.192
.129
.179
32.0
-0.
0.
0.
0.
0.
113
018
072
005
018
0
-0
-0
0
0
56.0
.101
.088
.151
.049
.088
1.00.0
-0
0
-0
0
0
.031
.034
.062
.034
.023
180.0
0.044
0.370
-0.560
-0.009
0.155
TABLE 7. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK1S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
XU>
-0.560
-0.299
-0.179
-0.176
-0.151
-0.149
-0.129
-0.113
-0.088
-0.062
-0.051
-0.031
-0.021
-0.009
-0.007
-0.001
0.005
0.009
0.009
0.018
i
21
22
. 23
24
25
26
27
28
29
30
3'1
32
33
34
35
36
37
38
39
40
0.018
0.023
0.025
0.034
0.034
0.035
0.039
! 0.042
0.044
0.049
0.072
0.082
0.082
0.088
0.101
0,155
' 0.192
0.235
0.263
0.370
Where X(i) is the ith ordered observation.
observations are listed in Table 7.
499
These ordered
-------
17.13.2.5.5 From Table 4, Appendix B, for the number of
observations, n, obtain the coefficients alf a2, . . . . , ak where k
is n/2 if n is even and (n-l)/2 if n is odd. For the data in
this example, n = 40 and k = 20. The a± values are listed in
Table 8.
17.13.2.5.6 Compute the test statistic, W, as follows:
W - -[Eax'"-1'1'-*'11)]
The differences x(n-i+1) - X(i) are listed in Table 8. For this data
in this example:
W = 1 (0.9230)2 = 0.918
0.9281
TABLE 8. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK1S EXAMPLE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0.3964 .
0.2737
0.2368
0.2098
0.1878
0.1691
0.1526
0.1376
0.1237
0.1108
0.0986
0.0870
0.0759
0.0651
0.0546
0.0444
0.0343
0.0244
0.0146
0.0049
0.930
0.562
0.414
0.368
0.306
0.250
0.217
0.195
0.170
0.134
0.100
0.075
0.063
0.048
0.042
0.035
0.029
0.016
0.014
0.000
x«o>
X(39)
X<36)
x<">
x(36)
X(35)
X<34>
X<33)
X<32>
X<31)
X(30)
X(29)
X(28)
X(27)
X<26>
X'25'
X(24,
X'23'
x(22)
X'21'
- x'1' •
- X'2'
- x(3)
- x(4)
- X'5'
- x(6>
- X'7'
- X'8'
- x(9)
- x<10'
- x(11)
- x<12>
- x<13'
- x(14)
- X(1S)
- x<16'
- X'17'
- X'18'
_ x(19)
- X'20'
500
-------
TABLE 9.
ASSIGNING RANKS TO THE CONTROL AND 5.15
CONCENTRATION LEVEL FOR STEEL'S MANY-ONE RANK TEST
Rep.
1
2
3
4
5
Rep.
1
2
3
4
5
Rank
1
2
3
4.5
4.5
6
7.5
.7.5
9
10
Control
1.233 (7.5,6.5,8.
1.217(6,5,6.5,7,
1.173 (4.5,3,5,5,
1.233(7.5,6.5,8.
1.266(9,10,10,10
18.0
1.217(6.5)
0.805(2)
1.146(4)
0.825(3)
0.775(1)
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 RAE
5,8.5,8.5,8.5,8.5)
7,7,7)
6,6,6)
5,8.5,8.5,8.5,8.5)
,10,10,10)
Concentration (/zg/L)
32.0 56.0
1.002(1) 1.159(5)
1.133(3.5) 0.970(2)
1.187(6) 0.907(1)
1.120(2) 1.107(3)
1.133(3.5) 1.146(4)
Concentration
5.6 /zg/L
5.6 jug/L
5 . 6 /zg/L
5.6 /zg/L
Control
Control
Control
.. Control
Control
5.6 /ig/L
IKS1 l
Concentration (/zg/L)
.5.6 , 10.0
1.133(2) ; 1.249(8.5)
0.835(1) 1.249(8.5)
1.159(3) 0.. 991(1)
1.369(10) 1.146(2)
1.173(4,5) 1.202(4)
(Continued)
100.0 180.0
0.948(2) 0.654(3)
1.013 (4.5) 0.980(5)
0.917(1) 0.050(1)
1.013(4.5) 0.601(2)
1.002(3) 0.765(4)
1Control 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 0.919. Since W = 0.918 is less
than the critical value, conclude that the data are not- 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 observations in order of size
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
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 /ig/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 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, the rank sum at that concentration
would be significantly lower than the rank sum of the control.
Thus compare the rank sums for the survival at each of the
various concentration levels with some "minimum" or critical rank
sum, at or below which the survival would be considered
significantly lower than the control. At a significance level of
0.05, the minimum rank sum in a test with seven concentrations
(excluding the control) and five replicates is 16 (See Table 5,
Appendix E).
502
-------
17.13.2.6.4 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 ng/Ii concentrations are less than the critical
value, the germination proportions in those concentrations are
considered significantly less than that in the control. Hence,
the NOEC and the LOEC are considered to be 18.0 M9/L and.32.0
respectively.
TABLE 11. RANK SUMS
Concentration Rank Sum
5
10
18
32
56
100
180
.6
.0
.0
. 0
•°
.0
.0
20
'24
16
16
15
15
15
.5
.0
.5
.0
. 0
.0
.0
17.13.2.7 Calculation of the ICp ; '
17.13.2.7.1 The germination data from Table 4 and Figure 2 are
utilized in this example. As can be seen fromithe figure, the
observed means are not monotonically non-increaising with respect
to concentration. Therefore, the means must be smoothed prior to
calculating the 1C. .. , . . ,
17.13.2.7.2 Starting with the observed control, mean, Yj. = 0.884
is less than the observed mean for the lowest effluent
concentration, Y2 = 0.804, so set Mx = 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: , .
M2 = M3 = (Y2 + Y3)/2 = 0.822
503
-------
17.13.2.7.5 Since M3 is larger than Y4 = 0.652, set M4 = 0.652.
Since Ts = 0.804 is larger than M4, these means must be smoothed.
17 . 13 . 2 . 7 . 6 Calculate the smoothed means :
M4 = M5 = (Mi + Y5)/2 = 0.728.
17.13.2.7.7 Since Ye = 0.754 is larger than M5, average T6 with
the two previous concentrations ;
M4 = M5 = M6 = (M4 + M5 + Y6)/3 = 0.737.
17.13.2.7.8 Since M6 > Y7 = 0.688 > Y8 = 0.372, set M7 = 0.688
and M8 = 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 Mj.(l-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 ^tg/L and C8 = 180.0
17.13.2.7.10 Using the equation in Section 4.2 from Appendix L,
the estimate of the IC25 is calculated as follows:
( c -C )
ICp * CfA
IC25 - 100.0 + [0.884(1 - 25/100) - 0.688] (180.0 - 100.0)
(0.372 - 0.688)
= 106.3 pg/L.
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 Mg/L. The empirical 95.0% confidence interval for the
true mean was 94.6667 /xg/L to 117.0588 ^ig/L. The computer
program output for the IC25 for this data set is shown in Figure 4
504
-------
4J
0)
CJ
o$
8
O
5-1
yy y
AA A
-S
0> OB h- <0 10 f
o o o o o o
NO uyodoyd NO IIVN
o
43
4J
O
10
Tl
0)
Cn
CO (Q
•o S,
* a
co a (0
H rH
-------
Cone . ID
Cone. Tested
Response
Response
Response
Response
Response
1
2
3
4
5
1
0
.89
.88
.85
.89
.91
2
5.6
.82
.55
.84
.96
.85
3
10
.90
.90
.70
.83
.87
4
18
.88
.52
.83
.54
.49
5
32
.71
.82
.86
.81.
.82
6
56
.84
.68
.62
.80
.83
7
100
.66
.72
.63
.72
.71
8
180
.37
.69
0
.32
.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
l
2
3
4
5
6
7
8
Number
Replicates
5
5
5
5
5
5
5
5
Concentration
ug/L
0.000
5.600
10.000
18.000
32.000
56.000
100.000
180.000
Response
Means
0 . 884
0.804 •
0.840
0.652
0.804
0.754
0.688
0.372
Std. Pooled
Dev. Response Means
0.022
0.152
0.083 v
0.187
0.056
0.098
0.041
0.252
0.884
0.822
0.822
0.737
0.737
0.737
0.688
0.372
The Linear Interpolation Estimate: 106.3291 Entered P Value: 25
Number of Resamplings: 80 •
The Bootstrap Estimates Mean: 105.8680 Standard Deviation: 5.6981
Original Confidence Limits: Lower: 94.6667 Upper: 117.0588'
Expanded Confidence Limits: Lower: 88.8354 Upper: 122.4237
Resampling time in Seconds: 0.28 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 statistical analysis of the growth data is
outlined in Figure 5. The response used in the statistical ' •
analysis is mean germ-tube length per replicate. An IC25 can be
calculated for the growth data via a point estimation technique
(see Section 9, Chronic Toxicity Test Endpoints and Data
Analysis). Hypothesis testing can be used to obtain the NOEC 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's Procedure, normality and homogeneity
of variance, are formally tested. The test for normality is the
Shapiro-WiIk's Test and Bartlett's Test is used to test for
homogeneity of variance. If either of these tegsts 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.
17.13.3.3 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.
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 provided in Figure 6.
17.13.3.5 Test for Normality ' ;
17.13.3.5.1 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.
507
-------
MEAN GERM-TUBE LENGTH
J>'" '
\
POINT ESTIMATION :
- :-^. -
-:••--—-- ... U
>\_ /, i
I
i ;,fl r
ENDPOJNT ESTIMATE'
IC25
;Alb>'iNE|q«l)yRE'ROOf
I
NORMAL DISTRIBUTION
-i'? ' -I
RIBUTION I
.>v-4.>j,.,":; '.^
hHOMOGENEOUS VARIANCE
NO
EQUAL NUMBER OF
REPLICATES?
YES
1 TEST WITH
BOMFERRONI
AOJOSTMENT'
DUNNETTS
TEST
BARTLETTSTEST
STEEL'S MANY-ONE
r „ , <~ tn .i.'-s^^'"1 ^^*- -, !•*.. ~,i
,,, 4, Jjl.,1,lg,l.r'si!,'^i«4 t'!' >-
.•.-'..--:-j-g4.. ^ r .i yajiggj.fe;BJfci?...::.
Figure 5. Flowchart for statistical analysis of giant kelp,
MacrocystiB pyrifera, growth data.
508
-------
TABLE 13. GIANT KELP, MACROCYSTIS PYRIFERA, GROWTH DATA
Copper Concentration
Rep
1
2
3
4
5
Meanf^)
si
i
Control
19.58
18.75
19.14
16.50
17.93
18.38
1.473
1
5.60
18.26
16.25
16.39
18.70
15.62
17.04
1.827
2
10.0
13.31
18.92
15.62
14.30
15.29
15.49
4.498
3
18
18
12
16
15
19
16.
.0
.59
.88
.28
.38
.75
58
7.327
4
32
12
10
15
12
1-1
12.
.0
.54
.67
.95
.54
.66
67
3.953
5
56
11
' 11
11
11
11
11.
.0
.44
.88
.88
.00
.55
55
0.133
6
, 100.0
7.92
7.59
, 8.25
, 9.13
8.80
8.34
0.396
7
180.0
6.49
7.25
--
7.63
8.13
7.38
0.478
8
TABLE 14. CENTERED OBSERVATIONS FOR SHAPIRO-WILK1S EXAMPLE
Copper Concentration
Rep Control
5.6
10.0
18.0
32.0
56.0
100.0
180.0
1
2
3
4
5
1.20
0.37
0.76
-1.88
-0,45
1.22
-0.79
-0.65
1.66
-1.42
-2.18
3.43
0.13
-1.19
-0.20
2.01
-3.70
-0.30
-1.20
3.17
-0.13
-2.00
3.28
-0.13
-1.01
-0.11
0.33
0.33
-0.55
0.00
-0.42
-0.75
-0.09
0.79
0.46
-0.89
-0.13
--
0.25
0.75
17.13.3.5.2 Calculate the denominator, D, of the statistic:
T;» 2
D = £ (XfX)
i-l
Where: X£ = 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
X(1) s X'2' <: . . . <; X(n)
where X(i> 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 ' X'1' i X'1'
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
-3.70
-2.18
-2.00
-1.88
-1.42
-1.20
-1.19
-1.01
-0.89
-0.79
-0.75
-0.65
-0.55
-0.45
-0.42
-0.30
-0.20
-0.13
-0.13
-0.13
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
-0.11
-0.09
0.00
0.13
0.25
0.33
0.33
0.37
0.46
0.75
0.76
0.79
1.20
1.22
1.66
2.01
3.17
3.28
3.43
510
-------
H-
IIS
5T i-1
0 %
3
O
I
rt rt
(D V
10
rt HI
CO O
rt
ff
rt
(D
en
in
1
I
GERM-TUBE LENGTH
-------
17.13.3.5.5 From Table 4, Appendix B, for the number of
observations, n, obtain the coefficients ax/ a2, ... ak where k is
n/2 if n is even and (n-l)/2 if n is odd. For the data in this
example, n ~ 39 and k = 19. The ai values are listed in
Table 16.
17.13.3.5.6 Compute the test statistic, W, as follows:
W. -
D
The differences x
X(34)
X<33>
X<32)
X(3D
X(30)
X(29)
X(28)
x(27)
X(26)
X(25)
X(24)
X(23)
X(22)
X(21)
- X'1'
- X121
- X'3'
- X'4>
- X'5'
- X'6'
- x(7>
- x<8)
- x(9)
- x<10)
- x(11)
- x(12>
- X'"'
- X'14'
- x(15>
- x<161.
- x(17)
- x(18)
- x(19)
512
-------
17.13.3.5.7 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
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 Bartlett's Test (Snedecor and
Cochran, 1980). The test statistic is as follows:
[ (EV..) In S2 - EV.. In S2]
B =
i-l
i-l
Where: Vi =
p =
degrees of freedom for each copper concentration
and control, V± = (n± - 1)
number of concentration levels including the
control
In = loge
i = 1,2, ..., p where p is the number of
concentrations including the control
n± = the number of replicates for concentration i.
(Ev.s2)
772 i-1 ,
E
i-l
fE
i-l
513
-------
17.13.3.6.2 For the data in this example (See Table 13), all
concentrations including the control have five replicates except
the 180 /zg/L concentration which has four replicates (ni = 5 for
i = 1 - 7; ns = 4) . Thus, V± = 4 for i = 1 - 7 and V8 = 3.
17.13.3.6.3 Bartlett's statistic is therefore:
p
B - [ (31) In (2. 5761) - I) VjlnCSJ?) ] / 1.0977
[31(0.9463) - [4ln(1.4729) + ... + 3ln(0l4780)] / 1.0977
= [29.3353 - 9.4481] / 1.0977
=18.12
17.13.3.6.4 B is approximately distributed as chi-square with p
- 1 degrees of freedom, when the variances are in fact the same.
Therefore, the appropriate critical value for this test, at a
significance level of 0.01 with seven degrees of 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's Adjustment ,
17.. 13.3.7.1 To obtain an estimate ,of the pooled variance for the
t test with Bonferroni's adjustment, construct an ANOVA table as
described in Table 17. . ... .
TABLE 17. ANOVA TABLE
Source df
Between p - 1
Within N - p
Sum of Squares
(SS)
SSB
ssw .
Mean Square (MS)
(SS/df)
2
SB = SSB/(p-l)
2
Sw = SSW/(N-p)
Total N - 1 SST
Where: p = number of concentration levels including the
control
514
-------
N = total number of observations nx + n2 ... + np
ni = number of observations in concentration i
SSB = Z,Tf/nrG2/N Between Sum of Squares
ssr = £Eir2.-G2/w Total Sum of Squares
ssw = SST-SSB Within Sum of Squeires
G = the grand total of all sample observations,
G = £ r_.
Ti = the total of the replicate measurements for
concentration i ' ' '
Yy = the jth observation for concentration i
(represents the mean length of the germ-tubes for
concentration i in test chamber j)
17.13.3.7.2 For the data in this example:
nx = n2 = n3 = n4 •= ns = ns = n7 = 5; ns = 4
N = 39 . v - - '•'•'•
TX = Ylx + Y12 + Y13 +'Y14 + 'Y16 = 91.90
T2 = Y2i + Y22 + Y23 + Y24 + YSS = 85 .22
T3 = Y31 + Y32 + Y33 -l- Y34 + Y3S. = 77.44
T4 = Y41 + Y42 + Y43 + Y44 + Y4S = 82.88
T5 = YS1 +. Y52 + Y53 + Y54 + Y5s = 63.36
T6 = Y61 + Y62 -i- Y63 + Y64 + Y6S = 57.75
T7 = Y71 + Y72 + Y73 + Y74 +.Y75 = 41.69
T8 = Y81 + Y82 + Y83 +-Y84 = 29.50
G = T! + T2 + T3 + .T4 + Ts + T6 + |T7 + T8 = 529.74
p
SSB = ETf/n.L-G2/N
515
-------
= 7749.905 - (529. 74) 2 = 554.406
.39
p
SST - £
= 7829.764 - (529.74)2 = 634.265
39
SSff . SST-SSB = 634.265 - 554.406 = 79.859
= SSB/(p-l) = 554,406/(8-l) = 79.201
g = 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
Between
Within
Total
df
7
31
38
Sum of Squares
(SS)
554.406
79.859
634.265
Mean Square
(SS/df)
79.201
2.576
(MS)
17.13.3.7.4 To perform the individual comparisons, calculate the
t statistic for each concentration,, and control combination as
follows :
t,
(I/n.)
516
-------
Where: Y± = mean length for concentration i
Y! = mean length for the control
Sw = square root of the within mean square
n-L = number of replicates for the control
nj = number of replicates for concentration i
17.13.3.7.5 Table 19 includes the calculated tlvalues for each
concentration and control combination. In this example,
comparing the 5.6 ptg/L concentration with the control, the
calculation'is as follows: .
(18.38 - 17.04 )
[ 1. 605 Vd/5) -(1/5) ]
= 1.320.
TABLE 19. CALCULATED t VALUES
Concentration (ptg/L)
5.6
10.0'
18.0
32.0
56.0
100.0
180.0
i
2.
3
4
5
6
1
8
" i tt
1.320
,2.847
,1.773
5 . 625
6.728
9.891
10.836
\
\
17.13.3.7.6 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 5, Appendix D. 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
weight for concentration "i" is considered significantly less
than the mean weight for the control if t£ is greater than the
critical value. Therefore, the 10.0 /ig/L, 32 /ug/L, 56.'0
e, '
I •
517
-------
100.0 /ig/L, 180.0 ^tg/L concentrations have significantly lower
mean length than the control. Because the 10.0 /zg/L
concentration shows signigicantly lower mean length than the
control while the higher 18.0 /ig/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 /Kj/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 1C
17.13.3.8.2 Starting with the observed control mean, Yx = 18.38
is greater than the observed mean for the lowest copper
concentration, Y2 = 17.044, so set Mx = 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: •
M3 = M4 = (Y3 + YJ/2 = 16.032 ,
17.13.3.8.5 Since M4 > Ys = 12.672 > Y6 = 11.550 > Y7 = 8.338 > Y8
= 7.375, set Ms = 12.672, M6 = 11.550, M7 = 8.338 and M8 = 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.
(Aig/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
YJ. (mm)
18.380
17.044
15.488
16.576
12.672
11.550
8.338
7.375
Smoothed
Means
Mi (ram)
18.380
17 . 044
16.032
16.032
12.672
11.550
8.3,38
7.375
17.13.3.8.7 Using the equation in Section 4.2 from Appendix L,
the estimate of the IC25 is calculated as follows:
ICp = Cj+[Ml(I-p/IOO)-Mj]
IC25 = 18.0 + [18.380(1 - 25/100) - 16.032] J (32.0 - 18.0)
1(12.672' -16.032)
= 27.36
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 M-td-
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 /zg/L and C5 = 32.0
519
-------
i
-H
tn •
o
, -H
CO H
A N
O Q<
- to
•nj -H
JJ -U
«l
§ p
rt ^
K< O
o
4->
O
•H
520
-------
17.13.3.8.8 When the ICPIN program was used to analyze this set
of data, requesting 80 resamples, the estimate ,of the XC25 was
27.3625 ng/li. The empirical 95.0% confidence interval for the
true mean was 20.8734 /ig/L to 42.3270 /xg/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 germination ECSOs
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 ECBOs 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 ICBOs 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 chloride are
provided in Table 23. The coefficient of variation of the ICSOs
for the germ-tube length endpoint ranged .betweeiti 8.4% and 55.5%
using copper chloride. The coefficient of variation of the IC50s
for the germination endpoint ranged between >!.!% 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
Cone . Tested
Response
Response
Response
Response
Response
1
2
3
4
5
19
18
19
16
17
1
0
.5818
.7516
.1416
.5018
.9315
2
5.6
.2613
.2518
.3915
.7014
.6215
3
10
.3118
.9212
.6216
.3015
.2919
4
18
.5912.
.8810.
.2815.
.3812.
.7511.
5
32
5411
6711
9511
5411
6611
6
56
.44
.88
.88
.00
.55
7
7
8
9
8
7
100
.92
.59
.25
.13
.80
8
180
6.49
7.25
7.63
*** 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
1
2
3
4
5
6
7
8
Number
Replicates
5
5
5
5
5
5
5
4
Concentration
ug/L
0.000
5.600
10.000
18.000
32.000
56.000
100.000
180.000
Response
Means
18.380
. 17.044
15.488
16.576
12.672
11.550
8.338
7.3.75
Std.
Dev. I
1.214
1.352
2.121
2.707
1.988
0.365
0.629
0,691
Pooled
lesponse Means
18.380
17.044
16.032
16.032
12.672
11.550
8.338
7.375
The Linear Interpolation Estimate:
27.3625 Entered P Value: 25
Number of Resamplings: 80
The Bootstrap Estimates Mean:
Original Confidence Limits:
Expanded Confidence Limits:
Resampling time in Seconds:
27.5292 Standard Deviation: 4.7812
Lower: 20.8734 Upper: 42.3270
Lower: 17.6289 Upper: 49.8093
0.28 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 (CU jiG/L) CHLORIDE AS THE REFERENCE
TOXICANT
Test Number
1
2
3
4
5
Mean
CV
Germ-Tube Length
NOEC
<5.6
10.0
18.0
5.6
32.0
IC40
122.7
43.1
70.7
88.0
124.7
89.8
38.8%
i
NOEC
10.0
18.0
18.0
56.0
56.0
r-
Germination
EC50
67.5
73.5
124.3
101.6
122.9
90.7
32.9%.
Data from Anderson et al., 1994 ;
TABLE 22. SINGLE LABORATORY PRECISION OF THE GIANT KELP,
MACROCYSTIS PYRIFERA GERMINATION AND GERM-TUBE LENGTH
TEST WITH SODIUM AZIDE (MG/L) AS THE REFERENCE
TOXICANT , .
Test Date
2/11/92
2/18/92
6/29/92
7/07/92
7/15/92
7/16/92
7/22/92
10/09/92
7/02/92
Mean
CV
NOEC
18.0
18.0
32.0
10.0
18.0
5.6
10.0
5.6
10.0
Germ- Tube Length
IC25 IC50
39.5 133.7
34.1 96.5
57.5 142.2-
33.1 92.5
42.8 138.9
25.0 68.4
30.2 80.6
25.1 80.0
24.8 80.1 .
34.7 101.4
30.8% 28.4%
Germination
NOEC
18.0
32.0
32.0
10.0
18.0
10.0
18.0
5.6
18.0
.'
EC50
52.3
72.6
132.1
79.2
117.8
48.3
62.4
60.3
84.0
78.8
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 (/iG/L) AS THE
REFERENCE TOXICANT
March 1990
May 1990
May 1990
December
1990
September
1990
September
1989
November
1989
May 1988
Lab
1
2
3
1
2
3
1
2
3
1
2
1
2
1
2
1
2
1
2
Germ- tube length
NOEC IC40
5.6 122.7
32.0 117.8
18.0 104.1
10.0 43.1
<5.6 99.1
18.0 68.7
'18.0 .70.7
18.0 91.3
32.0 134.2
5.6 88.0
5.6 45.3
32.0 124.7
18.0 54.4
<10.0 89.3**
<10.0 171.8**
32.0 >180.0
10.0 >180.0
<56.0 232.0***
<56.0 *
Germination
NOEC EC 50
10.0 46.9
32.0 46.2
32.0 *
18'. 0 112.0
32.0 164.2
18.0 67.9
18.0 112.0
56.0 64.5
32.0 158.0
56.0 77.7
18-. 0 *
56.0 127.4
56.0 114.8
56.0 115.5
56.0 327.7
<10.0 >180.0
18.0 >180.0
<56.0 211.0
56.0 100.7
CV
Germ- tube
8.4%
39 . 9%
45.3%
45.3%
55.5%
44.5%
nc
nc
CV
Germi'nat
ion
>!.!%
59.3%
nc
nc
7.4%
67.6%
nc
50.0%
*' No EC50 calculated because response was less than 50%.
** 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 ;
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. !
i
C. Prepare a. copper reference toxicant stock solution (10,000
p.g/L) by adding 0.0268 of copper chloride (CuCl2o2H2O) to 1
liter of reagent water. • '.,
i
D. Prepare copper reference toxicant solution of 0 (control)
5.6, 10, 18, 32, 100 and 180 /ig/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 filling to-1-L with dilution
water. . ' .[;
E. Sample effluent and reference toxicant solutions for
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, 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 concentration. Monitor the
temperature of the water bath or environmental chamber
continuously. j
I
I. At the end of the test, measure salinity, pH, and dissolved
oxygen concentration from each test concentration.
525 i
-------
PREPARATION AND ANALYSIS OF TEST ORGANISMS
A. Collect sporophylls and rinse in 0.2 pm 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. '
P. At 48 ± 3 hours, count the number of germinated and non-
germinated spores of the first 100 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: i
Sample Source: Microscope Model:
Sample Type: Solid Elutriate Pore Water Water Effluent RefTox Micrometer Conversion Factor:
Test
Cont.
#
1
2
3
4
S
6
7
8,
9
10
ii
12
is1
14
is
16
17
18
19
20
21
22
23
i4.
25
26
27
28
29
30
31
32
33
34
35
Station
Code
Number
of Spores ,
Germ.
Number
of Spores
Not Germ.
Length Measurements (in ocular micrometer units)
LI
L2
L3
L4
L5
L6
L7
L8
Computer Data Storage
Disk:
File:
L9
L10
Notes
i
527
-------
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!
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546
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USEPA. 1991b. Methods for aquatic toxicity identification
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APPENDICES
Page
A. Independence, Randomization, and Outliers 566
1. Statistical Independence 566
2. Randomization 566
3 Outliers 572
B. Validating Normality and Homogeneity of Variance
Assumptions 575
1. Introduction 575
2. Tests for Normal Distribution of Data 575
3. Test for Homogeneity of Variance 582
4. Transformations of the Data 584
C. Dunnett's Procedure 587
1. Manual Calculations 587
2. Computer Calculations 595
D. t test with the Bonferroni Adjustment . .602
E. Steel's Many-one Rank Test 609
P. 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. Probit Analysis 627
I. Spearman-Karber Method .631
J. Trimmed Spearman-Karber Method 638
K. Graphical Method 643
564
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L. Linear Interpolation Method ;...... .648
1. General Procedure i 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
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APPENDIX A
INDEPENDENCE, RANDOMIZATION, AND OUTLIERS
1. STATISTICAL INDEPENDENCE
1.1 Dunnett's Procedure and the t test with Bonferroni's
adjustment are parametric procedures based on the assumptions
that (1) the observations within treatments are independent and ,
normally distributed, and (2) that the variance of the
observations is homogeneous across all toxicant concentrations
and the control. Of the three possible departures from the
assumptions, non-normality, heterogeneity of variance, and lack •
of independence, those caused by lack of independence are the
most difficult to resolve (see Scheffe, 1959). For toxicity
data, statistical independence means that given knowledge of the
true mean for a given concentration or control, knowledge of the
error in any one actual observation would provide no information
about the error in any other observation. Lack of independence
is difficult to assess and difficult to test for statistically.
It may also have serious effects on the true alpha or beta level.
Therefore, it is of utmost importance to be aware of the need for
statistical independence between observations and to be
constantly vigilant in avoiding any patterned experimental
procedure that might compromise independence. One of the best
ways to help insure independence is to follow proper
randomization procedures throughout the test.
2. RANDOMIZATION
2.1 Randomization of the distribution of test organisms among
test chambers, and the arrangement of treatments and replicate
chambers is an important part of conducting a valid test. The
purpose of randomization is to avoid situations where t-est
organisms are placed serially into test chambers, or where all
replicates for a test concentration are located adjacent to one
another, which could introduce bias into the test results.
2.2 An example of randomization of the distribution of test
organisms among test chambers, and an example of randomization of
arrangement of treatments and replicate chambers are described
using the 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 follows: 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 numbers. An example of this
assignment is provided in Table A.I. 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.I, 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.
{
2.3.3 The next step is to read the double digit number to the
right of the first one. The second number identifies the
replicate chamber for the second fish taken from the tank.
Continuing the example, the second number read in row 2 of Table
A. 2 is 54. According to Table A.1!, this number corresponds to
replicate chamber 4 of the 50.0% effluent concentration. Thus,
the second fish taken from- the tank is ,to be placed in replicate
chamber 4 of the 50.0% effluent concentration.,
' i
2.3.4 Continue in this fashion until all the fish have been
randomly assigned to a replicate chamber. In order to fill each
replicate chamber with ten fish, the assigned numbers will be
used more than once. .If a number is read from the table that was
not assigned to a replicate chamber, then ignore it and continue
to the next number. If a replicate chamber becomes filled and a
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 double-
digit numbers) in order to make efficient use of the random
number table (Table A.2). To illustrate, consider the assignment
of only one double-digit number to each replicate chamber: the
first column of assigned numbers in Table A.I. Whenever the
numbers 00 and 31 through 99 are read from Table A.2, they will
be disregarded and the next number will be read.
TABLE A.I. RANDOM ASSIGNMENT OF FISH TO REPLICATE CHAMBERS EXAMPLE
ASSIGNED NUMBERS FOR EACH REPLICATE CHAMBER
Assigned Numbers
Replicate Chamber
01,
02,
03,
04,
05,
06,
07,
08,
09,
10,
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40,
41,
42,
43,
44,
45,
46,
47,
48,
49,
50,
51,
52,
53,
54,
55,
56,
57,
58,
59,
60,
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
Control ,
Control ,
Control ,
Control,
Control ,
6.25% effluent,
6.25% effluent,
6.25% effluent,
6.25% effluent,
6.25% effluent,
12.5% effluent,
12.5% effluent,
12.5% effluent,
12.5% ef fluent ,-
12.5% effluent,
25.0% effluent,
25.0% effluent,
25.0% effluent,
25.0% efffSfent,
25.0% eff Merit',
50.0% effluent,
50.0% effluent,
50.0% effluent,
50.0% effluent,
50.0% effluent,
100.0% effluent,
100.0% effluent,
100.0% effluent,
100.0% effluent,
100.0% effluent,
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
568
-------
TABLE A.2. TABLE OF RANDOM NUMBERS (Dixon and Massey, 1983)
10 09 73 25 33
37 54 20 48 05
08 42 26 89 53
99 01 90 25 29
12 80 79 99 70
66 06 57 47 17
31 06 01 08 05
85 26 97 76 02
63 57 33 21 35
73 79 64 57 53
98 52 01 77 67
11 80 50 54 31
83 45 29 96 34
88 68 54 02 00
99 59 46 73 48
65 48 11 76 74
80 12 43 56 35
74 35 09 98 17
69 91 62 68 03
09 89 32 05 05
91 49 91 45 23
80 33 69 45 98
44 10 48 19 49
12 55 07 37 42
63 60 64 93 29
61 19 69 04 46
15 47 44 52 66
94 55 72 85 73
42 48 11 62 13
23 52 37 83 17
04 49 35 24 94
00 54 99 76 54
35 96 31 53 07
59 80 80 83 91
46 05 88 52 36
32 17 90 05 97
69 23 46 14 06
19 56 54 14 30
45 15 51 49 38
94 86 43 19 94
98 08 62 48 26
33 18 51 62 32
80 95 10 04 06
79 75 24 91 40
18 63 33 25 37
74 02 94 39 02
54 17 84 56 11
11 66 44 98 83
48 32 47 79 28
69 07 49 41 38
76 52 01 35 86
64 89 47 42 96
19 64 50 93 03
09 37 67 07 15
80 15 73 61 47
34 07 27 68 50
45 57 18 24 06
02 05 16 56 92
05 32 54 70 48
03 52 96 47 78
14 90 56 86 07
39 80 82 77 32
06 28 89 80 83
86 50 75 84 01
87 51 76 49 69
17 46 85 09 50
17 72 70 80 15
77 40 27 72 14
66 25 22 91 48
14 22 56 85 14
68 47 92 76 86
26 94 03 68 58
85 15 74 79 54
11 10 00 20 40
16 50 53 44 84
26 45 74 77 74
95 27 07 99 53
67 89 75 43 87
97 34 40 87 21
73 20 88 98 37
75 24 63 38 24
64 05 18 81 59
26 89 80 93 45
45 42 72 68 42
.01 39 09 22 86
87 37 92 52 41
20 11 74 52 04
01 75 87 53 79
19 47 60 72 46
36 16 81 08 51
45 24 02 84 04
41 94 15 09 49
96 38 27 07 74
71 96 12 82 96
98 14 50 65 71
77 55 73 22 70
80 99 33 71 43
52 07 98 48 27
31 24 96 47 10
87 63 79 19 76
34 67 35 43 76
24 80 52 40 37
23 20 90 25 60
38 31 13 11 65
64 03 23 66 53
36 69 73 61 70
35 30 34 26 14
68 66 57 48 18
90 55 35 75 48
35 80 83 42 82
22 10 94 05 58
50 72 56 82 48
13 74 67 00 78
36 76 66 79 51
91 82 60 89 28
58 04 77 69 74
45 31 82 23 74
43 23 60 02 10
36 93 68 72 03
46 42 75 67 88
46 16 28 35 54
70 29 73 41 35
32 97 92 65 75
12 86 07 46 97
40 21 95 25 63
51 92 43 37 29
59 36 78 38 48
54 62 24 44 31
16 86 84 87 67
68 93 59 14 16
45 86 25 10 25
96 11 96 38 96
33 35 13 54 62
83 60 94 97 00
77 28 14 40 77
05 56 70 70 07
15 95 66 00 00
40 41 92 15 85
43 66 79 45 43
34 88 88 15 53
44 99 90 88 96
89 43 54 85 81
20 15 12 33 87
69 86 10 25 91
31 01 02 46 74
97 79 01 71 19
05 33 51 29 69
59 38 17 15 39
02 29 53 68 70
35 58 40 44 01
80 95 90 91 17
20 63 61 04 02
15 95 33 47 64
88 67 67 43 97
98 95 11 68 77
65 81 33 98 85
86 79 90 74 39
73 05 38 52 47
28 46 82 87 09
60 93 52 03 44
60 97 09 34 33
29 40 52 42 01
18 47 54 06 10
90 36 47 64 93
93 78 56 13 68
73 03 95 71 86
21 11 57 82 53
45 52 16 42 37
76 62 11 39 90
96 29 77 88 22
94 75 08 99 23
53 14 03 33 40
57 60 04 08 81
96 64 48 94 39
43 65 17 70 82
65 39 45 95 93
82 39 61 01 18
91 19 04 25 92
03 07 11 20 59
26 25 22 96 63
61 96 27 93 35
54 69 28 23 91
77 97 45 00 24
13 02 12 48 92
93 91 08 36 47
86 74 31 71 57
18 74 39 24 23
66 67 43 68 06
59 04 79 00 33
01 54 03 54 56
39 09 47 34 07
88 69 54 19 94
25 01 62 52 98
74 85 22 05 39
05 45 56 14 27
52 52 75 80 21
56 12 71 92 55
09 97 33 34 40
32 30 75 75 46
10 51 82 16 15
39 29 27 49 45
00 82 29 16 65
35 08 03 36 06
04 43 62 76 59
12 27 17 68 33
11 19 92 91 70
23 40 30 97 32
18 62 38 85 79
83 49 12 56 24
35 27 38 84 35
50 50 07 39 98
52 77 56 78 51
68 71 17 78 17
29 60 91 10 62
23 47 83 41 13
40 21 81 65 44
14 38 55 37 63
96 28 60 26 55
94 40 05 64 18
54 38 21 45 98
37 08 92 00 48
42 05 08 23 41
22 22 20 64 13
28 70 72 58 15
07 20 73 17 90
42 58 26 05 27
33 21 15 94 66
92 92 74 59 73
25 70 14 66 70
05 52 28 25 62
65 33 71 24 72
23 28 72 95 29
90 10 33 93 33
78 56 52 01 06
70 61 74 29 41
85 39 41 18 38
97 11 89 63 38
84 96 28 52 07
20 82 66 95 41
05 01 45 11 76
35 44 13 18 80
37 54 87 30 43
94 62 46 11 71
00 38 75 95 79
77 93 89 19 36
80 81 45 17 48
36 04 09 03 24
88 46 12 33 56
15 02 00 99 94
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 bath (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
Second
Third
Fourth
Fifth
Sixth
Seventh
Eighth
Ninth
Tenth
fish
fish
fish
fish
fish
fish
fish
fish
fish
fish
taken
taken
taken
taken
taken
taken
taken
taken
taken
taken
from
from
from
from
from
from
from
from
from
from
tank
tank
tank
tank
tank
tank
tank
tank
tank
tank
6.
50
25
25
100
25
12
50
25%
.0%
.0%
.0%
.0%
.0%
.5%
.0%
effluent,
effluent,
effluent,
effluent,
Control-,
Control ,
effluent,
effluent,
effluent,
effluent,
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
replicate
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
chamber
2
4
5
3
5
4
4
2
2
4
TABLE A.4. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS:
LABELLING THE POSITIONS WITHIN THE WATER BATH
EXAMPLE
1
7
13
19
25
2
8
14
20 ,
26
3
9
15
21
27
4
10
16
22
28
5
11
17
23
29
6
12
18
24
30
570
-------
2.4.2 The second step is to assign each of the 30 positions
three double-digit numbers. An example of this assignment is
provided in Table A.5. Note that the double digits 00 and 91
through 99 were not used. j
2.4.3 The random numbers used to carry out the random assignment
of replicate chambers to positions are provided in Table A.2.
The third step is to choose a starting position in Table A.2, and
read the first double-digit number. The first number,read
identifies the position for the first replicate chamber of the
control. For the example, the first entry in row 10 of Table A.2
was chosen as the starting position. The first, number in this
row was 73. According to Table A.5, this number corresponds to
position 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 second replicate chamber of the control.
Continuing the example, the second number read in row 10 of Table
A.2 is 79. According to Table A.5, this number corresponds to
position 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 jpositioris for the
lowest effluent concentration replicate chambers, and so on. If
a number is read from the table that was not assigned to a
position, then ignore that value and continue to the next number.
If a number is repeated in Table A.2, then ignore the repeats and
continue to the next number. The complete randomization of
replicate chambers to positions for the example is displayed in
Table A.6.
l
2.4.6 Three double-digit numbers were assigned to each position
(instead of one or two) in order to make efficient use of the
random number table (Table A.2). To illustrate, consider the
assignment of only sone double-digit number to eeich 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. OUTLIERS
3.1 An outlier is an inconsistent or questionable data point
that appears unrepresentative of the general trend exhibited by
the majority of the data. Outliers may be detected by tabulation
of the data, plotting, and by an analysis of the residuals. An
explanation should be sought for any questionable data points.
Without an explanation, data points should be discarded only with
extreme caution. If there is no explanation, the analysis should
be performed both with and without the outlier, and the results
of both analyses should be reported.
3.2 Gentleman-Wilk's A statistic gives a test for the condition
that the extreme observation may be considered an outlier. For a
discussion of this, and other techniques for evaluating outliers,
see Draper and John (1981).
572
-------
TABLE A.5. RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO POSITIONS:
EXAMPLE ASSIGNED NUMBERS FOR EACH POSITION
Assigned Numbers
01,
02,
'03,
04,
05,
06,
07,
08,
09,
10,
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40,
41.
42.,
43,
44,
45,
46,
47,
48,
49,
50,
51,
52,
53,
54,
55,
56,
57,
58,
59,
60,
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
Position
1
2
3
, 4
5
• •' i 6
7
8
1 9
i 10
; n
12
13
14
i 15
16
; 17
18
19
20
j 21
22
23
24
25
26
27
28
29
30
573
-------
TABLE A.6. EXAMPLE OF RANDOM ASSIGNMENT OF REPLICATE CHAMBERS TO
POSITIONS:
ASSIGNMENT OF ALL 30 POSITIONS
25.
25.
0%
0%
Control
Control
100.
0%
50
12
12
12
25
.0%
.5%
.5%
.5%
.0%
6.
50
100
100
25%
.0%
.0%
.0%
Control
Control
25
100
6.
50
.0%
.0%
25%
.0%
6.
50
6.
25%
.0%
25%
Control
50
.0%
100
12
6.
25
12
.0%
.5%
25%
.0%
.5%
574
-------
APPENDIX B
i ' ,.
VALIDATING NORMALITY AND HOMOGENEITY OF VARIANCE ASSUMPTIONS
1. INTRODUCTION
1.1 Dunnett's Procedure and the t test with Bonferroni' s .
adjustment are parametric procedures based on the assumptions
that the observations within treatments are independent and
normally distributed, and that the variance of the observations
is homogeneous across all toxicant concentrations and the
control. These assumptions should be checked prior to using
these tests, to determine if they have been met. Tests for
validating the assumptions are provided in the following
discussion. If the tests fail (if the data do not meet the
assumptions), a nonparametric procedure such as Steel's Many-one
Rank Test may be more appropriate. However, the decision on
whether to use parametric or nonparametric tests may be a
judgement call, and a statistician should be consulted 'in
selecting the analysis. !
2. TEST FOR NORMAL DISTRIBUTION OF DATA
2.1 SHAPIRO-WILK'S TEST ' :
2.1.1 One formal test for normality is the Shapiro-Wilk's Test
(Conover, 1980) . The test statistic is obtained by dividing the
square of an appropriate linear combination of the sample order
statistics by the usual symmetric estimate of variance. The
calculated W must be greater than zero and less than or equal to
one. This test is recommended for a sample size of 50 or less.
If the sample size is greater than 50, the Kolmogorov "D"
statistic (Stephens, 1974) is recommended. An example of the
Shapiro-Wilk1s 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 inj 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.I.
575
-------
TABLE B.I. MYSID, HOLMESIMYSIS COSTATA, GROWTH DATA
Concentration (%)
Replicate
1
2
3
4
5
Mean (Yi)
Si
i
Control
0.048
0.058
0.047
0.058
0.051
0.052
0.0000283
1
1.80
0.055
0.048
0.042
0.041
0.052
0.048
0.0000373
2
3.20
0.057
0.050
0.046
0.043
0.045
0.048
0.0000307
3
5.60
0.041
0.040
0.041
0.043
0.040
0.041
0.0000015
4
2.3 The first step of the test for normality is to center the
observations by subtracting the mean of all observations within a
concentration from each observation in that concentration. The
centered observations are listed in Table B.2.
TABLE B.2. CENTERED OBSERVATIONS FOR SHAPIRO-WILK"S EXAMPLE
'Concentration (%)
Replicate
1
2
3
4
5
Control
-0.
0.
-0.
0.
-0.
004
006
005
006
001
1
0
0
-0
-0
0
.80
.007
.000
.006
.007
.004
3.
0.
0.
-0.
-0.
. -o.
20
009
002
002
005
003
5
0
-0
0
0
-0
.60
.000
.001
.000
.002
.001
2.4 Calculate the denominator, D, of the statistic:
n
D = £ (X - X)
Where: XA = the ith centered observation
576
-------
X = the overall mean of the centered observations
n = the total number of centered observations
2.4.1 For this set of data, n = 20 :
X = 1 (0.001) = 0.000
20 '
D = 0.000393 '
2.5 Order the centered observations from smallest to largest
I
<: . . . <; X(n) '•
where X(i) denotes the ith ordered observation. The ordered
observations for this example are listed in Table B.3.
TABLE B.3. ORDERED CENTERED OBSERVATIONS FOR SHAPIRO-WILK'S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
XU>
-0.007
-0.006
-0.005
-0.005
-0.004
-0.003
-0.002
-0.001
-0.001
-0.001
i
11
12
13
14
15
16
17
18
19 .
20
X<"
0.000
0.000
0.000
0.002
0.002
0.004
0.006
0.006
0.007
0.009
2.6 From Table B.4, for the number of observations, n, obtain
the coefficients a17 a2, ... ak where k is n/2 if n is even and
(n-l)/2 if n is odd. For the data in this example, n = 20 and k
= 10. The a± values are listed in Table B.5.
577
-------
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (Conover, 1980)
\Number of
»
1
2
3
4
5
2
0.
-
-
-
-
7071
3
0.7071
0.0000
-
-
-
4
0.6872
0.1667
-
-
-
5
0.6646
0.2413
0.0000
-
-
6
0.6431
0.2806
0.0875
-
-
Observations
7
0.6233
0.3031
0.1401
0.0000
-
8
0.6052
0.3164
0.1743
0.0561
- .
9
0.5888
0.3244
0.1976
0.0947
0.0000
10
0.5739
0.3291
0.2141
,0.1224
0.0399
i
%
i\"
1
2
3
4
5
6
7
8
9
10
11
0.
0.
0.
0.
0.
0.
-
-
-
5601
3315
2260
1429
0695
0000
12
0.5475
0.3325
0.2347
0.1586
0.0922
0^0303
-
-
13
0.5359
0.3325
0.2412
0.1707
0.1099
0.0539
0.0000
-
-
14
0.5251
0.3318
0.2460
0.1802
0.1240
0.0727
0.0240
-
-
Number of
15
0.5150
0.3306
0.2495
0.1878
0;1353
0.0880
0.0433
0.0000
-
Observations
16
0.5056
0.3290
0.2521
0.1939
0.1447
0.1005
0.0593
0.0196
-
17
0.4968
0.3273
0.2540
0.1988
0.1524
0.1109
0.0725
0.0359
0.0000
18
0.4886
0.3253
0.2553
0.2027
0.1587
0.1197
0.0837
0.0496
0.0163
19
0.4808
0.3232
0.2561
0.2059
0.1641
0.1271
0.0932
0.0612
0.0303
0.0000
20
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
0.0711
0.0422
0.0140
.
\
A
i
2
3
4
5
6
7
8
9
10
11
12
13
14
15
21
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
-
-
-
.
4643
3185
2578
2119
1736
1399
1092
0804
0530
0263
0000
22
0.4590
0.3156
0.2571
0.2131
0.1764
0.1443
0.1150
0.0878
0.0618
0.0368
0.0122
-
-
-
-
23
0.4542
0.3126
0.2563
0.2139
0.1787
0.1480
0.1201
0.0941
0.0696
0.0459
0.0228
0.0000
-
-
-
24
0.4493
0.3098
0.2554
0.2145
0.1807
0.1512
0.1245
0.0997
0.0764
0.0539
0.0321
0.0107
-
-
-
Number of
25
0.4450
0.3069
0.2543
' 0.2148
0.1822
0.1539
0.1283
0.1046
0.0823
0.0610
0.0403
0.0200
0.0000
-'
-
Observations '• '
26
0.4407
0.3043
0.2533
0.2151
0.1836
0.1563
0.1316
0.1089
0.0876
0.0672
0.0476
0.0284
0.0094
-
27
0.4366
0.3018,
0.2522
0.2152
0.1848
0.1584
0.1346
0.1128
0.0923
0.0728'
0.0540
0.0358
0.0178
0.0000
-
28
0.4328
0.2992
0.2510
0.2151
0.1857
0.1601
0.1372
0.1162
0.0965
0.0778
0.0598
0.0424
0.0253
0.0084
-
29
0.4291
0.2968
0.2499
0.2150
0.1864
0.1616
0.1395
0.1192
0.1002
0.0822
0.0650
0.0483
0.0320
0.0159
0.0000
30
0.4254
0.2944
0.2487
0.2148
0.1870
0.1630
0.1415
0.1219
0.1036
0.0862,
0.0697
0.0537
0.0381
0.0227
0.0076
578
-------
TABLE B.4. COEFFICIENTS FOR THE SHAPIRO-WILK'S TEST (CONTINUED)
\
tV
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
31
0.4220
0.2921
0.2475
0.2145
0.1874
0.1641
0.1433
0.1243
0.1066
0.0899
0.0739
0.0585
0.0435
0.0289
0.0144
0.0000
-
-
-
-
32
0.4188
0.2898
0.2462
0.2141
0.1878
0.1651
0.1449
0.1265
0.1093
0.0931
0.0777
0.0629
0.0485
0.0344
0.0206
0.0068
-
-
-
-
33
0.
0.
0.
0.
0.
0.
4156
2876
2451
2137
1880
1660
0.1463
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
-
-
-
1284
1118
0961
0812
0669
0530
0395
0262
0131
0000
34
0.4127
. 0.2854
0.2439
0.2132
0.1882
0.1667
0.1475
0.1301
0.1140
0.0988
0.0844
0.0706
0.0572
0.0441
0.0314
0.0187
0.0062
-
-
-
Number of
35
0.4096
0.2834
0.2427
0.2127
0.1883
0.1673
0.1487
0.1317
0.1160
0.1013
0.0873
0.0739
0.0610
0.0484
0.0361
0.0239
0.0119
0.0000
-
-
Observations
36
0.4068
0.2813
0.2415
0.2121
0.1883
0.1678
0.1496
0.1331
0.1179
0.1036
0.0900
0.0770
0.0645
0.0523
0.0404
0.0287
0.0172
0.0057
-
•
37
0.4040
0.2794 '
0.2403
0.2116
0.1883
0.1683
0.1505
0.1344
0.1196
0.1056
0.0924
0.0798
0.0677
0.0559
0.0444
0.0331
0.0220
0.0110
0.0000
-
38
0.4015
0.2774
0.2391
0.2110
0.1881
0.1686
0.1513
0.1356
0.1211
0.1075
0.0947
0.0824
0.0706
0.0592
0.0481
0.0372
0.0264
0.0158
0.0053
-
39
0.3989
0.2755
0.2380
0.2104
0.1880
0. 1689
0.1520
0.1366
0.1225
0.1092
0.0967
0.0848
0.0733
0.0622
0.0515
0.0409
0.0305
0.0203
0.0101
0.0000
40
0.3964
0.2737
0.2368
0.2098
0.1878
0.1691
0.1526
0.1376
0.1237
0.1108
0.0986
0.0870
0.0759
0.0651
0.0546
0.0444
0.0343
0.0244
0.0146
0.0049
\
iV
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
41
0.3940
0.2719
0.2357
0.2091
0.1876
0.1693
0.1531
0.1384
0.1249
0.1123
0.1004
0.0891
0.0782
0.0677
0.0575
0.0476
0.0379
0.0283
0.0188
0.0094
0.0000
-
-
-
-
42
0.3917
0.2701
0.2345
0.2085
0.1874
0.1694
0.1535
0.1392
0.1259
0.1136
0.1020
0.0909
0.0804
0.0701
0.0602
0.0506
0.0411
0.0318
0.0227
0.0136
0.0045
-
-
-
-
43
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
-
-
-
3894
2684
2334
2078
1871
1695
1539
1398
1269
1149
1035
0927
0824
0724
0628
0534
0442
0352
0263
0175
0087
0000
44
0.3872
0.2667
0.2323
0.2072
0.1868
0.1695
0.1542
0.1405
0.1278
0.1160
0.1049
0.0943
0.0842
0.0745
0.0651
0.0560
0.0471
0.0383
0.0296
o:0211
0.0126
0.0042
-
- -
-
Number of
45
0.3850
0.2651
0.2313
0.2065
0.1865
0.1695
0.1545
0.1410
0.1286
0.1170
0.1062
0.0959
0.0860
0.0765
0.0673
0.0584
0.0497
0.0412
0.0328
0.0245
0.0163
0.0081
0.0000
-
-
Observations
46
0.3830
0.2635
0.2302
0.2058
0.1862
0.1695
tt. 1548
0.1415
0.1293
0.1180
0.1073
0.0972
0.0876
0.0783
0.0694
0.0607
0.0522
0.0439
0.0357
0.0277
0.0197
0.0118
0.0039
-
-
47
0.3808
0.2620
0.2291
0.2052
0.1859
0.1695
0.1550
0.1420.
0.1300
0.1189
0.1085
0.0986
0.0892
0.0801
0.0713
0.0628
0.0546
0.0465
0.0385
0.0307
0.0229
0.0153
0.0076
0.0000
-
48
0.3789
0.2604
0.2281
0.2045
0.1855
0.1693
0.1551
0.1423
0.1306
0.1197
0.1095
0.0998
0.0906
0.0817
0.0731
0.0648
0.0568
0.0489
0.0411
0.0335
0.0259
0.0185
0.0111
0.0037
-
49
0.3770
0.2589
0.2271
0.2038
0.1851
0.1692
0.1553
0.1427
0.1:512
0.11'OS
O.i'105
0.1010
0.0919
O.OU32
0.0748
0.0667
0.05I88
0.0311
0.0436
0.03161
0.0288
0.02',15
0.0143
0.0071
0.0000
50
0.3751
0.2574
0.2260
0.2032
0.1847
0.1691
0.1554
0.1430
0.1317
0.1212
0.1113
0.1020
0.0932
0.0846
0.0764
0.0685
0.0608
0.0532
0.0459
0.0386
0.0314
0.0244
0.0174
0.0104
0.0035
579
-------
2.7 Compute the test statistic, W, as follows
W- -[E
D.
*
a
The differences x("-i+1) - X(i) are listed in Table B.5. For this
set of data:
W = 1 (0.0194)2 =0.958
0.000393
TABLE B.5. COEFFICIENTS AND DIFFERENCES FOR SHAPIRO-WILK1S EXAMPLE
i
1
2
3
4
5
6
7
8
9
10
**
0.4734
0.3211
0.2565
0.2085
0.1686
0.1334
0.1013
0.0711
0.0422
0.0140
Y (n-i-t-1) _. v (i)
0.016
0.013
0.011
0.011
0.008
0.005
0.004
0.001
0.001
0.001
X(20)
X(19)
X<18>
XU7)
X(16)
X(1S)
XU4>
X!13)
X(12)
X<11,
- X'1'
- X<2'
- X<3>
- X(4)
- X'5'
- x(s)
- x(7) •
- X'8'
- X'9'
- x(10>
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 data, the critical value
at a significance level of 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.
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)
o.oi
0.02
0.05
0.10
0.50
0.90
0.95
,0.98
0.99
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20 .
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
0.753
0.687
0.686
0.713
0.730
0.749
0.764
0.781
0.792
0.805
0.814
0.825
0.835
0.844
0.851
0.858
0.863
0:868
0.873
0..878
0.881
0.884
0.888
0.891
0.894
0.896
0.898
0.900
0.902
0.904
0.906
0.908
0.910
0.912
0.914
0.916
0.917
0.919
0.920
0.922
0.923
0.924
0.926
0.927
0.928
0.929
0.929
0.930
0.756
0.707
0.715
0.743
0.760
0.778
0.791
0.806
0.817
0.828
0.837
0.846
' 0.855
0.863
0.869
0.874
0.879
0.884
0.888
0.892
0.895
0.898
0.901
0.904
0.906
0.908
0.910
0.912
0.914
0.915
0.917
0.919
0.920
0.922
0.924
0.925
0.927
0.928
0.929
0.930
0.932
0.933
0.934
0.935
0.936
0.937
0.937
0.938
0.767
0.748
0.762
0.788
0.803
0.818
0.829
0.842
0.850
0.859
0.866
0.874
0.881
0.887
0.892
0.897
0.901
0.905
0.908
0.911
0.914
0.916
0.918
0.920
0.923
0.924
0.926
0.927
0.929
0.930
0.931
0.933
0.934
0.935
0.936
0.938
0.939
0.940
0.941
0.942
0.943
0.944
0.945
0.945
0.946
0.947
0.947
0.947
0.789
0.792
0.806
0.826
0.838
0.851
0.859
0.869
0.876
0.883
0.889
0.895
0.901
0.906
0.910
0,914
0.917
0.920
0.923
0.926
0.928
0.930
0.931
0.933
0.935
0.936
0.937
0.939
0.940
0.941
0.942
0.943
0.944
0.945
0.946
0.947
0.948
0.949
0.950
0.951
0.951
0.952
0.953
0.953
0.954
0.954
0.955
0.955
0.959
0.935
0.927
0.927
0.928
0.932
0.935
0.938
0.940
0.943
0.945
0.947
0.950
0.952
0.954
0.956
0.957
0.959
0.960
0.961
0.962
0.963
0.964
0.965
0.965
0.966
0.966
0.967
0.967
0.968
0.968
0.969
0.969
0.970
0.970
0.971
0.971
0.972
0.972
0.972
0.973
0.973
0.973
0.974
0.974
0.974
0.974
0.974
0.998
0.987
0.979
0.974
0.972
0.972
0.972
0.972
0.973
0.973
0.974
0.975
0.975
0.976
0.977
0.978
0.978
0.979
0.980
0.980
0.981
0.981
0.981
0.982
0.982
0.982
0.982
0.983
0.983
0.983
0.983
0.983
0.984
0.984
0.984
0.984
0.984
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.985
0.98S
0.985
0.999
0.992
0.986
0.981
0.979
0.978
0.978
0.978
0.979
0.979
0.979
0.980
0.980
0.981
0.981
0.982
0.982
0.983
0.983
0.984
0.984
0.984
0.985
0.985
0.985
0.985
0.985
0.985
0.986
0.986
0.986
0.986
0.986
0.986
0.987
0.987
0.987
0.987
0.987
0.987
0.987
0.987
0.988
0.988
0.988
0.988
0.988
0.988
1.000
0.996
0.991
0.986
0.985
0.984
0.984
0.983
0.984
0.984
0.984
0.984
0.984
0.985
0.985
0.986
0.986
0.986
0.987
0.987
0.987
0.987
0.988
|0.988
0.988
0.988
0.988
0.988
0.988
0.988
0.989
0.989
0.989
0.989
0.989
0.989
0.989
p. 989
0.989
0.989
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
1.000
0.997
0.993
0.989
0.988
0.987
0.986
0.986
0.986
0.986
0.986
0.986
0.987
0.987
0.987
0.988
0.988
0.988
0.989
0.989
0.989
0.989
0.989
0.989
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.990
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
0.991
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, HOLMES IMYS IS COSTATA, GROWTH DATA
Concentration (%)
Replicate
l
2
3
4
5
Mean (Yi)
Sf
i
Control
0.048
0.058
0.047
0.058
0.051
0.052
0.0000283
1
1.80
0.055
0.048
0.042
0.041
0.052
0.048
0.0000373
2
3.20
0.057
0.050
0.046
• 0.043
• 0.045
0.048
0.0000307
3
5.60
0.041
0.040
0.041
0.043
0.040
0.041
0.0000015
4
3.3 The test statistic for Bartlett's Test (Snedecor and
Cochran, 1980) is as follows:
B
f p
(Ev) In S2 - Ev In S.2
i i i
i-1 1-1
Where: V± * degrees of freedom for each effluent concentration
and control, (Vi = n^. - 1)
p = number of levels of toxicant concentration
including the control
582
-------
In = loge i
i = 1, 2, ..., p where p is the number of
concentrations including the control
ni =the number of replicates for concentration i
Ev
i-1
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 (n.i - 5 for all
i) . Thus, Vi = 4 for all i. For this data, p = 4 , ~S2 =
0.0000245, and C = 1.104. Bartlett ' s statistic is therefore:
p , i
B= [(16) In (0.0000245) -41)1^(5^} ]/1.104 :
[16(-10.617) - 4 (-44.470) ] /I. 104
i
[-169.872 - (-177.880) ] /I L 104 '
..= 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-Wilk1 s and Bartlett's tests should be. performed on the
transformed observations to determine whether the assumptions of
normality and/or homogeneity of variance are met.
4.2 ARC SINE SQUARE ROOT TRANSFORMATION (USEPA, 1993).
4.2.1 For data consisting of proportions from a binomial
(response/no response; live/dead) response variable, the variance
within the ith treatment is proportional to Pi (1 - P±), where Pi
is the expected proportion for the treatment. This clearly
violates the homogeneity of variance assumption required by
parametric procedures such as Dunnett's Procedure or the t test
with Bonferroni's adjustment, since the existence of a treatment
effect implies different values of P± for different treatments,
i. Also, when the observed proportions are based on small
samples, or when P± is close to zero or one, the normality
assumption may be invalid. The arc sine square root (arc sine
Vp ) 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 1, a special modification of the arc
sine square root transformation must be used (Bartlett, 1937).
An explanation of the arc sine square root transformation and the
modification is provided below.
4.2.3 Calculate the response proportion (RP) at each effluent
concentration, in this case proportion surviving where:
584
-------
RP = (number of surviving or unaffected organisms)/(number
exposed).
Example: If 12 of 20 animals in a given treatment .replicate
survive: :
RP = 12/20 j
= 0.60
i
4.2.4 Transform each RP to its arc sine squares root, as follows:
4.2.4.1 For RPs greater than zero or less than one:
i
i
Angle (radians) =
Example: If RP = 0.60: .
i
Angle = arc sine JQ.QQ
= arc sine 0.7746
= 0.8861 radians
4.2.4.2 Modification of the arc sine square root when RP = 0
j
Angle (in radians) = arc sine
Where: N = Number of animals/treatment replicate
i
Example: If 20 animals are used:
I
Angle = arc sine
= 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
-------
APPENDIX C
DUNNETT'S PROCEDURE
1. MANUAL CALCULATIONS
1.1 Dunnett's Procedure (Dunnett, 1955; Dunnett, 1964) is used
to compare each concentration mean with the control mean to
decide if any of the concentrations differ from the control.
This test has an overall error rate of alpha, which accounts for
the multiple comparisons with the control. It is based on the
assumptions that the observations are independent and normally
distributed and that the variance of the observsitions is
homogeneous across all concentrations and control. (See Appendix
B for a discussion on validating the assumptions!) . Dunnett's
Procedure uses a pooled estimate of the variance, which is equal
to the error value calculated in an analysis of variance.
Dunnett's Procedure can only be used when the same number of
replicate test vessels have been used at each concentration and
the control. When this condition is not met, the t test with
Bonferroni's adjustment is used (see Appendix D).
1.2 The data used in this example are growth data from a Mysid
Survival and Growth Test, and are the same data used in Appendix
B. These data are listed in Table C.I.
TABLE C.I. MYSID, HOLMESIMYSIS CO3TATA, GROWTH DATA
Concentration (%)
Replicate
Mean
Total
i
1
2
3
4
5
(Yi)
(Tj.)
Control
0
0
0
0
0
0.
0.
1
.048
.058
.047
.058
.051
052
262
1
0
0
0
0
0
0.
0.
2
.80
.055
.048
.042
.041
.052
048
238
3
0
0
0
0
0
o.
0.
3
.20
.057
.050
.046
.043
.045
048
241
5
0
0
0
; 0
0
o.
0.
4
.60
.041
.040'
.041
.043
.040
041
205
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 df
Between p - l
Within N - p
Sum of Squares
(SS)
SSB
SSW
Mean
2
SB =
2
sw =
Square (MS)
(SS/df)
SSB/(p-l)
SSW/ (N-p)
Total N - l SST
Where: p = number of effluent concentrations including
the control:
N = the total sample size; N=.Eni
L
ni = the number of replicates for concentration "i"
SST=£ Y 2-G2/N Total Sum of Squares
SSB=Eri2/n^-G2/w Between Sum of Squares
i
SSW=SST-SSB Within Sum -of Squares
G = the grand total of all sample
observations; c
the total of the replicate measurements for
concentration i
588
-------
N = the total sample size; w=5inj
j
n± = the number of replicates for concentration i
Y±j = the jth observation for concentration i
• i .
1.4 For the data in this example:
n± = n2 =?= n3 = n4 = ns = 5 ;
N = 20 ]
TI = Yu + Y12 + Y13 + Y14 + Y15 = 0.262
T2 = Y21 + Y22 + Y23 + Y24 + Y25 = 0.238
T3 = Y31 + Y32 + Y33 + Y34 + Y35 = 0.241
T4 = Y41 + Y42 + Y43 + Y44 + Y45 = 0.205
G = T! + T2 + T3 + T4 ' = 0.946 '
SSB =
i_(Q.225) - (0.946)2- = 0.000254
5 20
P
SST =
= 0.0455 - (0.946)2 = 0.000754
20 i
'
SSW = SST-SSB = 0.000754 - 0.000254 = 0.000500
Si = SSB/(p-l) = 0.000254/(4-l) = 0.0000847
Si = SSW/(N-p) = 0.0005007(20-4) = ;0'.0000313
1.5 Summarize these data in the ANOVA table, ass shown in Table
C.3:
589
-------
TABLE C.3. COMPLETED ANOVA TABLE FOR DUNNETT'S PROCEDURE EXAMPLE
Source
df
Sum of Squares
(SS)
Mean Square (MS)
(SS/df)
Between
Within
Total
3
16
19
0.
0.
0.
000254
000500
000754
0.0000847
0.0000313
1.6 To perform the individual comparisons, calculate the t
statistic for each concentration and control combination, as
follows:
* (1/nJ
Where: Y^ = mean for the control
mean for each concentratioon i
Sw = square root of the within mean square
= number of replicates in the control
= 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
2
3
4
£i
1.131
1.131
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 the one-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± is greater than the critical value.
Comparing each of the calculated t 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
-------
m
u
3
I
'• 592
-------
1.9 To quantify the sensitivity of the test, the minimum
significant difference :(MSD) may be calculated,, The formula is
as follows: '. .
MSD*d sJd/n + (1/n)
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-L = number of replicates in the control
For example : ; • \ '
MSD = 2.23(O.Op559)yC{l/5)
.= 2.23 (0.00559) :(0. 632)
= 0.00788 . , ' -'•
1.10 Therefore, for this set of data, the minimum difference
between the control mean and a concentration mean that can be
detected as statistically significant is .0 . 00788 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 untransformed units.
1.11.2 Subtract the MSD from the transformed control mean. Call
this difference D. Next, obtain untransformed values for the
I • " ,
control mean and the difference, D. Finally, compute the
untransformed MSD as follows:
• ! "
.' ' 593 '•"••-
-------
MSBU = controlu ~ Du ,
Where: MSBU = the minimum significant difference for
untransformed data
Controlu = the untransformed control mean
Bu = the untransformed difference
1.11.3 Calculate the percent reduction from the control that
MSDU represents as:
MSBU
Percent Reduction = —: X 100
Controlu
1.11.3.1 An example of a conversion of the MSB to untransformed
units, when the arc sine square root transformation was used on
the data, follows.
Step 1. Subtract the MSB from the transformed control mean.
As an example, assume the data in Table C.I were
transformed by the arc sine square root
transformation. Thus:
0.052 - 0.00788 = 0.04412
Step 2. Obtain untransformed values for the control mean
(0.052) and the difference (0.04412) obtained in
Step 1, above.
[ Sine (0.052)]2 = 0.00270
[ Sine (0.04412)]2 = 0.00195
Step 3. The untransformed MSB (MSBU) is determined by .
subtracting the untransformed values obtained in
Step 2.
MSBU = 0.00270 - 0.00195 = 0.00075'
In this case, the MSB 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 mean (Dunnett's Procedure). The
ANOVA is used to obtain the error value;_ Dunnett' s Procedure
indicates which toxicant concentration means (if any) are
statistically different from the control mean at the 5% level of
significance. The program also provides the minimum difference
between the control and treatment means that could be detected as
statistically significant, and tests the validity of the
homogeneity of variance assumption by Bartlett's Test. The
multiple comparison is performed based on procedures described by
Dunnett (1955).
2.2 The source code for the Dunnett's program is structured into
a series of subroutines, controlled by a driver routine. Each
subroutine has a specific function in the Dunnett's Procedure,
such as data input, transforming the data, testing for equality
of variances, computing p values, and calculating the one-way
analysis of variance. • ;
2.3 The program compares up to seven toxicant concentrations
against the control, and can accommodate up to 50 replicates per
concentration. ;
2.4 If the number of replicates at each toxicant concentration
and control are not equal, a t test with the Bonferroni
adjustment is performed instead of Dunnett's Procedure (see
Appendix D).
2.5 The program was written in IBM-PC FORTRAN by Computer
Sciences Corporation, 26 W. Martin Luther King Drive, Cincinnati,
OH 45268. A compiled version of the program can be obtained from
EMSL-Cincinnati by sending a diskette with a written request.
2.6 DATA INPUT AND OUTPUT
2.6.1 The mysid growth data from Table C.I are used to
illustrate the data input and output for this program.
2.6.2 Data Input
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.I.
2.6.3. Program Output
2.6.3.1 When Option 3 (perform analysis on existing data set) is
selected from the menu, the user is asked to select the
transformation desired, and indicate whether they expect the
means of the test groups to be less or greater than the mean for
the control group (see Figure 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 proportion data)
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 cell counts,
weights, chlorophyll measurements, or turbidity measurements)
Enter "1", "2", (or "q" 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 ;
i
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 :
I
NO. 4? 0.058
NO. 5? 0.051
597
-------
Enter the data for cone. 2 one observation at a time.
NO. 1? 0.055
NO. 2? 0.048
NO. 3? 0.042
NO. 4? 0.041
NO. 5? 0.052
Number of observations for cone. 3 ? 5
Enter the data for cone. 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 cone. 4 ? 5
Enter the data for cone. 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 1.5
1) Create a data file
2) Edit a data file
3) Analyze an existing data set
4) Stop
Your choice ? 3
Pile name ? c:\mysid.dat
Available Transformations
1) no transform
2) square root
3) loglO
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 than ? L
Figure C.2. Example of Choosing Option 3 from the Main Menu 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
Cone. n Mean s.d. cv%
1
= control
2
3
4*
5
5
5-,
5
.0524
.0476 '
.0482
.0410
.0053
.0061
.0055
.0012
10.2
12.8
11 . 5
3.0
*) the mean for this cone, is significantly less' than
the control mean at alpha = 0.05 (1-sided) by Dunnett's test
Minimum detectable difference for Dunnett's test = -.006974
This difference corresponds to -13.31 percent of control
Between concentrations
sum of squares = .000333 with 3 degrees of freedom.
Error mean square = .000024 with 16 degrees of freedom.
Bartlett's test p-value for equality of variances = .060
Do you wish to restart the program ?
Figure C.3. Example of Program Output for the Dunnett's Program Using the
Data in Table C.I. ,
601
-------
APPENDIX D
t TEST WITH BONPERRONI'S ADJUSTMENT
1. The t test with Bonferroni's adjustment is used as an
alternative to Dunnett's Procedure when the number of replicates
is not the same for all concentrations. This test sets an upper,
bound of alpha on the overall error rate, in contrast to
Dunnett's Procedure, for which the overall error rate is fixed at
alpha. Thus, Dunnett's. Procedure is a more powerful test.
2. The t test with Bonferroni's adjustment is based on the same
assumptions of normality of distribution and homogeneity of
variance as Dunnett's Procedure (See Appendix B for testing these
assumptions), and, like Dunnett's Procedure, uses a pooled
estimate of the variance, which is equal to the error value
calculated in an analysis of variance.
3. An example of the use of the t test with Bonferroni's
adjustment is provided below. The data used in the example are 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 presented in Table D.I.
TABLE D.I. GIANT KELP, MACROCYSTIS PYRIFERA, GROWTH DATA
Copper Concentration (^g/L)
Rep
1
2
3
4
5
Yi
s|
i
Control
19
18
19
16
17
18.
.58
.75
.14
.50
.93
38
1.473
1
5.50
18.26
16.25
16.39
18.70
15.62
17.04
1.827
2
10.0
13.31
18.92
15.62
14.30
15.29
15.49
4.498
3
18. 0
18.59
12.88
16.28
15.38
19.75
16.58
7.327
4
32.0
12.54
10.67
15.95
12.54
11.66
12 .67
3.953
5
56
11
11
11
11
11
11.
.0
.44
.88
.88
.00
.55
55
0.133
6'
100.0
7
7
8
9
8
8.
0.
7
.92
.59
.25
.1.3
.80
34
396
180.0
6.49
7.25
--
7.63
8.13
7.38
0.478
8
602
-------
3.1 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 D.2: ' i
TABLE D.2. ANOVA TABLE
Source
Between
Within
df Sum of Squares
. (SS)
p - 1 SSB
N - p ' SSW
Mean Square (MS)
(SS/df)
SB - SSB/(p-l)
Sw = SSWV(N-p)
Where: p = number of effluent concentrations including
the control
N = the total sample size;
= the number of replicates for concentration i
SST=E
Total Sum of Squares
Between Sum of Squares
SSW=SST-SSB
Within Sum of Squares
Where:
G
The grand total of all sample
observations;
603
-------
The total of the replicate
measurements for concentration i
Yij = The jth observation for
concentration i
3 . 2 For the data in this example :
nj. = n2 = n3 = n4 = n5 = n6 = n7 = 5; n8 = 4
N = 39
TI - YU + Y12 + Y13 •+ Y14 + Y15 = 91.90
T2 = Y21 + Y22 + Y23 + Y24 + Y25 = 85.22
T3 = Y31 + Y32 + Y33 + Y34 + Y35 = 77.44
T4 = Y41 + Y42 + Y43 + Y44 + Y45 = 82.88
T5 = Y51 + Y52 + Y53 + Y54 + YS5 = 63.36
T6,= Y61 + Y62 + Y63 + Y64 + Y65 = 57.75
T7 = Y71 + Y72 + Y73 + Y74 + Y75 = 41.69
T8 = Y81 + Y82 + Y83 + Y84 = 29.50
G = T! + T2 + T3 .+ T4 + Ts + T6 + T7 + T8 = 529.74
p
SSB « Er^/n^-G2/!^
i-i
= 7749.905 - (529. 74)2 = 554.406
39
SST
= 7829.764 - (529. 74)2 = 634.265
39
SSff - SST-SSB
= 634.265 - 554.406 = 79.859
S§ = SSB/(p-l) = 554.406/(8-l) = 79.201
Sg = 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
Between
Within '
Total
df
7
31
38
Sura of Squares
-------
TABLE D.4. CALCULATED t VALUES
Concentration (^yg/L) i t.
5.6
10.0
18.0
32.0
56.0
100.0
180.0
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 "i" is considered significantly less than the mean
length for the control if t± is greater than the critical value.
Comparing each of the calculated t values in Table D.4 with the
critical value, the 10.0 ^ig/L, 32 /xg/L, 56.0 /xg/L, 100.0 /ig/L,
180.0 pg/li 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 /xg/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 /xg/L and 10.0 /xg/L, respectively.
606
-------
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APPENDIX E
STEEL'S MANY-ONE RANK TEST ;
1. Steel's Many-one Rank Test is a nonparametric test for
comparing treatments with a control. This test is an alternative
to Dunnett' s Procedure, and may be applied to daita when the
normality assumption has not been met. Steel's Test requires
equal variances across the treatments and the control, but it is
thought to be fairly insensitive to deviations from this
condition (Steel, 1959). The tables for Steel's Test require an
equal number of replicates at each concentration. If this is not
the case, use Wilcoxon's Rank Sum Test, with Bonferroni's
adjustment (See Appendix F).
2. For an analysis using Steel's Test, for each control and
concentration combination, combine the data and arrange the
observations in order of size from smallest to largest. Assign
the ranks to the ordered observations (1 to the smallest, 2 to
the next- smallest, etc.). If ties occur in the Cranking, assign
the average rank to the-observation. (Extensive; ties would
invalidate this procedure). The sum of the ranks within each
concentration is then calculated. To determine if the response
in a concentration is significantly less than the response in 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 excluding the
control and n equals the number of replicates for each
concentration and the control.
3. An example of the use of this test is provided below. The
test employs embryo-larval development data from a bivalve 48-
hour chronic test. The data are listed in Table; E.I.
4. For each control and concentration combination, combine the
data and arrange the observations in order of size from smallest
to largest. Assign the ranks (1, 2, 3, ..., 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 combineid data for the
control and 0.13 pig/L copper concentration is given in Table, E. 2.
609
-------
This ranking procedure is repeated for each control and
concentration combination. The complete set of rankings is
listed in Table E.3. The ranks are then summed for each toxicant
concentration, as shown in Table E.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 "minimum" or critical rank sum, at or below which the
survival would be considered to be significantly lower than the
control. At a probability level of 0.05, the critical rank sum
in a test with five concentrations and four replicates per
concentration, is 10 (see Table F.4).
7. Since the rank sums for the 0.50 /xg/L and 1.00 /xg/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 M9/L concentration shows
signigicantly lower normal development than the control while the
higher 2.00 ^tg/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 impairment versus the control is considered to the
LOEC for growth. Thus, for this test, the NOEC and LOEC would be
0.25 #g/L and 0.50 /xg/L, respectively.
610
-------
TABLE E.I. BIVALVE EMBRYO-LARVAL DEVELOPMENT DATA
Goooer Concentration (wa/L)
Repl
RAW
ARC SINE
SQUARE ROOT
TRANSFORMED
Mean (Y,)
s?
i
icate
A
B
C
D
A
B
C
D
Control
1.00
0.96
1.00
0.97
1.571
1.369
1.571
1.397
1.477
0.01191
1
0.13
0.96
0.97
1.00
0.96
1.369
1.397
1.571
1.369
1.427
0.00945
2
0.25
0.92
0.95
0.90
0.96
1.284
1.345
1.249
1.369
1.312
0.00303
3
0.50
0.91
0.93
0.88
0.93
1.266 i
1.303
1.217
1.303
1.272
0.00166
4
1 . 00
0.88
0.83
0.88
0.82
1.217
1 . 146
1.217
1.133 -
1.178
0.00203
5
2.00
1.0.0
0.67
0.75
0.60
1.571
0.959
1.047
0.886
1.116
0.09644
6
611
-------
TABLE E.2. ASSIGNING RANKS TO THE CONTROL AND 0.13 j/g/L CONCENTRATION
LEVEL FOR STEEL'S MANY-ONE RANK TEST
Transformed
Proportion
Rank
Normal
Concentration
2
2
2
4.5
4.5
7
7
7
1.369
1.369
1.369
397
397
571
571
1.571
0.13
0.13
Control
0.13 jug/L
Control
0.13 ug/l
Control
Control
612
-------
TABLE E.3. TABLE OF RANKS1
Copper Concentration (/vg/L)
Replicate
1
2
3
4 '
Repl i cate
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(7,7.5,7.5,7.5,7)
1.397(4.5,6,6,6,5)
Copper
0.50
1.266(2)
1.303(3.
1.217(1)
1.303(3.
0.13 ,
1.369(2)
1.397(4.5)
1.571(7)
1.369(2) ;
i
Concentration -(/vg/L)
1.00 !
1. 217(3. 5>
5) 1.146(2)
1.217(3.5);
5) 1.133(1)
0.25
1.284(2)
1.345(3)
1.249(1)
1.369(4.5)
(Continued)
2.00
1.571(7)
0.959(2)
1.047(3)
0.886(1)
Control ranks are given in the order of the concentration with which
they were ranked. ; •
TABLE E.4. RANK SUM'S
Concentration
/yg/L Copper) Rank Sum
0.13 15.5
0.25 10.5
0.50 10.0
1.00 10.0
2.00 . 13.0
613
-------
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)
n
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
2
11
18
15
27
23
37
32
49
43
63
56
79
71
97
87
116
105
138
125
161
147
186
170
213
196
241
223
272
252
304
k =
3
10
17
--
26
22
36
31
48
42
62
55
77
69
95
85
114
103
135
123
158
144
182
167
209
192
237
219
267
248
299
number
4
10
17
--
25
21
35
30
47
41
61
54
76
68
93
84
112
102
133
121
155
142
180
165
206
190
234
217
264
245
296
of treatments
5
10
16
25
21
35
30
46
40
60
53
75
67
92
83
111
100
132
120
154
141
178
164
204
188
232
215
262
243
294
6
10
16
--.
24
--
34
29
46
40
59
52
74
66
91
82
110
99
130
119
153
140
177
162
203
187
231
213
260
241
292
(excluding
• 7
--
16
--
,24
--
. 34
29
45
40
59
52
74
66
90
81
109
99
129
118
152
' 139
176
161
201
186
229
212
• 259
240
290
control )
8
--
16
--
24
--
33
29
45
39
58
51
73
65
90
81
108
98
129
117
151
138
175
160
200
185
228
211
257
239
288
9
--
15
--
23
--
33
29
44
39
58
51
72
65
89
80
108
98
128
117
150
137
174
160
199
184
227
210
256
238
287
614
-------
APPENDIX F
WILCOXON RANK SUM TEST
1. Wilcoxon's Rank Sum Test is a nonparametric 'test, to be used
as an alternative to Steel's Many-one Rank Test when the number
of replicates are not the same at each concentration. A
Bonferroni's adjustment of the pairwise error rate for comparison
of each concentration versus the control is used to set an upper
bound of alpha on the overall error rate, in contrast to Steel's
Many-one Rank Test, for which the overall error rate is fixed at
alpha. Thus, Steel's Test is a more powerful test.
2. The use of this test may be illustrated with devlopment data
from the red abalone test in Table F.I. 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 combination, combine the
data and arrange the values in order of size, from smallest to
largest. Assign ranks to the ordered observations, (a rank of 1
to the smallest, 2 to the next smallest, etc.). |If ties in rank
occur, assign the average rank to each tied observation.
4. An example of assigning ranks to the combined data for the •
control and effluent concentration 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 wou'ld be
considered to be significantly lower than the control. At a
' i
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) .
TABLE F.I. RED ABALONE, HALIOTUS RUFESCENS, SHELL DEVELOPMENT DATA
, Hi 1 1 if"? nrt
U 1 1 W U 1 VI 1
Replicate Control
RAW
ARC SINE
SQUARE ROOT
TRANSFORMED
Mean (Tt)
s?
1
A
B
C
D
E
A
B
C
D
E
0.99
0.99
0.99
1.00
1.471
1.471
1.471
1.521
, 1.484
0.000625
1
Effluent Concentration (%)
0.56
0.99
0.99
0.98
1.00
1.00
1.471
1.471
1.429
1.521
1.521
1.483
0.001523
2
1.00
0.99
1.00
0.99
0.99
1.00
1.471
1.521
1.471
1.471
1.521
1.491
0.000750
3
1.80
0.99
0.99
0.99
0.98
0.97
1.471
1.471
1.471
1.429
1.397
1.448
0.001137
4
3.20 5.6
0.39
0.57
0.61
0.65
0.80
0.674
0.856
0.896
0.938
1.107
0.894
0.024288
5
0
0
0
0
0
_
-
-
-
-
_
-
6
10.0
0
0
0
0
0
_
-
-
-
-
_
-
7
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
4
4
4
4
4
. 8
8
8
1.429
1.471
' 1.471
1.471
1.471
1.471
1.521
1.521
1.521
0.56 %
0.56 %
0.56 %
Control
Control
Control
0.56 %
0.56 %
Control
TABLE F.3. TABLE OF RANKS1
Repl i -
cate
1
2
3
4
5
Effluent Concentration (£)
Control
1.471(4,3.5,5.5,7)
1.471(4,3.5,5.5,7)
1.471(4,3.5,5.5,7)
1.521(8,8,9,9)
0.56
1.471(4)
1.471(4)
1.429(1)
1.521(8)
1.521(8)
1.00
1.471(3.5)
1.521(8)
1.471(3.5)
1.471(3,5)
1.521(8)
1.80
1.471(5.5)
1.471(5.5)
1.471(5.5)
1.429(2)
1.397(1)
3.20
0.674(1-)
0.856(2)
0:896(3)
0.938(4)
1.107(5)
Control 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 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.
TABLE F.4. RANK SUMS
Concentration
(£ Effluent) Rank Sum
0.56 25.0
1.00 26.5
1.80 19.5
3.20 15.0
618
-------
TABLE F.5. CRITICAL VALUES FOR WILCOXON'S RANK SUM TEST WITH •
BONFERRONI'S ADJUSTMENT OF ERROR RATE FOR COMPARISON OF "K"
TREATMENTS VERSUS A CONTROL FIVE PERCENT CRITICAL LEVEL
. (ONE-SIDED ALTERNATIVE: TREATMENT CONTROL)
K No. Replicates No. of Replicates' Per Effluent Concentration
r
in Control
1 3
4 .
5
6
7
8
9
10
2 3
4
5
6
7
8
9
10
3 3
4
5
6
7.
8
9
10
3
6
6
7
8
8
9
10
10
--
6
7
7
8
8
9
--
.
6
7
7
7
8
4
10
11
12
13
14
15
16
17
10
11
12
13
14
14
15
10
11
11
12
13
13
14
5
16
17
19
20
21
23
24
26
15
16
17
18
20
21
22
23
16
17
18
19
20
21
22
6
23
24
26
28
29
31
33
35
22
23
24
26
27
29
31
32
21
22
24
25
26
28
29
31
7
30
32
34
36
39
41
43
45
29
31
33
34
36
38
40
42
29
30
32
33
35
37
39
41
8
i
39
41
44
46
49
51
. 54
56
38
40
42
^44
46
49
51
53
:37
39
41
43
45
47
49
51
9
49
51
54
57 •
60
63
66
69
47
49 '
52
55
57
60
62
65
46
48
51
53
56
58
61 •
63
10
59
62
66
69
72
72
79
82
58
60
63
66
69
72
75
78
57
59
62
65
68
70
73
76
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 A CONTROL FIVE PERCENT CRITICAL LEVEL (ONE-SIDED
ALTERNATIVE: TREATMENT CONTROL) (CONTINUED)
K No. Replicates No. of Replicates Per Effluent Concentration
1
in Control
5 3
4
5
6
7
8
9
10
6 3
4
5
6
7
8
9
10
7 3
4
5
6
7
8
9
10
3
--
--
--
6
6
7
7
--
--
--
6
6
6
7
--
--
--
--
6
6
7
4
--
10
11
11
12
13
13
--
10
11
11
12
12
13
--
• --
10
11
11
12
13
5
15
16
17
18
19
20
21
15
16
16
17
18
19
20
--
15
16
17
18
19
20
6
22
23
24
25
27
28
29
21
22
24
25
26
27
29
21
22
23
25
26
27
28
7
28
29
31
32
34
35
37
39
28
29
30
32
33
35
37
38
29
30
32
33
35
36
38
8
36
38
40
42
43
45
47
49
36
38
39
41
43
45
47
49
36
37
39
41
43
44
46
48
9
46
48
50
52
54
56
59
61
45
47 •
49
51
54
56
58
60
45
47
49
51
53
55
58 .
60
10
56
58
61
63
66
68
71
74
56
58
60
63
65
68
70
73
56
58
60
62
65
67
70
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 A CONTROL FIVE PERCENT CRITICAL LEVEL (ONE-SIDED
ALTERNATIVE: TREATMENT CONTROL) (CONTINUED)
No. Replicates No. of Replicates Per Effluent Concentration
I
in Control
9 3
4
5
6
7
8
9
10
10 3
4
5
6
7
8
9
10
3
--
--
--
•
6
6
--
--
--
--
--
6
6
4
--
--
10
10
11
11
12
--
--
10
10
11
11
12
5
--
15
16
17
18
18
19
--
15
16
16
17
18
19
6
21
22
23
24
25
26
28
21
22
23
24
25
26
27
7
28
30
31
33
34
35
37
28
29
31
32
34
35
37
! 8
I
37
39
40
42
44
46
47
. 37
38
40
42
43
45
47
9
45
46
48
50
52
55
57
59
45
46
48 .
50
52
54
56
58
10
55
57
59
62
64
66
69
71
55
57
59
61
64
66
68
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 Appendix
F. Since a control and one concentration are being compared, the
K = 1 section of Table F.5 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.
a ,
where S, > S,
5. Compare F with the 0.005 level of a tabled F value with nx -
1 and n2 - 1 degrees of freedom, where nx and n2 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 test. The raw
data, mean and variance for the'two. controls are; given in Table
G.I. The data from each concentration meets the assumption of
normality. ;
TABLE G.I. MYSID, HOLMESIMYSIS COSTATA, GROWTH DATA FROM A SINGLE-
CONCENTATION EFFLUENT TEST
Replicate • Control Effluent •
RAW . A
B
C
. '• D
E
Mean (Y,) • •
Sf
i
,0.048
0.058
0.047
0.055
0.051 . -
. ' 0.052
0.0000217
1
0.041 . •
0.033
0.044
. 0.040
0 . 043
0.040 • .
•0.0000187
2
7. Since the variability of the control is greater than the
variability of the effluent concentration, S2 for the control is
placed in the numerator of the F statistic and S2 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, nA - 1, 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 distribution (Snedecor
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.
I
9'. Equal Variance t Test. ; , ,
623
-------
9.1 To perform the t test, calculate the following test
statistic:
s
"\
I
nl
1
"2
Where:
= mean for the control
Y2 = mean for the effluent concentration
^
(n2-i)s2
* ri2 - 2
Sf = estimate of the variance for the control
Sf = estimate of the variance for the effluent
concentration
n.! = number of replicates for the control
n2 = number of replicates for the effluent
concentration
9.2 Since we are 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 nx + 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 control 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^ + n2 - 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.I to illustrate the t -test, the
calculation of t is as follows:
t =
0.052 - 0.040
0.00449
\
= 4.226
(5-
1)
0.
0000217 +
(5-
1)
0.
0000187
5 - 2
0.00449
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
comparing 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 control, 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 two-tailed test is
the upper-tail value at the ct/2 level of significance.
10. UNEQUAL VARIANCE t TEST. j
10.1 If the F test for equality of variance fails, the t test is
still a valid test. However, the denominator of the t statistic
is adjusted as follows:
t =
\
Where:
= mean for the control
625
-------
Y2 = mean for the effluent concentration
Sf = estimate of the variance for the control
Si = estimate of the variance for the effluent
concentration • . . • ••
nx = number of replicates for the control
n2 = number of replicates for the effluent
concentration
10.2 Additionally, the degrees of freedom for the test are
adjusted using the following formula:
(n -1) (n -1)
df '=
C2+(l-C)2(nrl)
Where:
10.3 The modified degrees of freedom is usually not an integer.
Common practice is to round down to the nearest integer.
10.4 The t test is then conducted as the equal variance t test.
The calculated t is compared to the critical . t at the 0-.05
significance level with the modified degrees of freedom. If the
calculated t exceeds the critical t, the mean responses are found
to be statistically different.
626
-------
APPENDIX H
PROBIT ANALYSIS
1. This program calculates the EC1 and EC50 (or LCI and LC50),
and the associated 95% confidence intervals.
2. The program is written in IBM PC Basic for the IBM compatible
PC by Computer Sciences Corporation, 26 W. Martin Luther King
Drive, Cincinnati, OH 45268. A compiled, executable version of
the program and supporting documentation can be obtained from
EMSL-Cincinnati by sending a written request to EMSL at 3411
Church Street, Cincinnati, OH 45244.
2.1 A set of mortality data from a mysid survival and growth
test is given in Table H.I. The program's data input routine is
illustrated with this data in Figure H.I. The program begins
with a request for the following information:
1. Desired output of abbreviated (A) or full (F) output?
(Note: only abbreviated output is shown below.)
2. Output designation (P = printer, D = disk file).
3. Title for the output.
4. The number of exposure concentrations.
5. Toxicant concentration data. ;
TABLE H.I. DATA FOR PROBIT ANALYSIS
Concentration U)
Control 1.80 3.20 . 5.60 10.0 18.0
No. Dead
No. Exposed
1
25
0
25
3
25
9 i
25 .
24
25
25
25
2.2 The program output for the abbreviated output options, shown
in Figure H.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 LCI and LC50 values and associated
95% confidence intervals.
628
-------
Do you wish abbreviated (A) or full (F) input/output? A '
Output to printer (P) or disk file (D)? P ;
Title ? Example of Probit Analysis 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 concentration
Concentration = ? 1.80
Number responding = ? 0 '
Number exposed = ? 25
Concentration = ? 3.20 ;
Number responding = ? 3 i '
Number exposed = ? 25
Concentration = ? 5.60
Number responding = ? 9 !
Number exposed = ? 25 :
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
Number
Cone. Exposed
Control
1.8000
3.2000
5.6000
10.0000
18.0000
25
25
25
25
25
25
Number
Resp.
1
0
3
9
24-
25
Observed
Proportion
Respondi ng
0.0400
0.0000
0.1200
0.3600
' 0.9600
1.0000
Proportion
Responding
Adjusted for
Controls
0.0000
-.0306
0.0930
0.3404
0.9588
1.0000
Chi - Square for Heterogeneity (calculated)
Chi - Square for Heterogeneity
(tabular value at 0.05 level)
3.004
7.815
Example of Probit Analysis for Appendix H
Estimated LC/EC Values and Confidence Limits
Point
LC/EC 1.00
LC/EC 50.00
Exposure
Cone.
2.642
5.973
95% Confidence Limits
Lower Upper
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 nonparametrlc 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 Iog10 of the
tolerance. If the log tolerance distribution is symmetric, this
estimate of the mean is equivalent to an estimate of the median
of the log tolerance distribution.
2. If the response proportions are not monotonically non-
decreasing with increasing concentration (constant or steadily
increasing with concentration),.the data must be smoothed.
Abbott's procedure is used to "adjust" the concentration response
proportions for mortality occurring in the control replicates.
3. Use of the Spearman-Karber Method, is recommended when partial
mortalities occur in the test solutions, but the data do not fit
the Probit model.
4. To calculate the LC50 using the Spearman-Karber Method, the
following must be true: 1) the smoothed adjusted proportion
mortality for the lowest effluent concentration (not including
the control) must be zero, 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 below using a set
of mortality data from a Mysid Survival and Growth test. These
data are listed in Table I.I.
631
-------
TABLE I.I. EXAMPLE OF SPEARMAN-KARBER METHOD: MORTALITY DATA FROM
A MYSID SURVIVAL 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
0
3
16
25
Mortality .
Proportion
0.08
0.08
0.00
0.12
0.64
1.00
7. Let p0/ plf ..., pk denote the observed response proportion
mortalities for the control and k effluent concentrations. The
first step is to smooth the p± if they do not satisfy p0 s: px £
... s Pk- The smoothing process replaces'any adjacent Pi's that
do not conform to p0 £ p2 s ... & pk with their average. For
example, if pA is less than p^ then:
Where: pf = the smoothed observed proportion
mortality for effluent
.concentration i.
7.1 For the data in this example, because the observed mortality
proportions for the control and the 6.25% effluent concentration
are greater than the observed response proportions for ,the 12.5%
effluent concentration, the responses for these three groups must
be averaged:
, , , 0.08+0.08 + 0.00 0.16 n rtco
Po -Pi =P2 ; = —— • °-°53
632
-------
7.2 Since p3 = 0.12 is larger than pf, set pf = 0.12. Similarly,
p4 = Q..64 is larger than pf, so set p| = 0.64. Finally, p5 = 1.00
is larger than pf, so set pf = 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 A MYSID SURVIVAL AND GROWTH TEST
Effluent'
Concentration
-------
P3 - Po 0.120 - 0.053 0.067
1 _ « 1 - 0.053 0.947
P4 - Po 0.640 - 0.053 0.587
= = = 0.620
i • 1 - 0.053 0.947
PS - Po 1.000 - 0.053 0.947
1-Po
1 - 0.053 0.947
The smoothed, adjusted response proportions for the effluent
concentrations are shown in Table 1.2. A plot of the smoothed,
adjusted data is shown in Figure I.I.
9. Calculate the Iog10 of the estimated LC50, m, as follows:
m = !C-
i-l
Where: pf = the smoothed adjusted proportion mortality at
concentration i
Xi = the Iog10 of concentration i
k = the number of effluent concentrations tested, not
including the control.
9.1 For this example, the Iog10 of the estimated LC50, m, is
calculated as follows:
m = [(0.000 - 0.000) (0.7959 + 1.0969)1/2 +
[(0.071 - 0.000) (1.0969 + 1.3979)]/2 +
[(0.620 - 0.071) (1.3979 + 1.6990)1/2 +
[(1.000 - 0.620) (1.6990 + 2.0000)1/2
= 1.64147
634
-------
in
£_
O
CO
§
o
§-
Q.
O)
to
§
O.
to
O)
4->
to
"O
T3
E i—
rtJ fO
CU t_
-c rs
+-> to
o
to -t->
T3
- -40
-o to
> -8
03 Co
co -i—
o
51
§5
O)
635
-------
10. Calculate the estimated variance of m as follows:
V(m) = E— lfl ^—
i.2 4(^-1)
Where: X£ = the Ipg10 of concentration i
ni = the number of organisms tested at effluent
concentration i
pf = 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 '
11.1 For this example, the 95% confidence interval for m is
calculated as follows:
1.64147 ± 2^0.0011388 = (1.57398, 1.70896)
12. The estimated LC50 and a 95% confidence interval for the
estimated LC50 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 = antilog(m) = antilog(1.64147) = 43.8%.
12.2 The limits of the 95% confidence interval for the estimated
LC50 are calculated by taking the antilogs of the upper and lower
limits of the 95% confidence interval for m as follows:
lower limit: antilog(1.57398) = 37.5%
I
upper limit: antilog(1.70896) = 51.2%
637
-------
APPENDIX J
TRIMMED SPEARMAN-KARBER METHOD
1. The Trimmed Spearman-Karber Method is a modification of the
Spearman-Karber Method, a nonparametric statistical procedure for
estimating the LC50 and the associated 95% confidence interval
(Hamilton, et al, 1977). The Trimmed Spearman-Karber Method
estimates the trimmed mean of the distribution of the Iog10 of
the tolerance. If the log tolerance distribution is symmetric,
this estimate of the trimmed mean is equivalent to an estimate of
the median of the log tolerance distribution.
2. If the response proportions are not monotonically non-
decreasing with increasing concentration (constant or steadily
increasing with concentration), the data must be smoothed.
Abbott's procedure is used to "adjust" the concentration response
proportions for mortality occurring in the control replicates.
3. Use of the Trimmed Spearman-Karber Method is recommended only
when the requirements for the Probit Analysis and the Spearman-
Karber Method are not met.
4. To calculate the LC50 using the Trimmed Spearman-Karber
Method, the smoothed, adjusted, observed proportion mortalities
must bracket 0.5.
5. To calculate the 95% confidence interval for the LC50
estimate, one or more of the smoothed, adjusted, observed
proportion mortalities must be between zero and one.
6. Let p0/ Pi, ..., 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 p0 £ px s . ! . <: pk.
The smoothing process replaces any adj acent pA' s that do not
conform to p0 £ Pi '£ ... ^ Pk/ with their average. For example,
if Pi is less than p^ then:
P"-i = P! = (Pi + Pi-i)/2
Where: pf = the smoothed observed proportion mortality for
effluent concentration i.
638
-------
7. Adjust the smoothed observed proportion mortality in each
effluent concentration for mortality in the control group using
Abbott's formula (Finney, 1971). The -adjustment takes the form:
Pf = (PS : P§) / (1 - P^> :
Where: pjj = the smoothed .observed proportion mortality for the
control i
pf = the smoothed observed proportion mortality for
effluent concentration i.
8. Calculate the amount of trim to use in the estimation of the
LC50 as follows: :
Trim = max(pf, l-p|)
Where: p? = the smoothed, adjusted proportion mortality for
the lowest effluent concentration, exclusive of
the control ' ! .
pg = the smoothed, adjusted proportion mortality for
the highest effluent concentration
k = •• the number of effluent concentrations, exclusive
of the control. . .
The minimum trim should be calculated for each.data set rather
than using a fixed amount of trim for each, data set. .
9. Due to the intensive nature of the calculation for the
estimated LC50 and the calculation of the associated 95.%
confidence interval using the Trimmed Spearman-Kiarber Method, .it
is recommended that the data be analyzed by computer.
10. A, computer program which estimates the LC50 and associated
95% confidence interval using the Trimmed Spearman-Karber Method,
can be obtained through EMSL, 3411 Church Street, Cincinnati, OH
45244. The program can be obtained from EMSL-Cincinnati by
sending a written request to the above address. ',
639
-------
11. The Trimmed Spearman-Karber program automatically performs
the following functions:
a. Smoothing.
b. Adjustment for mortality in the control.
c. Calculation of the necessary trim.
d. Calculation of the LC50.
e. Calculation of the associated 95% confidence interval.
12. To illustrate the Trimmed Spearman-Karber method using the
Trimmed Spearman-Karber computer program, a set of data from a
Topsmelt Larval Survival and Growth .test will be used. The data
are listed in Table J.I.
TABLE J.I. EXAMPLE OF TRIMMED SPEARMAN-KARBER 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
0
2
1
5
25
25
Mortality
Proportion
0.00
0.08
0.04
0.20
1.00.
1.00
12.1 The program requests the following input (Figure J.I) :
a. Output destination (D = disk file or P = printer).
b. Control data.
c. Data for each toxicant concentration.
12.2 The program output includes the following (Figure J.2):
a. A table of the concentrations tested, number of
organisms exposed, and the mortalities.
b. The amount of trim used in the calculation.
c. The estimated LC50 and the associated 95% confidence
interval.
640
-------
A:>TSK
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5
ENTER DATE OF TEST:
1 I
ENTER TEST NUMBER:
2
WHAT IS TO BE ESTIMATED? -
(ENTER "L" FOR LC50 AND "E" 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 i
ENTER THE NUMBER OF MORTALITIES IN THE CONTROL: i
'° - - ' I
ENTER THE NUMBER OF CONCENTRATIONS
(NOT INCLUDING THE CONTROL; MAX = 10): '
5 ' |
ENTER THE 5 EXPOSURE CONCENTRATIONS (IN INCREASING ORDER):
6.25 12.5 25 50 100
ARE THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION EQUAL(Y/N)?
y
ENTER THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION:
25 ;
ENTER UNITS FOR DURATION OF EXPERIMENT I
(ENTER "H" FOR HOURS, "D" FOR DAYS, ETC.): !
Days - -•- .
ENTER DURATION OF TEST:
7 .
ENTER THE NUMBER OF MORTALITIES AT EACH EXPOSURE CONCENTRATION:
2 1 5 25 25
WOULD YOU LIKE THE AUTOMATIC TRIM CALCULATION(Y/N)?
Y ,
Figure J.I. Example input for Trimmed Spearman-Karber Method.
641
-------
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5
DATE : 1
TOXICANT:
SPECIES :
effluent
Topsmelt
RAW DATA: Concentration
.00
6.25
12.50
25.00
50.00
100.00
TEST NUMBER: 2
Number
Exposed
25
25
25
25
25
25
DURATION:
7 Days
Mortalities
0
2
1
5
25
25
SPEARMAN-KARBER TRIM:
6.00%
SPEARMAN-KARBER ESTIMATES: LC50:
95% LOWER CONFIDENCE:
95% UPPER CONFIDENCE:
30.98
27.17
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 LC50. It is a
mathematical procedure which estimates the LC50 by linearly
interpolating between points of a plot of observed percent
mortality versus the base 10 logarithm (Iog10) of percent
effluent concentration. This method does not provide a
confidence interval for the LC50 estimate and it's 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%. j
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.I. >
' TABLE K.I. 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
0
0
0
25
25
Mortality
Proportion
0.04
0.00
0.00
0.00
1.00
1.00
643
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4. Let p0/ px/ . .., pk denote the observed proportion mortalities
for the control and the k effluent concentrations. The first
step is to smooth the p± if they do not satisfy p0i ^ px £ ... <; pk.
The smoothing process replaces any adjacent PI'S that do not
conform to p0 £ p± z ... <: pk with their average. For example, if
Pi is less than p^ then:
P- = P • (P+P) /2
Where: pf = the smoothed observed proportion mortality for
effluent concentration i.
4.1 For the data in this example, because the observed mortality
proportions for the 6.25%, 12.5%, and 25.0% effluent
concentrations are less than the observed response proportion for
the control, the values for these four groups must be averaged:
s s s s 0.04 + 0.00 + 0.00 + 0.00 0.04
Po -Pi - P2 * P3 - I • —— = °-01
4.2 Since p4 = ps = 1.00 are larger then 0.01, set pf = pf =
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:
Pi =
Where: pf = the smoothed observed proportion mortality for the
control
pf = the smoothed observed proportion mortality for
effluent concentration i.
644
-------
5.1 Because the smoothed observed proportion mortality for the
control group is greater than zero, the responses must be
adjusted using Abbott's formula, as follows: - • .
a Pi - Po 0.01 - 0.01 0.0
Po = Pi = Pz = P3 = = ' = = 0.0
° -, • 1 - 0.0125 0.139
1 " Po
P4 - Po 1.00 - 0,01 0.99
= s
1 _ ps 1 - 0.01 0.99
A table of the smoothed, adjusted response proportions for the
effluent concentrations is shown in Table K.2.
TABLE K.2. EXAMPLE OF GRAPHICAL METHOD: SMOOTHED, ADJUSTED
MORTALITY DATA FROM A TOPSMELT LARVAL SURVIVAL AND
GROWTH TEST . ',
Effluent
Concentration
*
Control
6.25
12.5
25.0
50.0
100.0
Mortality
Proportion
0.04
0.00
0.00
0.00
1.00
1.00
i
Smoothed
Mortality
Proportion
0.01
' 0.01
0.01
0.01
i.oo •;
1.00
Smoothed ,
Adjusted
Mortality
Proporti on
0.00
0.00
0.00
. 0.00
1.00
1.00
5.2 Plot the smoothed, adjusted data on 2-cycle:semi-log graph
paper with the logarithmic axis (the y axis) used for percent
effluent concentration and the linear axis (the x axis) used for
observed percent mortality. A plot of the smoothed, adjusted
data is shown in Figure K.I.
i
645 i
-------
100
LU
LLJ
LL
Li.
LU
LU
O
LU
Q.
50
10
1
Figure K.
0 10 20 30 40 50 60 70 80 90 100
PERCENT MORTALITY
Plot of the smoothed adjusted response proportions for
topsmelt, Atherinops affinis, survival data.
646
-------
6. Locate the two points on the graph which bracket 50%
mortality and connect them with a straight line.
7. On the scale for percent effluent concentration, read the
value for the point where the plotted line and the 50% mortality
line intersect. This value is the estimated LC50 expressed as a
percent effluent concentration. '
7.1 For this example, the two points on the graph which bracket
the 50% mortality line (0% mortality at 25% effluent, and 100%
mortality at 50% effluent) are connected with a straight line;
The point at which the plotted line intersects the 50% mortality
line is the estimated LC50. The estimated LC50 = 35% effluent.
647
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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 1C). The procedure was designed for general
applicability in the analysis of data from short-term chronic
toxicity tests, and the generation of an endpoint from a
continuous model that allows a traditional quantitative
assessment of the precision of the endpoint, such as confidence
limits for the endpoint of a single test, and a mean and
coefficient of variation for the endpoints of multiple tests.
1.2 The Linear Interpolation Method assumes that the responses
(1) are monotonically non-increasing, where the mean response for
each higher concentration is less than or equal to the mean
response for the previous concentration, (2) follow a piecewise
linear response function, and (3) are from a random, independent,
and representative sample of test data. If the data are not
monotonically non-increasing, they are adjusted by smoothing
(averaging). In cases where the responses ,at the low toxicant
concentrations are much higher than in the controls, the
smoothing process may result in a large upward adjustment in the
control mean. Also, no 'assumption is made about the distribution
of the data except that the data within a group being resampled
are independent and identically distributed.
2. DATA SUMMARY AND PLOTS
2.1 Calculate the mean responses for the control and each
toxicant concentration, construct a summary table, and plot the
data.
3. MONOTONICITY
3.1 If the assumption of monotonicity of test results is met,
the observed response means (Yj should stay the same or decrease
648
-------
as the toxicant concentration increases. If the means do not
decrease monotonically, the responses are "smoothed" by averaging
(pooling) adjacent means.
3.2 Observed means at each concentration are considered in order
of increasing concentration, starting with the control mean (Yx) .
If the mean observed response at the lowest toxicant
concentration (Y2) is equal to or smaller than the control mean
(Yj , it is used as the response. If it is larger than the
control mean, it is averaged with the control, and this average
is used for both the control response (Mj.) and the lowest
toxicant concentration response (M2). This mean is then compared
to the mean observed response for the next higher toxicant
concentration (Y3) . Again, if the mean observed response for the
next higher toxicant concentration is smaller than the mean of
the control and the lowest toxicant concentration, it is used as
the response. If it is higher than the mean of the first two, it
is averaged with the first two, and the mean is used as the
response for the control and 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 Yi decrease monotonically, the Y± become M±
without smoothing. I
i
4. LINEAR INTERPOLATION METHOD
4.1 The method assumes a linear response from one concentration
to the next. Thus, the ICp is estimated by linear interpolation
between two concentrations whose responses bracket the response
of interest, the (p) percent reduction from the control'.
.
4.2 To obtain the estimate, determine the concentrations Cj and
GJ+I which bracket the response Mx (1 - p/100) , where HL is the
smoothed control mean response and p is the percent reduction in
.response relative to the control response. These calculations
can easily be done by hand or with a computer program as
described below. The linear interpolation estimate is
calculated as follows: I
649
-------
(C - C )
ICp - Cr + [ M (1 - p/100) - AT ] —2-li —
Where: Cj = tested concentration whose observed mean
response is greater than Mx(l - p/100).
Cj + ! = tested concentration whose observed mean
response is less than'Mi (1 - p/100).
M! = smoothed mean response for the control.
Mj = smoothed mean response for concentration
J.
Mj + j. = smoothed mean response for concentration
J + 1.
percent reduction in response relative
to the control response.
ICp = estimated concentration at which there
is a percent reduction from the smoothed
mean control response. The ICp is
reported for the test, together with the
95% confidence interval calculated by
the ICPIN.EXE program described below.
4.3 If the Cj is the highest concentration tested, the ICp would
be specified as greater than Q. If the response at the lowest
concentration tested is used to extrapolate the ICp' value, the
ICp should be expressed as a less than the lowest test
concentration.
5. CONFIDENCE INTERVALS
5.1 Due to the use of a linear interpolation technique to
calculate an estimate of the ICp, standard statistical -methods
for calculating confidence intervals are not applicable for the
ICp. This limitation is avoided by use a technique known as the
bootstrap method as proposed by Efron (1982) for deriving point
estimates and confidence intervals.
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 true mean. In the bootstrap method, the test
data Yji is randomly resampled with replacement to produce a new
set of data Y^*, that is statistically equivalent to the
original data, but a new and slightly different estimate of the
ICp (ICp*) is obtained. This process is repeated at least 80
times (Marcus- and Holtzman, 1988) resulting in multiple "data"
sets, each with an associate ICp* estimate. The distribution of
the ICp* estimates derived from the sets of resampled data
approximates the sampling distribution of the ICp estimate. The
standard error of the ICp is estimated by the standard deviation
of the individual ICp* estimates,. Empirical confidence intervals
are derived from the quantiles of the ICp* empirical
distribution. For example, if the test data are resampled a
minimum of 80 times, the empirical .2.5% and the 97.5% confidence
limits are approximately the second smallest and second largest
ICp* estimates (Marcus and Holtzman, 1988).
5.3 The width of the confidence intervals calculated by the
bootstrap method is related to the variability qf the data. When
confidence intervals are. wide, the reliability of the 1C estimate
is in question. However, narrow intervals do not necessarily
indicate, that the estimate is highly reliable, because of
undetected violations .of assumptions and the fact that the
confidence limits based on the empirical quantiles of a bootstrap
distribution of 80 samples may be unstable.
5.4 The bootstrapping method of calculating confidence intervals
is computationally intensive. For this reason, all of the
calculations associated with,determining the confidence .intervals
for the ICp estimate have been incorporated into a computer
program. Computations are most easUly done with a computer,
program such as the revision of the BOOTSTRP program (USEPA,
1988; USEPA, 1989) which is now called "ICPIN" and is described
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.I includes the raw
data and the mean growth for each concentration. A plot of the
data is provided in Figure L.I.
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 Y± and the
smoothed means by M±. Table L.2 contains the smoothed means and
Figure L.I 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,
where Mid-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 C5 =
10.0%.
039,
TABLE L.I. MYSID, HOLMESIMYSIS COST ATA, GROWTH DATA
Replicate Control
Concentration (%)
1.80
3.20
5.60
10.0
1
2
3
4
5
Mean(Yi)
i
0.048
0.058
0.047
0.058
0.051
0.052
1
0,055
0.048
0.042
0.041
0.052
, 0.048
2
0.057
0.050
0.046
0.043
0.045
0.048
3
0.041
0.040
0.041
0.043
0.040
0.041
4
0.033
0.000
0.000
0.000
0.000
0.007
5
652
-------
CO
O)
II
•X-
co
fO
O)
•a
a>
i
co
•a
co to
c: +->
ra (a
a> -a
> 5
5_ S_
O) O)
co
to
ra O
"O O
O)
1H3I3M
NV3W
653
-------
TABLE 1.2. MYSID, HOLMESIMYSIS COSTATA. MEAN
GROWTH RESPONSE AFTER SMOOTHING
Toxicant
Cone.
(*)
Control
1.80
3.20
5.60
10.00
18.00
i
1
2
3
4
5
6
Response
Means
YI (mg)
0.052
0.048
0.048
0.041
0.007
0.000
Smoothed
Means
Mi (mg)
0.052
0.048
0.048
0.041
0.007
0.000
6.3.2 Using the equation from section 4.2, the estimate of the
IC25 is calculated as follows:
ICp • CJ * [ M1 (I - p/100) - MJ ]
IC25 - 5.60 + [0.052 (1 - 25/100} - 0.041]
= 5.86%
(10.0 - 5.60)
(0.007 - 0.041)
6.4 CONFIDENCE INTERVALS
6.4.1 Confidence intervals for the ICp are derived using the
bootstrap method. As described above, this method involves
randomly resampling the individual observations and recalculating
the ICp at least 80 times, and determining the mean ICp, standard
deviation, and empirical 95% confidence intervals. For this
reason, the confidence intervals are calculated using a computer
program called ICPIN. This program is described below and is
available to carry out all the calculations of both the
interpolation estimate (ICp) and the confidence intervals.,
654
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7. COMPUTER CALCULATIONS
7.1 The computer program, ICPIN, prepared for the Linear
Interpolation Methods was written in TURBO PASCAL for IBM
compatible PCs. The program (version 2.0) has been modified by
Computer Science Corporation, Duluth, MN with funding provided by
the Environmental Research Laboratory, Duluth, MN (Norberg-King,
1993). The program was originally developed by Battelle
Laboratories, Columbus, OH through a government;contract
supported by the Environmental Research Laboratory, Duluth, MN
(USEPA, 1988) . A compiled, executable version of the program and
supporting documentation can be obtained by sending a written
request to EMSL-Cincinnati, 3411 Church Street,\Cincinnati, OH
45244. , !
7.2 The ICPIN.EXE program performs the following functions: 1)
it calculates the observed response means (Yj (response means);
2) it calculates the standard.deviations; 3) checks the
responses for monotonicity; 4) calculates smoothed means (Mi)
(pooled response means) if necessary; 5) uses the means., Mi, to
calculate the initial ICp of choice by linear interpolation; 6)
performs a user-specified number of bootstrap resamples between
80 and 1000 (as multiples o£ 40); 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). j •
i
7.3 For the ICp calculation, up to twelve treatments can be
input (which includes the control). There can be up to 40
replicates per concentration, and the program does not require an
equal number of replicates per concentration. The value of p can
range from 1% to 99%. : .
7.4 DATA INPUT • j '
I
7.4.1 Data is entered directly into the program onscreen. A
sample data entry screen in shown in Figure L.2. The program
documentation provides guidance on the entering and analysis of
data for the Linear Interpolation Method.
655
-------
ICp Data Entry/Edit Screen
Current File:
Cone. ID
Cone. Tested
Response 1
Response 2
Response 3
Response 4
Response 5
Response 6
Response 7
Response 8
Response 9
Response 10
Response 11
Response 12
Response 13
Response 14
Response 15
Response 16
Response 17
Response 18
Response 19
Response 20
1
2
3
4
5
6
F10 for Command Menu
Use Arrow Keys to Switch Fields
Figure L.2. ICp data entry/edit screen. Twelve concentration identifications can be
used. Data for concentrations are entered in columns 1 through 6. For
concentrations 7 through 12 and responses 21-40 the data is entered in
additional fields of the same screen.
656
-------
7.4.2 The user selects the ICp estimate desired (e.g., IC25 or
IC50) and the number of resamples to be taken for the bootstrap
method of calculating the confidence intervals. The program has
the capability of performing any number of resamples from 80 to
1000 as multiples of 40. However, Marcus and Holtzman (1988)
recommend a minimum of 80 resamples for the bootstrap method be
used and at least 250 resamples are better (Norberg-King, 1993).
7.5 DATA OUTPUT |
7.5.1 The program output includes the. following (see Figure L.3)
1. A table of the;concentration identification, the
concentration tested and raw data response for each
replicate and concentration. .
2. A table of test concentrations, number of replicates,
concentration (units), response means (Yj , standard
deviations for each response mean, and the pooled
response means (smoothed means; M±) .
3. The linear interpolation estimate of the IGp using the
means (Mi) . Use this value for the ICp estimate.
4. The mean IGp and standard deviation from the .bootstrap
resampling.
5. The confidence intervals calculated by the bootstrap
method for the ICp. Provides an original 95% confidence
intervals to be used with the initial ICp when the number
of replicates per concentration is over six and provides
both original and expanded confidence intervals when the
number of replicates per concentration are less than '
seven.
•
7.6 ICPIN program output for the analysis of the mysid growth
data in Table L.I is provided in Figure L.3. ;
7.6.1 When the ICPIN program was used to analyze this 'set of
data, requesting 80 resamples, the estimate of the IC25 was
5.8174%. The empirical 95% confidence intervals for the true
mean was 4.9440% to 6.2553%.
657
-------
Cone . ID
Cone . Tested
Response
Response
Response
Response
Response
1
2
3
4
5
1
0
.048
.058
.047
.058
.051
2
1.80
.055
.048
.042
.041
.052
3'
3.20
.057
.050
.046
.043
.045
4
5.60
.041
.040
.041
.043
.040
5
10.0
.033
0
0
iO
0
6
18.0
0
0
0
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
ID Replicates
1
2
3
4
5
6
5
5
5
5
5
5
Concentration
%
0
1
3
5
10
18
.000
.800
.200
.600
.000
.000
Response
Means
0
0
0
0
0
0
.052
.048
.048
.041
.007 -
.000
Std.
Dev .
0.
0.
0.
0.
0.
0.
005
006
006
001
015
000
Pooled
Response Means
0.
0.
0.
; o.
0.
0.
052
048
048
041
007
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 L.3. Example of ICPIN program output for the IC25.
658
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I
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i
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«U.S. GOVEMMENT PRINTING OFFICE:! 99 5-650-006 / 22 074
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